PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR

ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)

BY

NABIL Y KHADDURI

A thesis submitted to the Graduate School

in partial fulfillment of the requirements

for the degree

Master of Science

Major subject Horticulture

New Mexico State University

Las Cruces New Mexico

May 2002

Percussion as an Alternative Seed Treatment for Robinia neomexicana (New Mexico

Locust) a thesis by Nabil Y Khadduri in partial fulfillment of the requirements for

the degree Master of Science has been approved and accepted by the following

Interim Dean of the Ora uate School

Chair ofExamining Committee

Date

Committee in charge

Dr John T Harrington Chair

Dr David R Dreesen

Dr Leigh Murray

Dr Rolston St Hilaire

ACKNOWLEDGMENTS

I would like to thank Dr John Harrington for his guidance sense ofhumor

and flexibility throughout my studies I would also like to thank the members ofmy

committee for their support especially Dr Murray who patiently taught me the

subtleties of categorical analysis of variance

I am also grateful to Dr Anne Wagner and Molycorp Mine who provided

funding for this research Thanks to Dr Bob Karrfali for his assistance with x-ray

radiography and to Mohamed Murshed and Dr Soumitra Ghoshroy for their

assistance with scanning electron microscopy Special thanks go to my colleague Lee

Rosner who assisted me in all phases of this project Finally I thank my family for

their enduring love Jill Walid Amin Rolla Khalid Piet Zach Charlie and above

all mywife Lucie

111

VITA

June 3 1972

June 1990

May 1994

July 1995

August 1994- March 1998

March 1998-August 2000

August 2000-May 2002

Born in Beirut Lebanon

Graduated from Georgetown Day High School Washington DC

Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science

Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree

6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM

Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM

Graduate Research Assistant at New Mexico State University

FIELD OF STUDY

Horticulture

iv

ABSTRACT

PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR

ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)

BY

NABIL Y KHADDURI

Master of Science in Horticulture

New Mexico State University 2002

Las Cruces New Mexico

Dr John T Harrington Chair

New Mexico locust (Robinia neomexicana A Gray) a small tree native to

montane regions of the southwestern United States fills a successional role in postshy

disturbance situations The ability to colonize rapidly and ameliorate harsh sites

contributes to the importance ofNew Mexico locust in reclamation efforts Seedling

production for outplanting on disturbed sites has been hampered by poor germination

This research sought to improve seed propagation techniques in New Mexico locust

Nine seed sources were collected across a range of latitudes within New

Mexico Seed treatments included a control three levels of hot water and four levels

of percussion scarification Hot water treatments included immersion at 100degC and

boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1

2 4 and 8 minutes A total of four lOO-seed samples were used to test each source

by scarification treatment combination Categorical Analysis of Variance was used to

determine main effects and interactions Pairwise comparisons were conducted to

compare non-control treatment means using a conservative alpha value of 005

divided by the number of comparisons

Averaged over all sources and for each source percussion scarification

durations of4 or 8 minutes yielded greatest germination In addition to consistently

bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or

faster than all other treatments Averaged over all sources germination increased

with increasing durations of percussion then leveled off for the 4- and 8-minute

treatments Immersion and 30-second boiling scarification maximized germination

for the hot water treatments with a decrease in germination when seeds were boiled

for 60 seconds

Percussion scarification specifically weakens the strophiole the natural

dormancy barrier in many papilionoid legumes without excessively damaging the

seed coat As with New Mexico locust papilionoid legumes often play an integral

role in the revegetation ofdisturbed lands The percussion scarification technique

developed in this research has the potential to facilitate the use of these valuable

species in reclamation projects

VI

CONTENTS

LIST OF TABLES ix

LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x

INTRODUCTION 1

LITERATlTRE REVIEW 4

Seed Donnancy 4

Physical Donnancy 5

Evolutionary Role ofPhysical Donnancy 6

Methods for Breaking Donnancy 6

Acid Scarification 6

Hot Water Scarification 8

Mechanical Scarification 11

Alternative Method for Breaking Donnancy Percussion 12

Objectives 15

Hypothesis 15

MATERIALS AND METHODS 16

Seed Collection and Cleaining 16

Experimental Design and Treatment Structure 16

Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20

G T ennlnatlon estmg 21

X-ray Radiography 22

Scanning Electron Microscope Photography 22

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Percussion as an Alternative Seed Treatment for Robinia neomexicana (New Mexico

Locust) a thesis by Nabil Y Khadduri in partial fulfillment of the requirements for

the degree Master of Science has been approved and accepted by the following

Interim Dean of the Ora uate School

Chair ofExamining Committee

Date

Committee in charge

Dr John T Harrington Chair

Dr David R Dreesen

Dr Leigh Murray

Dr Rolston St Hilaire

ACKNOWLEDGMENTS

I would like to thank Dr John Harrington for his guidance sense ofhumor

and flexibility throughout my studies I would also like to thank the members ofmy

committee for their support especially Dr Murray who patiently taught me the

subtleties of categorical analysis of variance

I am also grateful to Dr Anne Wagner and Molycorp Mine who provided

funding for this research Thanks to Dr Bob Karrfali for his assistance with x-ray

radiography and to Mohamed Murshed and Dr Soumitra Ghoshroy for their

assistance with scanning electron microscopy Special thanks go to my colleague Lee

Rosner who assisted me in all phases of this project Finally I thank my family for

their enduring love Jill Walid Amin Rolla Khalid Piet Zach Charlie and above

all mywife Lucie

111

VITA

June 3 1972

June 1990

May 1994

July 1995

August 1994- March 1998

March 1998-August 2000

August 2000-May 2002

Born in Beirut Lebanon

Graduated from Georgetown Day High School Washington DC

Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science

Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree

6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM

Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM

Graduate Research Assistant at New Mexico State University

FIELD OF STUDY

Horticulture

iv

ABSTRACT

PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR

ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)

BY

NABIL Y KHADDURI

Master of Science in Horticulture

New Mexico State University 2002

Las Cruces New Mexico

Dr John T Harrington Chair

New Mexico locust (Robinia neomexicana A Gray) a small tree native to

montane regions of the southwestern United States fills a successional role in postshy

disturbance situations The ability to colonize rapidly and ameliorate harsh sites

contributes to the importance ofNew Mexico locust in reclamation efforts Seedling

production for outplanting on disturbed sites has been hampered by poor germination

This research sought to improve seed propagation techniques in New Mexico locust

Nine seed sources were collected across a range of latitudes within New

Mexico Seed treatments included a control three levels of hot water and four levels

of percussion scarification Hot water treatments included immersion at 100degC and

boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1

2 4 and 8 minutes A total of four lOO-seed samples were used to test each source

by scarification treatment combination Categorical Analysis of Variance was used to

determine main effects and interactions Pairwise comparisons were conducted to

compare non-control treatment means using a conservative alpha value of 005

divided by the number of comparisons

Averaged over all sources and for each source percussion scarification

durations of4 or 8 minutes yielded greatest germination In addition to consistently

bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or

faster than all other treatments Averaged over all sources germination increased

with increasing durations of percussion then leveled off for the 4- and 8-minute

treatments Immersion and 30-second boiling scarification maximized germination

for the hot water treatments with a decrease in germination when seeds were boiled

for 60 seconds

Percussion scarification specifically weakens the strophiole the natural

dormancy barrier in many papilionoid legumes without excessively damaging the

seed coat As with New Mexico locust papilionoid legumes often play an integral

role in the revegetation ofdisturbed lands The percussion scarification technique

developed in this research has the potential to facilitate the use of these valuable

species in reclamation projects

VI

CONTENTS

LIST OF TABLES ix

LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x

INTRODUCTION 1

LITERATlTRE REVIEW 4

Seed Donnancy 4

Physical Donnancy 5

Evolutionary Role ofPhysical Donnancy 6

Methods for Breaking Donnancy 6

Acid Scarification 6

Hot Water Scarification 8

Mechanical Scarification 11

Alternative Method for Breaking Donnancy Percussion 12

Objectives 15

Hypothesis 15

MATERIALS AND METHODS 16

Seed Collection and Cleaining 16

Experimental Design and Treatment Structure 16

Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20

G T ennlnatlon estmg 21

X-ray Radiography 22

Scanning Electron Microscope Photography 22

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

ACKNOWLEDGMENTS

I would like to thank Dr John Harrington for his guidance sense ofhumor

and flexibility throughout my studies I would also like to thank the members ofmy

committee for their support especially Dr Murray who patiently taught me the

subtleties of categorical analysis of variance

I am also grateful to Dr Anne Wagner and Molycorp Mine who provided

funding for this research Thanks to Dr Bob Karrfali for his assistance with x-ray

radiography and to Mohamed Murshed and Dr Soumitra Ghoshroy for their

assistance with scanning electron microscopy Special thanks go to my colleague Lee

Rosner who assisted me in all phases of this project Finally I thank my family for

their enduring love Jill Walid Amin Rolla Khalid Piet Zach Charlie and above

all mywife Lucie

111

VITA

June 3 1972

June 1990

May 1994

July 1995

August 1994- March 1998

March 1998-August 2000

August 2000-May 2002

Born in Beirut Lebanon

Graduated from Georgetown Day High School Washington DC

Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science

Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree

6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM

Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM

Graduate Research Assistant at New Mexico State University

FIELD OF STUDY

Horticulture

iv

ABSTRACT

PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR

ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)

BY

NABIL Y KHADDURI

Master of Science in Horticulture

New Mexico State University 2002

Las Cruces New Mexico

Dr John T Harrington Chair

New Mexico locust (Robinia neomexicana A Gray) a small tree native to

montane regions of the southwestern United States fills a successional role in postshy

disturbance situations The ability to colonize rapidly and ameliorate harsh sites

contributes to the importance ofNew Mexico locust in reclamation efforts Seedling

production for outplanting on disturbed sites has been hampered by poor germination

This research sought to improve seed propagation techniques in New Mexico locust

Nine seed sources were collected across a range of latitudes within New

Mexico Seed treatments included a control three levels of hot water and four levels

of percussion scarification Hot water treatments included immersion at 100degC and

boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1

2 4 and 8 minutes A total of four lOO-seed samples were used to test each source

by scarification treatment combination Categorical Analysis of Variance was used to

determine main effects and interactions Pairwise comparisons were conducted to

compare non-control treatment means using a conservative alpha value of 005

divided by the number of comparisons

Averaged over all sources and for each source percussion scarification

durations of4 or 8 minutes yielded greatest germination In addition to consistently

bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or

faster than all other treatments Averaged over all sources germination increased

with increasing durations of percussion then leveled off for the 4- and 8-minute

treatments Immersion and 30-second boiling scarification maximized germination

for the hot water treatments with a decrease in germination when seeds were boiled

for 60 seconds

Percussion scarification specifically weakens the strophiole the natural

dormancy barrier in many papilionoid legumes without excessively damaging the

seed coat As with New Mexico locust papilionoid legumes often play an integral

role in the revegetation ofdisturbed lands The percussion scarification technique

developed in this research has the potential to facilitate the use of these valuable

species in reclamation projects

VI

CONTENTS

LIST OF TABLES ix

LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x

INTRODUCTION 1

LITERATlTRE REVIEW 4

Seed Donnancy 4

Physical Donnancy 5

Evolutionary Role ofPhysical Donnancy 6

Methods for Breaking Donnancy 6

Acid Scarification 6

Hot Water Scarification 8

Mechanical Scarification 11

Alternative Method for Breaking Donnancy Percussion 12

Objectives 15

Hypothesis 15

MATERIALS AND METHODS 16

Seed Collection and Cleaining 16

Experimental Design and Treatment Structure 16

Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20

G T ennlnatlon estmg 21

X-ray Radiography 22

Scanning Electron Microscope Photography 22

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

VITA

June 3 1972

June 1990

May 1994

July 1995

August 1994- March 1998

March 1998-August 2000

August 2000-May 2002

Born in Beirut Lebanon

Graduated from Georgetown Day High School Washington DC

Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science

Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree

6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM

Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM

Graduate Research Assistant at New Mexico State University

FIELD OF STUDY

Horticulture

iv

ABSTRACT

PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR

ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)

BY

NABIL Y KHADDURI

Master of Science in Horticulture

New Mexico State University 2002

Las Cruces New Mexico

Dr John T Harrington Chair

New Mexico locust (Robinia neomexicana A Gray) a small tree native to

montane regions of the southwestern United States fills a successional role in postshy

disturbance situations The ability to colonize rapidly and ameliorate harsh sites

contributes to the importance ofNew Mexico locust in reclamation efforts Seedling

production for outplanting on disturbed sites has been hampered by poor germination

This research sought to improve seed propagation techniques in New Mexico locust

Nine seed sources were collected across a range of latitudes within New

Mexico Seed treatments included a control three levels of hot water and four levels

of percussion scarification Hot water treatments included immersion at 100degC and

boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1

2 4 and 8 minutes A total of four lOO-seed samples were used to test each source

by scarification treatment combination Categorical Analysis of Variance was used to

determine main effects and interactions Pairwise comparisons were conducted to

compare non-control treatment means using a conservative alpha value of 005

divided by the number of comparisons

Averaged over all sources and for each source percussion scarification

durations of4 or 8 minutes yielded greatest germination In addition to consistently

bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or

faster than all other treatments Averaged over all sources germination increased

with increasing durations of percussion then leveled off for the 4- and 8-minute

treatments Immersion and 30-second boiling scarification maximized germination

for the hot water treatments with a decrease in germination when seeds were boiled

for 60 seconds

Percussion scarification specifically weakens the strophiole the natural

dormancy barrier in many papilionoid legumes without excessively damaging the

seed coat As with New Mexico locust papilionoid legumes often play an integral

role in the revegetation ofdisturbed lands The percussion scarification technique

developed in this research has the potential to facilitate the use of these valuable

species in reclamation projects

VI

CONTENTS

LIST OF TABLES ix

LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x

INTRODUCTION 1

LITERATlTRE REVIEW 4

Seed Donnancy 4

Physical Donnancy 5

Evolutionary Role ofPhysical Donnancy 6

Methods for Breaking Donnancy 6

Acid Scarification 6

Hot Water Scarification 8

Mechanical Scarification 11

Alternative Method for Breaking Donnancy Percussion 12

Objectives 15

Hypothesis 15

MATERIALS AND METHODS 16

Seed Collection and Cleaining 16

Experimental Design and Treatment Structure 16

Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20

G T ennlnatlon estmg 21

X-ray Radiography 22

Scanning Electron Microscope Photography 22

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

ABSTRACT

PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR

ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)

BY

NABIL Y KHADDURI

Master of Science in Horticulture

New Mexico State University 2002

Las Cruces New Mexico

Dr John T Harrington Chair

New Mexico locust (Robinia neomexicana A Gray) a small tree native to

montane regions of the southwestern United States fills a successional role in postshy

disturbance situations The ability to colonize rapidly and ameliorate harsh sites

contributes to the importance ofNew Mexico locust in reclamation efforts Seedling

production for outplanting on disturbed sites has been hampered by poor germination

This research sought to improve seed propagation techniques in New Mexico locust

Nine seed sources were collected across a range of latitudes within New

Mexico Seed treatments included a control three levels of hot water and four levels

of percussion scarification Hot water treatments included immersion at 100degC and

boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1

2 4 and 8 minutes A total of four lOO-seed samples were used to test each source

by scarification treatment combination Categorical Analysis of Variance was used to

determine main effects and interactions Pairwise comparisons were conducted to

compare non-control treatment means using a conservative alpha value of 005

divided by the number of comparisons

Averaged over all sources and for each source percussion scarification

durations of4 or 8 minutes yielded greatest germination In addition to consistently

bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or

faster than all other treatments Averaged over all sources germination increased

with increasing durations of percussion then leveled off for the 4- and 8-minute

treatments Immersion and 30-second boiling scarification maximized germination

for the hot water treatments with a decrease in germination when seeds were boiled

for 60 seconds

Percussion scarification specifically weakens the strophiole the natural

dormancy barrier in many papilionoid legumes without excessively damaging the

seed coat As with New Mexico locust papilionoid legumes often play an integral

role in the revegetation ofdisturbed lands The percussion scarification technique

developed in this research has the potential to facilitate the use of these valuable

species in reclamation projects

VI

CONTENTS

LIST OF TABLES ix

LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x

INTRODUCTION 1

LITERATlTRE REVIEW 4

Seed Donnancy 4

Physical Donnancy 5

Evolutionary Role ofPhysical Donnancy 6

Methods for Breaking Donnancy 6

Acid Scarification 6

Hot Water Scarification 8

Mechanical Scarification 11

Alternative Method for Breaking Donnancy Percussion 12

Objectives 15

Hypothesis 15

MATERIALS AND METHODS 16

Seed Collection and Cleaining 16

Experimental Design and Treatment Structure 16

Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20

G T ennlnatlon estmg 21

X-ray Radiography 22

Scanning Electron Microscope Photography 22

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

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N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

2 4 and 8 minutes A total of four lOO-seed samples were used to test each source

by scarification treatment combination Categorical Analysis of Variance was used to

determine main effects and interactions Pairwise comparisons were conducted to

compare non-control treatment means using a conservative alpha value of 005

divided by the number of comparisons

Averaged over all sources and for each source percussion scarification

durations of4 or 8 minutes yielded greatest germination In addition to consistently

bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or

faster than all other treatments Averaged over all sources germination increased

with increasing durations of percussion then leveled off for the 4- and 8-minute

treatments Immersion and 30-second boiling scarification maximized germination

for the hot water treatments with a decrease in germination when seeds were boiled

for 60 seconds

Percussion scarification specifically weakens the strophiole the natural

dormancy barrier in many papilionoid legumes without excessively damaging the

seed coat As with New Mexico locust papilionoid legumes often play an integral

role in the revegetation ofdisturbed lands The percussion scarification technique

developed in this research has the potential to facilitate the use of these valuable

species in reclamation projects

VI

CONTENTS

LIST OF TABLES ix

LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x

INTRODUCTION 1

LITERATlTRE REVIEW 4

Seed Donnancy 4

Physical Donnancy 5

Evolutionary Role ofPhysical Donnancy 6

Methods for Breaking Donnancy 6

Acid Scarification 6

Hot Water Scarification 8

Mechanical Scarification 11

Alternative Method for Breaking Donnancy Percussion 12

Objectives 15

Hypothesis 15

MATERIALS AND METHODS 16

Seed Collection and Cleaining 16

Experimental Design and Treatment Structure 16

Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20

G T ennlnatlon estmg 21

X-ray Radiography 22

Scanning Electron Microscope Photography 22

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

CONTENTS

LIST OF TABLES ix

LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x

INTRODUCTION 1

LITERATlTRE REVIEW 4

Seed Donnancy 4

Physical Donnancy 5

Evolutionary Role ofPhysical Donnancy 6

Methods for Breaking Donnancy 6

Acid Scarification 6

Hot Water Scarification 8

Mechanical Scarification 11

Alternative Method for Breaking Donnancy Percussion 12

Objectives 15

Hypothesis 15

MATERIALS AND METHODS 16

Seed Collection and Cleaining 16

Experimental Design and Treatment Structure 16

Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20

G T ennlnatlon estmg 21

X-ray Radiography 22

Scanning Electron Microscope Photography 22

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Analysis 22

RESULTS 24

DISCUSSION 36

Detennining the Optimal Treatment 36

Strophiole and Seed Coat Behavior 37

CONCLUSION 44

Appendices

A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47

B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64

LITERATURE CITED 72

Vlll

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

LIST OF TABLES

Table Page

1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18

2 Mean seed weight and standard error by source 19

3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25

4 Effect of seed source and treatment on G50 (days to 50 germination) 32

B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

LIST OF FIGURES

Figures Page

1 Seedsourcecollectionmap 17

2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26

3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27

4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28

5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29

6 Timecourse of New Mexico locust germination following scarification treatments 31

7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33

8 Seed damage close-ups 34

9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35

10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38

11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40

12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42

New Mexico locust a primary invader following disturbance 50

Implementation of hot water and percussion scarification 52

Effect of scarification on germination response for New Mexico locust 55

x

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56

AS Effect of scarification on germination response for black locust 57

A6 Effect of selected scarification treatments on germination speed for black locust 59

A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60

AS New Mexico locust seeds following percussion treatment at time of germination counts 61

A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

INTRODUCTION

New Mexico locust (Robinia neomexicana A Gray) a small tree native to the

southwestern United States occurs at elevations from 1200 to 2800 meters This

species fills a successional role in post-disturbance situations As a primary invader

New Mexico locust quickly establishes on burned areas arid flood banks as well as

road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root

suckering favor the successful establishment ofNew Mexico locust on disturbed sites

(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust

well suited for steep-slope revegetation where erosion is a problem

New Mexico locust is a nitrogen-fixing legume that tolerates and improves

nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium

(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and

Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust

(Klemmedson 1994)

The ability to colonize rapidly and ameliorate harsh sites contributes to New

Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable

site and creates an environment suitable for successional plant establishment New

Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas

ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a

disturbance such as wildfire New Mexico locust and Gambel oak colonize and

dominate the site until shaded out by ponderosa pine Once shaded these species

become understory shrubs a process taking an average of 15-20 years (Dick-Peddie

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with

small dense patches averaging about 005 ha beneath pine until the next disturbance

once again offers it a competitive advantage (Klemmedson 1994)

Gottfried (1980) expecting ponderosa pine regeneration to be greater where

New Mexico locust had been eradicated found that survival of planted pine seedlings

was greater where locust had not been removed In a follow-up study Gottfried

(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites

as compared to grass or 20-year old locust sites He concluded that managing an

appropriate cover ofNew Mexico locust could help regenerate pine the later

successional species

The ability ofNew Mexico locust to improve harsh sites also makes it a

candidate for reclaiming disturbances associated with mining Black locust (Robinia

pseudoacacia 1) has been used for decades to reclaim mine spoils and other

disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman

and Carpenter 1980) Ashby et al (1985) described similar positive attributes of

black locust also mentioned previously for New Mexico locust quick cover for

stabilization supply ofN and nutrient-rich litter to the soil and site improvement for

establishment of later successional trees Referring specifically to mine reclamation

the authors note the ability of black locust to grow on a wide range ofmine soil

conditions including extremely acid soils Black locust also shows some tolerance to

soils compacted by grading and topsoiling practices (Ashby et aL 1985) New

2

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Mexico locust holds promise as a native southwe~tem counterpart to black locust in

mine reclamation but it has been used infrequently to date

Natural invasion and succession occur slowly on most mine sites (Monsen

1984) While New Mexico locust often colonizes sites naturally there is no

assurance a seed will reach a particular site and establish in a reasonable time frame

The challenge is to introduce New Mexico locust as mother plants to facilitate

colonization Seedling production for outplanting on disturbed sites has been

hampered by poor germination (Lin et al 1996) The goal then is to develop

techniques to overcome poor germination in New Mexico locust thereby facilitating

macropropagation

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

LITERATURE REVIEW

Seed Dormancy

Low gennination in New Mexico locust is primarily the result of seed

donnancy the failure of an intact viable seed to genninate under favorable

conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the

embryo to genninate because of some inherent constraint (Bewley and Black 1994)

The constraint may originate within the embryo referred to as endogenous or embryo

donnancy or from the tissues surrounding the embryo known as exogenous or coatshy

imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black

1994)

New Mexico locust seed is donnant when the impenneable seed coat does not

allow movement ofwater to the embryo This specific fonn of coat-imposed

donnancy where the restraining tissue involved is an impenneable seed coat is

known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)

Physical donnancy is relatively uncommon in seeds less than 10 of all woody

species exhibit this condition (USDA Forest Service 1948) However it is

characteristic of many genera of the Fabaceae (bean) family For example all

successional trees in the genera Gleditsia and Robinia (of which New Mexico locust

is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin

4

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Physical Dormancy

Seeds that exhibit physical dormancy are said to be hard seeded referring to

the thick impermeable seed coat (Leadem 1997) The first line of protection of the

testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle

consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have

thick lignified secondary walls The most common type of sclereid cell in palisade

layers is the macrosclereid The macrosclereids contain water-repellent substances

including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the

primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh

1984)

All seed coat openings must be blocked to effectively prevent water uptake

The natural opening in a seed where it was attached to the mother plant is the weak

link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam

1988) Anatomical structures associated with the seed coat opening vary from

family to family In the Papilionoideae the subfamily of the Fabaceae family

containing New Mexico locust the site ofwater entry is the strophiole The

strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)

Kelly and van Staden (1987) refer to the strophiole site as the lens where

macrosclereid cells must separate and raise before water can pass through Since

middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole

permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to

5

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

genninating New Mexico locust artificially would be to loosen the strophiolar cells

and allow water to pass through to the embryo

Evolutionary Role of Physical Dormancy

Why is physical dormancy a mechanism that blocks germination

ecologically advantageous When a plant produc~s seeds with different degrees of

dormancy seeds that are dispersed at one time will germinate over a range of time

(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds

are exposed to changing environmental conditions improving the overall chances of

survival of the seed line (Bewley and Black 1994) Varying expression of dormancy

is under genetic control which indicates that this may be an evolved survival strategy

(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in

the southwestern US where extreme environmental variations exist

Methods for Breaking Dormancy

There are three common approaches to breaking dormancy in New Mexico

locust seed acid scarification hot water soaks and mechanical treatments In each

case the objective of the treatment is to scarify or abrade the seed coat to make it

permeable to water

Acid Scarification

Acid scarification consists of soaking seeds in concentrated sulfuric acid

rinsing thoroughly with water upon removal followed by subsequent drying before

PlaJlltmg Olson (1974) reports that this scarification method produces the highest

middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937

6

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

study conducted by HG Meginnis This stud~ t~ough conducted on black locust

seed is regularly cited in the suggested use of acid to treat New Mexico locust seed

as well (Olson 1974 Vines 1960)

Thee published studies document the specific use of sulfuric acid to treat

New Mexico locust seed Cox and Klett (1984) report that soaking seeds in

concentrated sulfuric acid for thirty minutes improved germination to 34 over an

untreated control of4 Lin et al (1996) found that for two seed lots acid

scarification for twenty-five to eighty minutes produced germination of 80 A third

lot however resulted in less than 20 germination at all durations of 10 to 80

minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10

minutes resulted in less than 20 germination across five sources However there

was an increasing trend in germination with acid soak duration and acid treatments

were not imposed for as long a time as the previous two studies (30 to 80 minutes)

The mixed results obtained by these investigators were probably due to the

widely varying germination response of seed lots to acid treatment For this reason

Olson (1974) recommends correlating duration of acid treatment to each seed lot If

treatment duration is too long the embryo will be permanently damaged If soak

duration is too short the seed will remain impermeable to water and fail to germinate

Compounding the problem of treatment duration is the negative effect acid

have on seedling growth Any type of scarification can injure seeds to some

by disrupting cells essential for growth leading to fungal invasion and

middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected

7

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)

showed more than 50 seedling mortality after gennination (Kelly and van Staden

1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication

of leakage of sugars organic acids amino acids and proteins

Perhaps the greatest drawback to acid scarification is the dangerous nature of

the treatment itself Difficulties associated with storage and trained use of

concentrated acid may discourage many propagators from using this option (Dreesen

and Harrington 1997) Consequently hot water and to a lesser extent mechanical

scarification are more commonly used and practical treatments for legume seeds

Most southwestern container growers use either of these two treatments to scarify

New Mexico locust seed in particular (Hine et al 1997)

Hot Water Scarification

The widespread use ofhot water scarification to treat locust seed is probably

due to its ease of application and efficacy in comparison to other scarification

treatments Although this practice had been in use for some time Wilson (1944) was

one of the first to report on its systematic application for black locust The treatment

consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)

removing the vessel from the heat source and allowing the water to cool and seeds to

soak for 24 hours

Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined

the specific application ofhot water scarification on New Mexico locust seed Each

these authors compared hot water to acid scarification Lin et aL (1996) reported

8

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

60 gennination for two hot water-treated seed lots while a third lot germinated

around 50 (as reported above acid scarification germination ranged from 20 to

80 for these lots) It should be noted that the authors only soaked the seeds in water

from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox

and Klett (1984) stated that using the standard 24-hour hot water treatment improved

germination over an acid pre-treatment (which produced less than 20 germination)

but they did not give specific gennination numbers for the hot water treatment Hine

et al (1997) found that across five seed lots germination was 66 for the standard

hot water treatment (as compared to less than 20 germination for acid scarification)

Variations in treatment intensity and duration and their subsequent effects on seed lot

appear to contribute to conflicting conclusions as to whether acid scarification or hot

water treatment is a more successful treatment

Given mixed results ease ofapplication is an important argument for use of

hot water application over acid soaks Safety concerns and material costs for hot

water treatments are low (Dreesen and Harrington 1997) An extension of this is the

practicality ofhot water for treating large batches of seed where a relative increase in

water volume is all that is needed However increases in water and seed even when

inthe same 4 1 proportion can result in dramatically different rates of cooling than in

atest tube experiment Different cooling rates can lead to contrasting results for the

treatment temperature duration and seed lot (USDA Forest Service 1948)

As with acid scarification parameters ofhot water treatment application may

to be optimized to maximize germination Hine et al (1997) found that as

9

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

temperature was increased from 70degC to 100degC overall germination in New Mexico

locust seed increased The 100degC treatment maximized germination for each of five

sources tested with germination usually above 60

Prolonged exposure of seeds ofmany woody legumes to boiling temperatures

also may improve germination (Briscoe 1996) This approach has not been tested on

New Mexico locust seed Exposing seed to prolonged boiling temperatures may

result in fungal infection and subsequent seedling mortality Marunda (1990) noted

severe fungal infections on both acid and hot water treated Acacia holoserica Cunn

ex G Don seeds attributing the infections to seed damage resulting from the

treatments Identifying an optimal hot water treatment level that does not result in a

high rate of seedling mortality is a logical goal

With hot water treatments as with acid there is also a danger of overtreating

Seeds and reducing germination (Gosling et al 1995) The legume Leucaena

leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to

water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In

addition acceptably high germination was obtained from treatment at 70degC from 3

minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2

minutes Perhaps along with a greater margin of error for treatment duration treating

~ew Mexico locust seeds at a lower temperature may result in a comparatively lower

of seedling mortality In practice however nursery growers tend to use the

and quickest treatment available The short treatment length at near-boiling

whether recommended or not may be what is used most frequently

10

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Fine-tuning a treatment in this range of treatment temperature and duration may be

the most logical approach

Mechanical Scarification

This treatment involves scratching the seed coat with an abrasive material

(USDA Forest Service 1948) One system employs a rotating drum lined with

sandpaper A paddle spinning at a given number ofrevolutions per minute throws the

seed against the sandpaper (Hine et al 1997) A second method dry tumbling

tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)

These approaches have the advantage that control of temperature is not required as in

water treatments nor is safety an issue as with add scarification

Hine et al (1997) mechanically treated New Mexico locust seed using 100shy

grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than

10 They concluded that the seed coats were too thin for their apparatus and

proposed that a finer grit sandpaper would produce more even abrasion Although

germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry

tumbling technique for 5 to 7 days produced gennination rates as high as 90

As with acid and hot water treatments treatment duration must be specifically

shy

correlated to seed lot Treated seeds are also susceptible to fungal infection

iHopkinson 1997) In addition equipment is specialized and may not always be

ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned

degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al

11

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the

strophiole has not been studied

Alternative Method for Breaking Dormancy Percussion

Strophiole degradation has been examined in depth using an impaction

treatment more commonly known as percussion Hamly (1932) first proposed that

shaking or impacting seeds could split the macrosc1ereid cells at the strophiole

Hamly successfully released tension ofbent strophiolar cells of impermeable

Melilotus alba Medik (White sweet clover) seeds by delivering physical blows

resulting in cells that had split apart and were only slightly bent Ninety-two percent

oftreated seeds became permeable as compared to 05 of control seeds Barton

(1947) showed that percussion significantly increases permeability in black locust

seed Ballard (1976) reasoned that the integrated nature of the strophiole within the

seed coat means a blow on any part of the seed weakens the strophiole There are no

published studies on the use of percussion on New Mexico locust

Percussion methods vary and often seem SUbjective or ill-defined Hamly

(1932) placed seeds in a glass bottle and shook them three times a second for 20

minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking

seeds in a closed metal container with at least fifty seeds per container at 850-900

JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into

sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250

~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of

55 after 3 hours 60 after 6 hours and 100 after 12 hours

12

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

There are several potential advantages of percussion over acid hot water and

mechanical scarification Kelly and van Staden (1987) note that a percussion

treatment only alters macrosclereid cells of the strophiole by loosening separating

and raising them They emphasize that minimal damage is done to the overall

anatomical structure of the seed In acid scarification of the same seed lot radicle

and cotyledon damage occurred Based on photographic evidence the authors

believe that this morphological damage was due to uneven pressure during

imbibition with water entering haphazardly wherever the acid had sufficiently

dissolved the coat This ultimately led to uneven hydration of the embryo In

percussed seeds water only entered through the s~ophiole when the entire seed coat

had been saturated resulting in controlled and even embryo hydration (Kelly and van

Staden 1987)

Although all successful scarification treatments may focus at least in part on

the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be

less damaging to seed coat structures particularly when compared with acid Kelly

and van Staden did not compare hot water or mechanical scarification methods

against percussion However the fungal infection associated with hot water and

mechanical scarification as with acid scarificatio~ have been directly associated with

seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in

has been observed in hot-water scarified legume seeds (Rehman et al 1998

13

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

By limiting embryo damage percussion scarification reduces seedling

mortality compared to other treatments Kelly and van Staden (1987) noted that

seedlings from the percussion treatment were morphologically sound and vigorous

when compared to acid treatment seedlings The authors attribute this improved

seedling vigor to the controlled water uptake that limited anatomical damage

Despite a 12-hour percussion treatment morphological damage was not substantially

greater than untreated seeds In addition seedling mass measured 10 days following

germination was not significantly different between percussed and untreated seeds

A final advantage ofpercussion may be the ability to adequately treat the most

dormant seeds while avoiding harm to seeds that have already been made permeable

by the treatment or that were permeable to begin with Instead ofcorroding scalding

or scratching the seed coat percussion appears to be just a loosening of the

strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to

increase germination with a lengthy treatment (12 hours) Figuring out whether a

substantial cushion oftreatment durations exist for percussion ofNew Mexico locust

seed would be a significant improvement to current scarification methods where seed

damage and seedling health is an issue

The greatest challenge for applying percussion to New Mexico locust seeds is

standardizing a treatment that is both effective and readily reproducible by nurseries

with mechanical scarification this may require the development of specific

~UllDment to properly quantify and execute the treatment

14

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

f

Objectives

The first objective of the study is to detennine which scarification treatment

results in optimal gennination speed and percentage ofNew Mexico locust The

second objective is to describe treatment interactions with nine seed sources collected

in New Mexico

Hypothesis

A percussion treatment acts exclusively on the main area ofwater uptake in

New Mexico locust seed loosening the strophiole so that imbibition can occur This

direct mechanism ofdonnancy release will result in improved gennination rate and

overall gennination percentage for New Mexico locust seed compared to standard

treatments Standard treatments including acid hot water and mechanical

scarification while eventually weakening the strophiolar cleft cause excessive

morphological damage through generalized seed coat degradation These treatments

are therefore prone to inconsistency due to over and under-treatment

15

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

MATERIALS AND METHODS

Seed Collection and Cleaning

Seeds were collected from August 25 through September 15 2000 from nine

locations along a latitudinal gradient in New Mexico Seed source locations and

descriptive information are given in Figure 1 and Tables I and 2 Stands were

separated by at least 45 km

Pods were hand picked as they began to ripen and dehisce (open) Collections

were made from ground level to approximately 3 m from several trees at each site

Pods were dried in open-ended brown paper bags for two to four weeks at room

temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth

sack which was pounded against the ground to separate seeds from pods For pods

still containing seeds hand maceration (crunching pods with hands) completed seed

separation Debris was removed by threshing with the aid of a greenhouse fan A

South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove

remaining chaff and light seeds by air density separation Seeds were placed in dry

storage in sealed quart-size zip-lock bags within a sealed plastic container Storage

temperatures ranged from 2-4oC Seeds remained in storage for approximately 7

months before the experiment

Experimental Design and Treatment Structure

The experiment compared the impact ofhot water bath and percussion

l11aLl11 on germination properties ofNew Mexico locust seed A single

16

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

-l

Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II

Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources

Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date

Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500

Holman 36deg0306 105deg2429 HolmanNM 2476 911500

Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00

Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600

Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200

Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900

Capitan 33deg2728 105deg4325 Alto NM 2189 911200

Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

f

Table 2 Mean seed weight and standard error by source

a

Source Mean seed weight (g) for Standard error 100 seeds n = 5

Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila

2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab

00103 00403 00276 00301 00370 00374 00458 00279 00348

a Mean seed weights followed by the same letter are not significantly

different

19

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

experiment was conducted utilizing a randomized complete block design blocked by

time with a factorial treatment structure Experimental factors were seed source and

scarification treatment Nine seed sources from throughout New Mexico were used

(Figure 1 Table 1) Scarification treatments included a control three levels ofhot

water and four levels of percussion Hot water treatments included immersion at

100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment

levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test

each treatment combination Four blocks of seed were tested one at a time (all seed

sources included in each run) in close chronological sequence (within 6 weeks) A

total of four 100-seed samples were used to test each source by scarification treatment

combination

Treatments

Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml

distilled water Each test tube was used to treat one 100-seed sample at a time

resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed

in a beaker with the beaker filled with tap water to the same level as the water within

test tubes Nine test tubes (one per seed source) were placed in the beaker

simultaneously so that one replication of each seed sourcescarification treatment

cotnblnatlon was conducted simultaneously Hot water treatments include immersion

boiling For immersion treatments seeds were added to test tubes with the water

(boiling) and the test tubes were immediately removed from the beaker to

For the boiling treatments test tubes remained in the beaker with water at

20

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

boiling temperature for a set duration after the addition of seeds Following all

treatments test tubes with seeds cooled 24 hours in a test tube rack

Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic

model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa

(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were

placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured

with duct tape Each can was used to treat one 100-seed sample To take advantage

ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to

position the soil tin at the end of the shaking arm This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular

to the direction of shaking motion allowing seeds to impact at greatest force by

hitting flat surfaces of the soil tin

Germination Testing

Before gennination testing all seeds were soaked 24 hours in reverse

osmosis-treated water (this was included in the process ofhot water treatment

jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined

90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant

WI) One Petri dish was used for each treatment by source combination

papers were initially moistened with 5 mL reverse osmosis-treated water then

~VU1CUlJl moistened as needed to ensure adequate conditions for gennination

were monitored daily (at 300 PM) for gennination for 14 days in accordance

~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species

21

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)

Germination was classified as protrusion of the radicle from the embryo by at least

1mm as viewed with the naked eye

X-ray Radiography

On August 132001 three lOO-seed samples from the Sacramento seed source

were treated for 14 or 8-minute percussion durations The seeds were sent

immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling

Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging

Radiographs were made at SX with a Faxitron Specimen Radiography System Model

MX-20

Scanning Electron Microscope Photography

On February 192002 a Hitachi S-3200N Variable Pressure Scanning

Electron Microscope (Mountain View CA) was used to examine strophiolar cells of

untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally

igt their entire length using a razor blade splitting the strophiolar region roughly in

Cross sections of seed samples magnifying the strophiolar region were viewed

600X under the microscope

Analysis

Categorical Analysis of Variance was used to determine treatment source and

(Le time) main effects and interactions The response variable was total

~vlI This procedure is a Chi-square test of homogeneity using the natural log

of germinated to non-germinated seed for each treatment Maximumshy

22

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

likelihood analysis was used to calculate Chi-square test statistics with their observed

significance levels A single contrast was conducted to cempare treatment means

against the control Observed significance levels less than or equal to 005 were

considered significant Pairwise comparisons were conducted between non-control

treatment mean percents using a conservative alp~a value of 005 divided by the

number of comparisons (Bonferroni approach) and approximate pairwise z statistics

Categorical analyses were performed using Proc Catmod (SAS Institute 1989)

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

RESULTS

Scarification seed source block and all interactions between factors

influenced germination (Table 3) The main effects are reported in the presence of

these interactions because the overall robustness of treatments and seed sources are of

interest Background levels ofnaturally occurring variation in seed sources are

seen in control germination percentages in Figure 2 In particular time variability

is unlikely to be under the control of the producer Appendix B details experimental

data for source by treatment gennination percents for each of the four blocks

Averaged over all treatments germination by source ranged from 40 to

79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4

5) percussion scarification durations of4 or 8 minutes yielded greatest germination

Averaged over all sources germination increased with increasing durations of

percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion

and 30-second boiling scarification maximized germination for the hot water

treatments with a decrease in germination when seeds were boiled for 60 seconds

For all but two sources there is a similar ipcreasing trend in germination from

1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination

1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little

1 to 4 minutes of treatment As treatment duration increased from 4 to 8

mutes five sources decreased in germination (Figure 4a) whereas three sources

24

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors

Source of DF Chi-Square Observed Variability Significance Level

Source 8 98370 lt00001 Trt 7 233876 lt00001

contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001

SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001

SourceTrtBlock 168 34283 lt00001

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

S 4)

8 ~

0

sectg ~ 40 ~

N 0 0

20

o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b

80

60 -I-- - ~- middotmiddotmiddotmiddotmiddot1

r -] Northern NM Sources

Central NM Sources

Southern NM Sources

sect-o ~

~b-v(i

SOURCE

Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

80

~ cd ~ ltl) () 60 ~ ~

N

t 0shyrti tshySltl)

40

0

20

o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min

Hot Water Treatments Percussion Treatments

Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

100~----------------------~--~----------------~

80

60

ltU

~

40 IJ

sectgsI

l100

80

60

40

a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E

_~iii-bull _ _ bull f --~--

t-- -~ ---- -shy ---- shyI ~ ---- - shy

L

fiII

~

b

- shy ---r

- Holman

Sacramento Sandia Gila Jemez

c ~

ltgt ~

Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I

j~=~=-~~-1

I middotmiddot ~ bull r

t tr

Y -------M-a-gd-a-Ie-n-a--

1 _ _

fCapitan

---- Manzano ~y -_ Raton

2 4 8

Time interval ofpercussion scarification (min)

Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b

28

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

tv 0

~ d

~ A s o

1 c3

80

60

40

20

o

TIT

t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1

I

Raton Holman

-_ Jemez

--- Sandia Manzano Magdalena Capitan Sacramento Gila

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

continued to increase in germination (Figure 4b) There was no difference between

the two treatment levels for the Manzano source (Figure 4b)

The effect ofhot water scarification also varied among sources with few

overall differences between hot water treatments (Figure 5) However boiling for 60

seconds yielded highest germination for the Jemez source whereas increasing

durations ofboiling decreased germination for the Raton source

In addition to consistently high germination 4- and 8-minute percussionshy

treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)

050 measured as the number of days needed to complete 50 ofeventual

germination averaged 2 days for both ofthese treatments

X-ray radiographs were taken of seeds collected from the Sacramento source

percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes

ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4

minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting

of the seed coat and chips in seed coat structure (Figure 8b-d)

Scanning electron microscope (SEM) images at 600X reveal the direct effect

of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas

untreated seeds retain cellular organization in the strophiolar region strophiolar cells

seeds treated for 4 minutes ofpercussion separate and raise resulting in a

~-~e~U appearance

30

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -

050= gt1

-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control

2 4 6 8 10 12

Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification

treatments G50 is the number of days to 50 of eventual germination

31

14

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Table 4 Effect of seed source and treatment on G50 (days to 50 germination)

Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2

Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2

w tv Manzano 5 2 2 3 3 2 2 2 3

Magdalena 4 2 2 3 4 3 2 1 3

Capitan 5 4 2 2 3 2 2 2 3

Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation

Note Columns and rows may not agree due to rounding

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes

33

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d

34

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Untreated Treated

Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

DISCUSSION

Determining the Optimal Treatment

Averaging results of scarification treatmems across seed sources is a useful

tool to predict which treatment is most likely to be successful for an unknown seed

source Averaged over all sources four- and eight-minute percussion treatments

resulted in the highest germination 815 and 790 respectively and produced

germination that was as fast or faster than all other treatments Although there was no

difference between these two treatments on average an argument for 4 minutes of

percussion as a standard protocol for treating New Mexico locust seeds can be made

Descriptively 4 minutes of percussion improved germination over 8 minutes

ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion

improved germination for three ofnine sources with no difference for a fmal source

For individual sources 4 minutes ofpercussion was always the best or secondmiddot best

treatment and was always equal to or better than all hot water treatments

Germination at 8 minutes ofpercussion was lower than all other percussion durations

for the Jemez and Gila sources and was lower than at least one hot water treatment for

these sources as well

The x-ray radiographs of the Sacramento seed source illustrate the excessive

internal and external seed damage that can occur with 8 minutes of percussion

germination counts 8-minute percussed seeds from Holman and Capitan

ClUll signs of damage that were visible to the naked eye (see below)

r~evertheless some of these seeds still fit the criteria for germination Neither x-ray

36

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

photography nor visible observations during seed counts revealed seed damage at the

4-minute percussion level In operational trials seeds percussed for 4 minutes

developed into healthy seedlings (Khadduri and Harrington unpublished data)

Strophiole and Seed Coat Behavior

Four minutes ofpercussion successfully breaks seed dormancy in a large

percentage ofNew Mexico locust seeds without excessively damaging the

surrounding seed coat or internal seed coat structures Most papilionoid legume

seeds including seeds ofNew Mexico locust have a specific region of the seed coat

known as the strophiole or lens (Hamly 1932) The strophiole is located on the

cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately

damaged the strophiole is the first point of water entry to the embryo (Hopkinson

1997) When a papilionoid seed is percussed for the appropriate amount of time

repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar

region without excessively damaging the rest of the seed coat (Ballard 1976) SEM

photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells

ofNew Mexico locust seeds when compared with constrained organized strophiolar

cells of untreated seeds (Figure 9) Research-has shown that water passes to the

embryo through the treated strophiole in a controlled manner and regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues

(Kelly and van Staden 1987)

37

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)

38

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

In contrast hot-water treatments can excessively degrade the seed coat

(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a

proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)

damage was observed in hot-water treated seeds from six sources during germination

counts Damage included seeds where the radicle failed to elongate (Figure 11 b)

seeds where the radicle elongated but was delayed in freeing itself from the seed coat

(Figure 11c) and seeds where the radicle completely broke off from the embryo

(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged

fIrst) If the radicle emerged completely from the seed coat they were counted as

germinated

While nearly all seeds treated for 8 minutes ofpercussion imbibed water

damage was evident in x-ray evaluations (Sacramento seed source) and in

germination counts (Holman and Capitan) at this treatment level Seed damage

during germination counts was similar to levels found in hot-water treated seeds

suggesting that over-treatment by both hot water and longer durations of percussion

may result in similar damage to seed structure

The Sacramento and Holman seed sources were two of the fIve sources that

opcrplltlpn in germination from the 4- to 8-minute durations of percussion

the benefIts ofloosening the strophiole as evidenced by imbibition of

all 8-minute treated seeds were outweighed by external and internal seed coat

39

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

a

b c d

11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was

-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo

40

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

damage (which also may be contributing to high imbibition) For the three sources

for which 8 minutes of percussion resulted in maximum germination extended

percussion of seeds necessary to loosen the strophiole may not have excessively

damaged the seed anatomy The Manzano source did not significantly change in

germination from 4 to 8 minutes of percussion suggesting that an increase in

permeable seeds at the 8-minute treatment duration was balanced by the number of

seeds that failed to germinate due to seed damage To summarize the 4-minute

percussion treatment improved germination over 8 minutes ofpercussion for a

majority of sources (five ofnine) the 8-minute percussion treatment improved

germination over 4-minutes ofpercussion for three sources and there was no

difference with the Manzano source Notably for the Raton source 8 minutes of

percussion increased germination by 235 percentage points over 4-minutes of

percussion (7875 vs 5525 respectively) the next best treatment Raton was the

only source to result in germination less than 75 for 4 minutes ofpercussion so a

grower may wish to apply longer percussion durations to lots that do not respond well

to this treatment level

Finally there was an interesting trend in the Jemez and Raton sources seed

that significantly contributed to the source by treatment interaction (Figure 12) A

~u test showed that the Jemez source had a significantly lighter seed weight than

other sources while the Raton source had an average seed weight For the Jemez

the longest duration of boiling increased germination Conversely only short

U4UVll) of percussion were needed to achieve high germination and the longest

41

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

80 _ Jemez a Raton

iD

~ C iD e iD

Q c 0 ~

c ~ iD 0

20

0------shy

Immersion Boiling 30 sec Boiling 60 sec

Hot Water Scarification

10degTr========~--------~------------------------------

80

60

40

20

_ Jemez Raton

0------shy

1 min 2 min 4 min 8 min

Percussion Scarification

Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources

42

b

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations

ofboiling consistently decreased germination whereas longer percussion treatments

consistently increased germination

A possible explanation is that a thick seed coat is associated with a loose

strophiole structure and that a thin seed coat is associated with a constrained

strophiole This contrast merits further study and may suggest an ecological

adaptation Further investigation ofthis phenomenon may contribute to successful

pre-screening ofunknown seed sources to determine optimal treatment leveL

43

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

CONCLUSION

Based on results obtained in pilot study (see Appendix) and thesis

experiments 4 minutes ofpercussion is recommended as a standard protocol for

treating an unknown source ofNew Mexico locust seed In addition to laboratory

results the 4-minute percussion treatment has resulted in germination greater than

80 and healthy seedling grow-outs for production purposes (unpublished data) In

laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were

percussed at a time whereas for production we have treated up to five hundred seeds

per tin For large-scale production ofNew Mexico locust seedlings growers may

wish to experiment with larger quantities of seed and larger-size soil tins

Seed storage trials following percussion treatments were not conducted

However germination greater than 80 and healthy seedlings were obtained when

seeds were percussion-treated and subsequently stored at room-temperature (22degC)

for one week (unpublished data) Seed storage trials merit further study as the

standard hot water treatment leaves seeds in a softened condition that is not

conducive to storage

Hydroseeding is an effective method for direct seeding in reclamation

situations Percussion treatment may be conducive to hydro seeding as seeds remain

a dry relatively undamaged yet permeable condition following treatment Sulfuric

seeds also remain dry following treatment but extensive damage to seed coat

poor seedling vigor have been reported with this treatment (Kelly and van Staden

A grow-out study using a hydro seeder on a reclamation site testing percussion

44

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

against other treatments such as sulfuric acid and mechanical scarification also

merits further study

Papilionoids are the largest subfamily oflegumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust these legumes often play an integral role in the revegetation of

disturbed lands The pilot study (Appendix) indicates that percussion scarification

successfully treats the widely used black locust as well Developing superior

scarification methods to the standard hot water mechanical scarification and acid

treatments may facilitate the use ofpapilionoid legumes in reclamation projects

45

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

APPENDICES

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

APPENDIX A

PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR

NEW MEXICO LOCUST AND BLACK LOCUST SEEDS

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4

Hot water and sulfuric acid soaks are traditional treatments for seeds of many

temperate woody legwnes including locusts However these scarification techniques

often produce inconsistent germination Percussion scarification where seeds are

repeatedly propelled against a hard surface was compared with hot water

scarification to evaluate treatment efficacy for New Mexico locust (Robinia

neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment

seeds were placed in a 98degC water bath which was immediately removed from the

heat source For percussion scarification seeds were placed in a soil sample tin and

agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the

control were followed by 24-hour water soaks Hot water baths resulted in 56 and

41 germination for New Mexico locust and black locust respectively For both

species nearly all durations ofpercussion increased germination over the hot water

treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3

4 and 5 minutes for black locust resulted in at least 90 germination Traditional

scarification treatments randomly degrade the entire seed coat which can lead to

tissue damage during water uptake Percussion scarification specifically weakens the

strophiole the natural source ofwater entry to the seed in papilionoid legwnes

INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom

2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031

48

Following percussion imbibition is controlled through the strophiole and underlying

tissue is protected

Keywords New Mexico locust black locust scarification percussion germination

Introduction

Locust trees (Robinia spp) are aggressive pioneer species that quickly

colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that

are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)

Recognition of these attributes has contributed to increased demand for locusts in

reclamation projects Locust seeds however exhibit physical dormancy where a

thick seed coat prevents the movement of water and gasses to the embryo (Leadem

1997)

Sulfuric acid or hot water soaks have traditionally been used to break seed

dormancy in macro-propagation of locusts Many growers are moving away from the

use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In

addition to being dangerous acid soak durations must be specifically correlated to

seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size

seed weight and hard seededness etc This variability limits the utility of sulfuric

acid soaks as even within a given lot some seeds may be damaged whereas others

are under-treated and fail to imbibe (take up water) A survey of Southwestern

container growers indicates that hot water baths are currently the most common

scarification method for locust seeds (Hine et al 1997) However hot water baths

also produce inconsistent germination (Lin et al 1996)

49

b

Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM

50

Percussion scarification where seeds are repeatedly propelled against a hard

surface is an alternative dormancy-breaking method that has proven successful in

legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative

treatment to hot water scarification to evaluate treatment efficacy for New Mexico

locust and black locust seeds

Materials and Methods

Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)

were collected fall 2000 in Huerfano County CO Black locust seeds were collected

September 2000 from Taos County NM by harvesting ripe pods from several trees at

a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry

for 3 weeks and were threshed and separated from large chaffusing a greenhouse

fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff

from both seed lots Seeds were storedmiddotat 2-4C until the germination study was

conducted in March 2001

This study compared hot water scarification with percussion scarification

using treatment and species as experimental factors Along with a control

scarification treatments included immersion of seed in boiling water as well as

percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used

to test each species by scarification treatment combination

For the hot water treatment test tubes were filled with 30 mL water and

placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at

51

b

c

Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann

52

Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a

test tube and each test tube was immediately removed to cool at room temperature

Percussion scarification was implemented using a pneumatic paint shaker

(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed

standardization of treatment intensity Compressed air pressure was maintained at

530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per

minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To

maximize lateral movement of paint shaker a paint can was used as a spacer to place

soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Following all treatments

including the control seeds were soaked for twenty-four hours in distilled water prior

to plating out

Gennination was evaluated on lab benches at room temperature Each

replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with

humidity maintained by enclosing the petri dishes in ziplock bags Gennination was

monitored daily for 14 days in accordance with International Seed Testing

Association standards for Robinia species (ISTA 1999) Gennination was defined as

protrusion of the radicle from the embryo by at least 1mm as viewed by the naked

eye

Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)

was used to detennine treatment differences in germination percentages for each

source This procedure is an extension of a Chi-square test of homogeneity using the

53

natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy

values less than 005 were considered significant A limited set of pairwiseshy

comparisons was conducted to compare treatment mean percents using a

conservative alpha value of 005 divided by the number of comparisons

Results

Percussion maximized germination for both New Mexico locust and black

locust There were three germination response levels for New Mexico locust (Figure

A3) First there was a low response in the control indicating a dormant seed lot

The hot water treatment and I-minute duration of percussion share a second

intermediate response Germination was highest at percussion levels from 2 to 10

minutes

A selected percussion treatment intermediate in time (5 minutes) produced the

steepest germination curve indicating the fastest germination rate (Figure A4) The

hot water treatment produced an intermediate curve or relatively slower germination

rate The control curve was very flat indicating the slowest germination rate for New

Mexico locust

Control seeds germinated poorly for black locust as well (Figure A5) Hot

water scarification improved germination over the control but all levels of percussion

improved germination over hot water The optimal durations of percussion treatment

were bracketed with a significant increase from the 1 to 2 minute percussion level a

high germination response from 2 to 5 minutes of percussion and finally a significant

54

~ d 25 CL)

p t1 0 ~ t1

g Vl ~ Vl

100~---------------------------------------------------------------

80

60

40

20

o Control Hot Water Percussion

I min Percussion

2 min Percussion

3 min

Scarification

Percussion 4 min

Figure A3 Effect of scarification on germination response for New Mexico locust

Percussion 5 min

=significant difference between adjacent treatment bars ns = no significant difference

Percussion 10 min

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

b

Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM

50

Percussion scarification where seeds are repeatedly propelled against a hard

surface is an alternative dormancy-breaking method that has proven successful in

legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative

treatment to hot water scarification to evaluate treatment efficacy for New Mexico

locust and black locust seeds

Materials and Methods

Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)

were collected fall 2000 in Huerfano County CO Black locust seeds were collected

September 2000 from Taos County NM by harvesting ripe pods from several trees at

a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry

for 3 weeks and were threshed and separated from large chaffusing a greenhouse

fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff

from both seed lots Seeds were storedmiddotat 2-4C until the germination study was

conducted in March 2001

This study compared hot water scarification with percussion scarification

using treatment and species as experimental factors Along with a control

scarification treatments included immersion of seed in boiling water as well as

percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used

to test each species by scarification treatment combination

For the hot water treatment test tubes were filled with 30 mL water and

placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at

51

b

c

Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann

52

Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a

test tube and each test tube was immediately removed to cool at room temperature

Percussion scarification was implemented using a pneumatic paint shaker

(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed

standardization of treatment intensity Compressed air pressure was maintained at

530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per

minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To

maximize lateral movement of paint shaker a paint can was used as a spacer to place

soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Following all treatments

including the control seeds were soaked for twenty-four hours in distilled water prior

to plating out

Gennination was evaluated on lab benches at room temperature Each

replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with

humidity maintained by enclosing the petri dishes in ziplock bags Gennination was

monitored daily for 14 days in accordance with International Seed Testing

Association standards for Robinia species (ISTA 1999) Gennination was defined as

protrusion of the radicle from the embryo by at least 1mm as viewed by the naked

eye

Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)

was used to detennine treatment differences in germination percentages for each

source This procedure is an extension of a Chi-square test of homogeneity using the

53

natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy

values less than 005 were considered significant A limited set of pairwiseshy

comparisons was conducted to compare treatment mean percents using a

conservative alpha value of 005 divided by the number of comparisons

Results

Percussion maximized germination for both New Mexico locust and black

locust There were three germination response levels for New Mexico locust (Figure

A3) First there was a low response in the control indicating a dormant seed lot

The hot water treatment and I-minute duration of percussion share a second

intermediate response Germination was highest at percussion levels from 2 to 10

minutes

A selected percussion treatment intermediate in time (5 minutes) produced the

steepest germination curve indicating the fastest germination rate (Figure A4) The

hot water treatment produced an intermediate curve or relatively slower germination

rate The control curve was very flat indicating the slowest germination rate for New

Mexico locust

Control seeds germinated poorly for black locust as well (Figure A5) Hot

water scarification improved germination over the control but all levels of percussion

improved germination over hot water The optimal durations of percussion treatment

were bracketed with a significant increase from the 1 to 2 minute percussion level a

high germination response from 2 to 5 minutes of percussion and finally a significant

54

~ d 25 CL)

p t1 0 ~ t1

g Vl ~ Vl

100~---------------------------------------------------------------

80

60

40

20

o Control Hot Water Percussion

I min Percussion

2 min Percussion

3 min

Scarification

Percussion 4 min

Figure A3 Effect of scarification on germination response for New Mexico locust

Percussion 5 min

=significant difference between adjacent treatment bars ns = no significant difference

Percussion 10 min

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Percussion scarification where seeds are repeatedly propelled against a hard

surface is an alternative dormancy-breaking method that has proven successful in

legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative

treatment to hot water scarification to evaluate treatment efficacy for New Mexico

locust and black locust seeds

Materials and Methods

Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)

were collected fall 2000 in Huerfano County CO Black locust seeds were collected

September 2000 from Taos County NM by harvesting ripe pods from several trees at

a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry

for 3 weeks and were threshed and separated from large chaffusing a greenhouse

fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff

from both seed lots Seeds were storedmiddotat 2-4C until the germination study was

conducted in March 2001

This study compared hot water scarification with percussion scarification

using treatment and species as experimental factors Along with a control

scarification treatments included immersion of seed in boiling water as well as

percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used

to test each species by scarification treatment combination

For the hot water treatment test tubes were filled with 30 mL water and

placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at

51

b

c

Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann

52

Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a

test tube and each test tube was immediately removed to cool at room temperature

Percussion scarification was implemented using a pneumatic paint shaker

(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed

standardization of treatment intensity Compressed air pressure was maintained at

530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per

minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To

maximize lateral movement of paint shaker a paint can was used as a spacer to place

soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Following all treatments

including the control seeds were soaked for twenty-four hours in distilled water prior

to plating out

Gennination was evaluated on lab benches at room temperature Each

replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with

humidity maintained by enclosing the petri dishes in ziplock bags Gennination was

monitored daily for 14 days in accordance with International Seed Testing

Association standards for Robinia species (ISTA 1999) Gennination was defined as

protrusion of the radicle from the embryo by at least 1mm as viewed by the naked

eye

Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)

was used to detennine treatment differences in germination percentages for each

source This procedure is an extension of a Chi-square test of homogeneity using the

53

natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy

values less than 005 were considered significant A limited set of pairwiseshy

comparisons was conducted to compare treatment mean percents using a

conservative alpha value of 005 divided by the number of comparisons

Results

Percussion maximized germination for both New Mexico locust and black

locust There were three germination response levels for New Mexico locust (Figure

A3) First there was a low response in the control indicating a dormant seed lot

The hot water treatment and I-minute duration of percussion share a second

intermediate response Germination was highest at percussion levels from 2 to 10

minutes

A selected percussion treatment intermediate in time (5 minutes) produced the

steepest germination curve indicating the fastest germination rate (Figure A4) The

hot water treatment produced an intermediate curve or relatively slower germination

rate The control curve was very flat indicating the slowest germination rate for New

Mexico locust

Control seeds germinated poorly for black locust as well (Figure A5) Hot

water scarification improved germination over the control but all levels of percussion

improved germination over hot water The optimal durations of percussion treatment

were bracketed with a significant increase from the 1 to 2 minute percussion level a

high germination response from 2 to 5 minutes of percussion and finally a significant

54

~ d 25 CL)

p t1 0 ~ t1

g Vl ~ Vl

100~---------------------------------------------------------------

80

60

40

20

o Control Hot Water Percussion

I min Percussion

2 min Percussion

3 min

Scarification

Percussion 4 min

Figure A3 Effect of scarification on germination response for New Mexico locust

Percussion 5 min

=significant difference between adjacent treatment bars ns = no significant difference

Percussion 10 min

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

b

c

Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann

52

Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a

test tube and each test tube was immediately removed to cool at room temperature

Percussion scarification was implemented using a pneumatic paint shaker

(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed

standardization of treatment intensity Compressed air pressure was maintained at

530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per

minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To

maximize lateral movement of paint shaker a paint can was used as a spacer to place

soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Following all treatments

including the control seeds were soaked for twenty-four hours in distilled water prior

to plating out

Gennination was evaluated on lab benches at room temperature Each

replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with

humidity maintained by enclosing the petri dishes in ziplock bags Gennination was

monitored daily for 14 days in accordance with International Seed Testing

Association standards for Robinia species (ISTA 1999) Gennination was defined as

protrusion of the radicle from the embryo by at least 1mm as viewed by the naked

eye

Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)

was used to detennine treatment differences in germination percentages for each

source This procedure is an extension of a Chi-square test of homogeneity using the

53

natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy

values less than 005 were considered significant A limited set of pairwiseshy

comparisons was conducted to compare treatment mean percents using a

conservative alpha value of 005 divided by the number of comparisons

Results

Percussion maximized germination for both New Mexico locust and black

locust There were three germination response levels for New Mexico locust (Figure

A3) First there was a low response in the control indicating a dormant seed lot

The hot water treatment and I-minute duration of percussion share a second

intermediate response Germination was highest at percussion levels from 2 to 10

minutes

A selected percussion treatment intermediate in time (5 minutes) produced the

steepest germination curve indicating the fastest germination rate (Figure A4) The

hot water treatment produced an intermediate curve or relatively slower germination

rate The control curve was very flat indicating the slowest germination rate for New

Mexico locust

Control seeds germinated poorly for black locust as well (Figure A5) Hot

water scarification improved germination over the control but all levels of percussion

improved germination over hot water The optimal durations of percussion treatment

were bracketed with a significant increase from the 1 to 2 minute percussion level a

high germination response from 2 to 5 minutes of percussion and finally a significant

54

~ d 25 CL)

p t1 0 ~ t1

g Vl ~ Vl

100~---------------------------------------------------------------

80

60

40

20

o Control Hot Water Percussion

I min Percussion

2 min Percussion

3 min

Scarification

Percussion 4 min

Figure A3 Effect of scarification on germination response for New Mexico locust

Percussion 5 min

=significant difference between adjacent treatment bars ns = no significant difference

Percussion 10 min

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a

test tube and each test tube was immediately removed to cool at room temperature

Percussion scarification was implemented using a pneumatic paint shaker

(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed

standardization of treatment intensity Compressed air pressure was maintained at

530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per

minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To

maximize lateral movement of paint shaker a paint can was used as a spacer to place

soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at

the greatest distance from the pivot point (fulcrum) Following all treatments

including the control seeds were soaked for twenty-four hours in distilled water prior

to plating out

Gennination was evaluated on lab benches at room temperature Each

replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with

humidity maintained by enclosing the petri dishes in ziplock bags Gennination was

monitored daily for 14 days in accordance with International Seed Testing

Association standards for Robinia species (ISTA 1999) Gennination was defined as

protrusion of the radicle from the embryo by at least 1mm as viewed by the naked

eye

Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)

was used to detennine treatment differences in germination percentages for each

source This procedure is an extension of a Chi-square test of homogeneity using the

53

natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy

values less than 005 were considered significant A limited set of pairwiseshy

comparisons was conducted to compare treatment mean percents using a

conservative alpha value of 005 divided by the number of comparisons

Results

Percussion maximized germination for both New Mexico locust and black

locust There were three germination response levels for New Mexico locust (Figure

A3) First there was a low response in the control indicating a dormant seed lot

The hot water treatment and I-minute duration of percussion share a second

intermediate response Germination was highest at percussion levels from 2 to 10

minutes

A selected percussion treatment intermediate in time (5 minutes) produced the

steepest germination curve indicating the fastest germination rate (Figure A4) The

hot water treatment produced an intermediate curve or relatively slower germination

rate The control curve was very flat indicating the slowest germination rate for New

Mexico locust

Control seeds germinated poorly for black locust as well (Figure A5) Hot

water scarification improved germination over the control but all levels of percussion

improved germination over hot water The optimal durations of percussion treatment

were bracketed with a significant increase from the 1 to 2 minute percussion level a

high germination response from 2 to 5 minutes of percussion and finally a significant

54

~ d 25 CL)

p t1 0 ~ t1

g Vl ~ Vl

100~---------------------------------------------------------------

80

60

40

20

o Control Hot Water Percussion

I min Percussion

2 min Percussion

3 min

Scarification

Percussion 4 min

Figure A3 Effect of scarification on germination response for New Mexico locust

Percussion 5 min

=significant difference between adjacent treatment bars ns = no significant difference

Percussion 10 min

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy

values less than 005 were considered significant A limited set of pairwiseshy

comparisons was conducted to compare treatment mean percents using a

conservative alpha value of 005 divided by the number of comparisons

Results

Percussion maximized germination for both New Mexico locust and black

locust There were three germination response levels for New Mexico locust (Figure

A3) First there was a low response in the control indicating a dormant seed lot

The hot water treatment and I-minute duration of percussion share a second

intermediate response Germination was highest at percussion levels from 2 to 10

minutes

A selected percussion treatment intermediate in time (5 minutes) produced the

steepest germination curve indicating the fastest germination rate (Figure A4) The

hot water treatment produced an intermediate curve or relatively slower germination

rate The control curve was very flat indicating the slowest germination rate for New

Mexico locust

Control seeds germinated poorly for black locust as well (Figure A5) Hot

water scarification improved germination over the control but all levels of percussion

improved germination over hot water The optimal durations of percussion treatment

were bracketed with a significant increase from the 1 to 2 minute percussion level a

high germination response from 2 to 5 minutes of percussion and finally a significant

54

~ d 25 CL)

p t1 0 ~ t1

g Vl ~ Vl

100~---------------------------------------------------------------

80

60

40

20

o Control Hot Water Percussion

I min Percussion

2 min Percussion

3 min

Scarification

Percussion 4 min

Figure A3 Effect of scarification on germination response for New Mexico locust

Percussion 5 min

=significant difference between adjacent treatment bars ns = no significant difference

Percussion 10 min

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

~ d 25 CL)

p t1 0 ~ t1

g Vl ~ Vl

100~---------------------------------------------------------------

80

60

40

20

o Control Hot Water Percussion

I min Percussion

2 min Percussion

3 min

Scarification

Percussion 4 min

Figure A3 Effect of scarification on germination response for New Mexico locust

Percussion 5 min

=significant difference between adjacent treatment bars ns = no significant difference

Percussion 10 min

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

~ ~ () u agt

Il-t d o ~

~ ~ c3

100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control

80-J=middot======

6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1

40

20

-----~-----------

0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0

0 --~-----y-----~------_---~--- T-----~

o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown

Day 7

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

80

~ cd

5 60 ()

[) Cl d 0 1t1 d

40sect dUl

-J

20

o

ns

Scarification

Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion

over-treats seeds from this lot of-black locust

A selected percussion treatment intermediate in time (5 minutes) also had the

fastest germination rate for black locust (Figure A6)_ The hot water curve is

somewhat flatter indicating a slower germination rate The control curve is very flat

indicating a slow germination rate

A qualitative difference between hot water and percussion-treated seeds was

also observed Along with healthy germinants the hot water treatment produced

over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds

where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated

but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the

radicle broke off completely from the embryo (Figure A7d) In addition to these

examples of over-treatment there were numerous small dark seeds that failed to

imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)

Germination was rapid and uniform with little damage evident (Figure A8b)

To summarize both New Mexico locust and black locust seed lots were

dormant The standard hot water scarification treatment improved germination but

only to 56 and 41 for New Mexico locust and black locust respectively Nearly

all percussion durations improved total germination as well as germination rate over

the hot water treatment Germination was greater than 90 for percussion durations

of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust

58

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

CI)

sgt t CI) 0

igt 0

sect ttl t E

Vl 0

c3

100r==~==~~=r------------------------------------~ ___ Percussion 5 min

middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control

80r=========~--~~----------------------------~

60

40

20 -~-~---~---------- ---~------------I

omiddot

O

_~_----------lIf-----~bull -----_ shy---shy I

Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6

Time

Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown

Day 7

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

a

b c d

Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo

60

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

a

b

Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling

61

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Discussion

Papilionoid seeds have a specific anatomical feature known as the strophiole

or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is

located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid

legume seed is percussed for the appropriate amount of time repeated hits on the

integrated seed coat loosen the constrained cells of the strophiolar region without

excessively damaging the rest of the seed coat (Ballard 1976)

When a percussed seed is soaked water enters exclusively through the

strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of

water to the embryo is associated with even pressure on underlying seed tissues This

contrasts with seeds that have been hot water or acid scarified treatments that can

randomly degrade the seed coat Unlocalized cracks in the seed coat can promote

irregular water uptake associated with uneven pressure on underlying seed tissues and

subsequent seed damage (Rehman et al 1998 Marunda 1990)

Papilionoids are the largest subfamily of legumes covering almost all

legumes occurring in temperate climates (Baskin and Baskin 1998) As with New

Mexico locust and black locust these legumes often play an integral role in the

revegetation ofdisturbed lands Developing superior scarification methods to the

standard hot water and acid treatments should fadlitate the use ofthese legumes in

restoration projects

62

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)

63

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

APPENDIXB

GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE

BLOCK AND TREATMENT FOR NEW MEXICO LOCUST

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust

Source Block Treatment Germination Percentage Standard Error

Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min

470

Capitan Percussion I min 39 488

Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan

2

Percussion 2 min Percussion 2 min

71 65

454 477

Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila

Immersion 75 433

Gila 4 Immersion 72 449

Gila I Boiling 30 sec 68 466

Gila 2 Boiling 30 sec 81 392

Gila 3 Boiling 30 sec 54 498

Gila 4 Boiling 30 sec 63 483

Gila I Boiling 60 sec 55 497

Gila 2 Boiling 60 sec 68 466

Gila 3 Boiling 60 sec 57 495

Gila 4 Boiling 60 sec 70 458

65

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407

Holman 4 Control 24 427

Holman Percussion 1 min 77 421

Holman 2 Percussion 1 min 85 357

Holman 3 Percussion 1 min 80 400

Holman 4 Percussion 1 min 89 313

Holman 1 Percussion 2 min 88 325

Holman 2 Percussion 2 min 96 196

Holman 3 Percussion 2 min 93 255

Holman 4 Percussion 2 min 96 196

Holman 1 Percussion 4 min 92 271

Holman 2 Percussion 4 min 97 171

66

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

I

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466

67

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Table B 1 Continued

Source Block Treatment Germination Percentage Standard Error

Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500

68

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Table B 1 Continued

Source Block Treatment Gerrni~ation Percentage Standard Error

Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495

69

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Table B 1 Continued

-Source Block Treatment Germination Percentage Standard Error

Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255

70

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Table B 1 Continued

Source Block Treatment Gennination Percentage Standard Error

Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

LITERATURE CITED

Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p

Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303

___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469

Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362

Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p

Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066

Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p

Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p

Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p

Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10

Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563

Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p

Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery

72

Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88

Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO

Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532

Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375

Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29

Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382

Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO

Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8

---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4

ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement

Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404

73

Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85

Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p

Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980

Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144

Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52

Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1

Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28

Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p

Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772

Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68

Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450

Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557

Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396

74

SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p

SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840

Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p

Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia

USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p

Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p

Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA

Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454

Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia

Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85

Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p

Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980

Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144

Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52

Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1

Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28

Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p

Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772

Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68

Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450

Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557

Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396

74

SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p

SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840

Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p

Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia

USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p

Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p

Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA

Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454

Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia

SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p

SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840

Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p

Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia

USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p

Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p

Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA

Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454

Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia