ASPECTS OF VEGETATIVE AND REPRODUCTIVE …

ASPECTS OF VEGETATIVE AND REPRODUCTIVE PHYSIOLOGY

OF DWARF PHARBITIS NIL

HAROLD ADDRICK SIMMONS, B.S., M.S.

A DISSERTATION

IN

BOTANY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

DOCTOR OF PHILOSOPHY

Approved

c Accepxea

December, 1979

' mi

'A/-'* ^ ' ACKNOWLEDGMENTS /^' . ' ^

I am appreciative to Drs. Thomas Brady, Joe Goodin,

Philip Morey and Daniel Krieg for serving on my doctoral

committee and for their aid and constructive criticism

of this research. I also appreciate the initial guidance

of Dr- Jerry Berlin.

Kim Christie, Susan Erwin, Cal Hoffman and Byron

Stephens were most helpful as laboratory assistants. I

thank Phil Keller for his many discussions concerning

this research.

To Dr. Murray Coulter, who guided and directed this

research, I am especially thankful for his investment of

time, interest, and talent.

Most of all, I thank my wife, Ann, for her support

during this work and for typing this manuscript.

11

CONTENTS

ACKNOWLEDGMENTS ii

ABSTRACT iv

LIST OF TABLES vii

LIST OF FIGURES viii

I. INTRODUCTION 1

II. APEX DEVELOPMENT OF DWARF PHARBITIS IN

RESPONSE TO GIBBERELLIN AND PHOTOPERIOD . . 7

Materials and Methods 8

Results 1^

Discussion 2 + III. A COMPARISON OF THE EFFECTS OF PHOTOPERIOD

AND GIBBERELLIN ON THREE DIFFERENT PROCESSES OF GROWTH AND DEVELOPMENT IN PHARBITIS NIL 32

Materials and Methods 33

Results 3^

Discussion 39

IV. SUMMARY ^7

LIST OF REFERENCES ^8

111

ABSTRACT

Developmental and growth responses resulting from

differences in the status of the endogenous rhythm, phyto-

chrome, and gibberellin (GA). were analyzed in Pharbitis

nil. The effects of these three components on floral in

duction, floral development, stem elongation and leaf

expansion were assayed independently to determine if the

action of each regulatory component is the same or different

in growth and development processes. The status of the

endogenous rhythm and/or phytochrome was effected by

application of specific photoperiodic treatments known

to influence these two components differently. In order

to assay effects of both a change in phytochrome status

and light perception during a light sensitive phase of the

endogenous rhythm, responses were analyzed following

exposure of plants to a diurnal (24 hour) short day with

a light break at the eighth hour of darkness. In order to

show the developmental regulation resulting only from

light impingement on the endogenous rhythm, responses

were analyzed following exposure of plants to a bi-

diurnal (48 hour) short day with a light break at the

eighth hour of darkness. This light break is followed

by a long dark period of sufficient length to allow

iv

phytochrome to become innocuous in reference to flowering.

The influence of GA status was evaluated for each of the

photoperiodic conditions above. The effects of GA were

studied by comparing the growth and development of a GA

deficient dwarf (strain Kidachi), to that of the dwarf

treated with exogenous GA.,, and to that of a normal strain

(Violet) which contains abundant endogenous GA.

Normal and dwarf Pharbitis exposed to diurnal long

days and diurnal short days with a light break remained

vegetative due to combined inhibition imposed by the

phytochrome status and light impingement during the photo-

phobe (light sensitive) phase of the endogenous rhythm.

However, plants exposed to diurnal light break cycles

exhibited development of axillary buds which did not occur

in plants subjected to diurnal long days. Application of

GAo caused increased stem elongation, increased leaf area,

and axillary bud development of plants exposed to diurnal

long days or diurnal light break cycles.

Both normal and dwarf Pharbitis subjected to diurnal

or bidiurnal short days were induced to flower. Shoot apex

analysis of dwarf plants exposed to bidiurnal short days

revealed subapical elongation and development of axillary

buds, characteristics found only on GA-treated plants from

other photoperiods. Both exogenous and endogenous GA

V

increased flowering, stem elongation, and leaf area of

plants treated with diurnal or bidiurnal short days.

Applied GA^ was sufficient to overcome the stem growth

deficiency of the dwarf; however, flower production of the

dwarf strain equaled that of the normal only if it re

ceived the extended dark period of a bidiurnal short day

in conjunction with applied GA-. Therefore, the extended

dark period must facilitate synthesis and/or utilization

of some factor which enhances flowering.

Pharbitis exposed to bidiurnal short days with a

light break were induced to flower; however, the level of

flowering was repressed compared to plants that received

short day treatments. The length of darkness following

the light break was sufficient for phytochrome reversion;

therefore, the repression of flowering was due to light

effect during a light sensitive phase of the endogenous

rhythm. GA^ applied in conjunction with bidiurnal light

break cycles increased flowering, stem elongation, and leaf

area, but had no effect on the size or shape of developing

floral primordia.

These data show that the effects of the endogenous

rhythm, phytochrome status, and GA, may be analyzed

separately and in combination, and that these components

affect development of Pharbitis nil in different ways.

vi

LIST OF TABLES

T a b l e Page

1. Summary From the Literature Concerning Effect of Photoperiodic Treatments 5

2. Number of Terminal and Axillary Apical

Meristems Per Shoot Apex 19

3. Relative Area of Shoot Apex 20

4. Area of Vegetative Apical Meristems Per Shoot Apex 21

5- Combined Width of Floral Apical Meristems Per Shoot Apex , 22

6. Width of Terminal Meristem 23

7 . Flowering Responses of Dwarf and Normal Pharbitis Following Seven Cycles of Designated Photoperiodic Treatments . . . . 35

8. Stem Elongation of Dwarf and Normal Pharbitis Following Seven Cycles of Designated Photoperiodic Treatments . . . . 36

9. Relative Leaf Area of Dwarf and Normal Pharbitis Following Seven Cycles of Designated Photoperiodic Treatments . . . . 37

Vll

LIST OF FIGURES

Figure Page

1. Photoperiodic Treatments 10

•2. Percentage Flowering 13

3. Representative Drawings From Control Plants . I6

4. Representative Drawings From GA., Treated-P l a n t s -? 18

5 . M u l t i v a r i a t e Analysis of Developmental Responses 45

V l l l

CHAPTER I

INTRODUCTION

The transition of a plant bud from a vegetative state

to a flowering state remains one of the most intriguing

problems in the study of plant growth and development

(Chailakhyan, 1979). Although an array of environmental

factors have been shown to influence this transition, the

flowering of photoperiodic plants can be controlled by

manipulation of light/dark cycles; consequently, vegetative

and reproductive development may be selectively studied

(Bernier, 1971). Three components affecting regulation

of development were considered in this project: (1)

responses of plants to gibberellic acid; (2) responses

of plants resulting from the status of the photoreceptive

pigment phytochrome; and (3) responses of plants to an

endogenous rhythm which plays a role in the photoperiodic

response (Evans, 1969)-

Responses of plants to applied gibberellic acid

(GA^) are as follows: (1) bolting and flowering in some

long day (LD) rosette plants (Lang, 1957), but not floral

induction of short day (SD) plants under non-inductive

conditions (Jones, 1973); (2) increased stem elongation

by influencing cell elongation, or cell division, or both

(Sachs, 1965); (3) alteration of leaf area (Gray, 1957;

1

2

Humphries, 1958); and, (4) acceleration of leaf development

(Okuda, 1959) Maksmowych et al., 1976). GA., may influence

flowering indirectly by altering stem growth (Lang and

Reinhard, 196I) and enhancing mitotic activity of a meri

stem to render it more responsive to a floral stimulus •

(Ogawa and Zeevaart, I967). Extraction of vegetative and

flowering Pharbitis nil revealed no auxin differences and

hormone application studies with Pharbitis nil indicated

that GA., is more intricately involved in flowering than

other plant hormones (Ogawa and Zeevaart, I967).

Phytochrome, a photoreceptive pigment involved in

developmental regulation, exists in two forms, Pr. and P .

The far-red absorbing form, P., , is the predominant form

resulting from exposure to red or white light. At the

onset of darkness, P disappears as the red-absorbing

form, P , reappears. Reversion of P ^ to P during a

dark period must occur for floral induction to proceed in

SD plants (Hendricks, I963; Vince-Prue, 1975). In the SD

plant Pharbitis nil, this reversion begins about the sixth

hour of darkness and the proportion of P ^ declines rapidly

until the eighth to tenth hour (Evans and King, 1969). A

light interruption (LB) at the eight hour point of the

dark period of a 24 hour SD photoperiodic treatment re

establishes a high PfpiP^ ratio and results in an inhibi

tion of flowering for SD plants (Vince-Prue, 1975) since

3

the length of darkness after the light break is not suffi

cient for phytochrome reversion (Vince-Prue, 1976).

Biinning (1936) first proposed that an endogenous

rhythm was involved in photoperiodic timing and that it

was comprised of two twelve hour alternating phases, one

termed "photophil," or light-loving and the other

"scotophil," or dark-loving (=light sensitive). This

hypothesis has undergone many revisions (Dunning, 1973),

but experimental evidence shows that an endogenous rhythm

plays a major role in the photoperiodic flowering process

for many SD and LD plants (Hillman, 1976). The endogenous

rhythm also plays a role in the regulation of flowering of

Pharbitis (Hamner and Takimoto, 1964). The light period

of a LD extends into the light sensitive phase and a

light flash during the middle of the dark period of a SD

occurs during a light sensitive phase; therefore, when

light is perceived during a light sensitive phase of the

endogenous rhythm, flowering in a SD plant is inhibited

(Hamner and Hoshizaki, 1974).

The inhibition of flowering, which is seen follow

ing exposure of SD plants to a 24 hour short day with a

light break in the middle of the dark period (24 hour LB),

is therefore due to the combined influence of phytochrome

change and light perception during a light sensitive

phase of the endogenous rhythm (Vince-Prue, 1975). In

4

this paper, floral inhibition resulting in plants exposed

to a 24 hour LB treatment, will be termed floral inhibition

due to the effects of both phytochrome and the endogenous

rhythm.

Using bidiurnal (48 hour) photoperiodic treatments,

Hamner and Takimoto (1964) demonstrated that an endogenous

rhythm functions as a time measuring component for floral

induction in Pharbitis. In a SD plant, such as Pharbitis.

a light interruption eight hours after the beginning of

the dark period in a bidiurnal photoperiodic treatment

(48 hour LB) caused the greatest decrease in flowering

(Hamner and Takimoto, 1964). Since the length of darkness

after the light break was of sufficient duration for phyto

chrome reversion (Evans and King, 1969). this inhibition

of flowering was due to light perception during a light

sensitive phase of the endogenous rhythm (Hamner and

Takimoto, 1964). In this paper, floral repression occurring

in plants subjected to a 48 hour LB treatment, will be

termed floral repression due to the effect of the endoge

nous rhythm only. Some of the more pertinent historical

background for choosing each photoperiodic treatment is

summarized in Table 1.

Unfortunately, there is no defined experimental

treatment in which only phytochrome inhibition can be in

voked without involving inhibition due to light perception

5

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The extent of phytochrome inhibition alone can, however,

be extrapolated since the effects of combined inhibition

from both phytochrome and the endogenous rhythm (24 hour

LB treatment) and the effects of inhibition due to the

endogenous rhythm alone (48 hour LB treatment) can be

compared.

This study compares the effects and interactions of

the endogenous rhythm, phytochrome, and gibberellin, on

flower induction and development, stem elongation, and

leaf expansion of dwarf and normal Pharbitis • Light

break treatments given in either diurnal and bidiurnal

photoperiodic cycles have been used to separate the effects

of phytochrome and the endogenous rhythm in these processes

of growth and development. The effects of these treatments

were studied at both the cellular and organismal levels

using plants with and without applied GAo-

CHAPTER II

APEX DEVELOPMENT OF DWARF PHARBITIS IN RESPONSE

TO GIBBERELLIN AND PHOTOPERIOD

Studies of shoot apex responses to photoperiod have

been limited almost exclusively to LD and SD treatments

(Vince-Prue, 1975)- Pharbitis nil is a qualitative SD

plant, remaining vegetative under LDs and flowering

readily under SDs, and this facilitates comparisons

between vegetative and reproductive main shoot apices.

Marushige (1965a, 196512) has compared buds of normal

Pharbitis in successive LDs and SDs, while Bhar and

Radforth (1969) have compared apex development of plants

under LDs to those exposed to a single inductive SD.

Once floral induction occurred, reproductive development

was essentially the same in response to a single 24 hour

SD or a series of SDs (Bhar and Radforth, I969).

Studies detailing shoot apex responses to applied

GA- have also been documented (Sachs, 19651 Cutter, 1971:

Maksymowych et al., 1976). Applied GA- promotes flowering

if applied to Pharbitis before, during, or just after an

inductive long night (Takimoto, 1969).

This study utilizes dwarf Pharbitis for comparisons

of apex development of plants exposed to photoperiodic

treatments which distinguish between the floral inhibition

7

8

due to the effects of both phytochrome and the endogenous

rhythm to floral repression due to the effect of the

endogenous rhythm only. The interaction of these photo

periodic effects and those of GA- was considered by com

paring the buds of dwarf Pharbitis from each photoperiodic

treatment treated with exogenous GA., to those of plants

given the same photoperiods without GA-.

Materials and Methods

Seeds of dwarf Pharbitis were scarified with con

centrated HpSO^ and germinated in a 1:1 mixture of

vermiculite and sterile soil (Takimoto, 1969). The

experimental treatments were carried out in Percival

Growth Chambers with cool white fluorescent tubes supple

mented with 10?5 of the illumination as incandescent light

to provide a minimum illumination of 100 lux (measured as

17-3 - 18.5 |iW cm'^ nm"-*- with an IL Plant Growth Photo

meter at the cotyledonary surface). Seedlings were

grown in continuous light until five days old, then sub

jected to the photoperiodic treatments shown in Figure 1.

The temperature was maintained at 18.5 - 1.5 C for all

experiments (Hamner and Takimoto, 1964).

A preliminary experiment was conducted to determine

the number of cycles of each photoperiodic treatment re

quired to induce maximum flowering in dwarf Pharbitis.

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After each of ten successive cycles of photoperiodic

treatment, ten plants were removed from the growth chambers

and maintained under continuous light. Ten days after each

set of treatments, the flowering response was measured as

the percentage of plants- possessing floral buds (Takimoto,

1969). The number of cycles required to produce maximum

flowering was seven (Figure 2), therefore seven cycles were

used for comparison of control and GA--treated plants.

One lot (10 plants) of each photoperiodic treatment

served as a control. Another lot received 10 }xg of GA-

per plant. This was applied as 2 ^g in 10 p.1 of O.OO^fo

Tween 20 to the cotyledons during the main light period

of each of the first five photoperiodic cycles.

After seven photoperiodic cycles, the plants were

placed in continuous light until harvested. Ten days after

seven repetitive cycles of 24 hour photoperiodic treat

ments, and three days after seven repetitive cycles of

48 hour photoperiodic treatments, shoot apices were har

vested so that plants were chronologically the same age.

The terminal shoot tip of each plant was harvested and

fixed in FAA, dehydrated with ethanol, infiltrated with

xylene, embedded in paraffin, and mounted. Ten ;im sections

were serially mounted and stained with safranin and fast

green (Jensen, 1962). Five to eight repetitions were

made for each lot of plants. Median or near median

sections were selected for analysis.

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A count of the number of terminal and axillary apical

meristems per shoot apex was initially made. In order to

estimate and compare total size of shoot apices from each

treatment, the "cut and weigh" method of Evans (1972) was

used. Outlines of whole apices (50X) were cut from uni

form paper, weighed, and matched against a standard curve

to estimate relative area. Additional analysis included

an area measurement for all apical meristems per shoot apex

for vegetative plants and width measurements for all apical

meristems per shoot apex for flowering plants. A final

assessment of response was made by measuring width of

only terminal meristems.

Results

Representative camera lucida drawings (25X) of shoot

apices for control plants are shown in Figure J, and those

for GA-treated plants are shown in Figure 4. The number

of terminal and axillary apical meristems per shoot apex

of plants from each photoperiod is summarized in Table 2.

The relative area of the shoot apex as calculated by the

"cut and weigh" method is presented in Table 3. Estimated

areas of apical meristems, calculated from height and

width as shown in Figure 3a for vegetative plants, is in

cluded in Table 4. Width comparisons of floral apical

meristems, calculated as shown in Figure 3b, are presented

Figure 3. Representative drawings from control plants of each photoperiodic treatment. All are 25X. The arrows (h=height and w=width) show how area was measured for vegetative development (e.g. 24 Hour LD) and how width was measured for floral development (e.g. 24 Hour SD). See also Table 2 in relation to Figures 3 and 4.

Figure 4. Representative drawings from GA3 treated plants of each photoperiodic treatment. Note that axillary bud development is present on all except the plants from 48 Hour LB.

18

24 Hour LD a

24 Hour SD b

48 Hour SD d

48 Hour LB e

19

TABLE 2

NUMBER OF TERMINAL AND AXILLARY APICAL

MERISTEMS PER SHOOT APEX

Photoperiodic

Treatment

24 Hour LD

24 Hour LB

24 Hour SD

48 Hour SD

48 Hour LB

Response

vegetative

vegetative

floral

floral

floral

(Number Shoot

No GA-

1/4*

2/1

2/2

3/6

1/5

2/2

3/6

1/5

of Tips

Meristems/ Examined)

Plus 10 ;ig GA-

3/8

2/2

3/5

3/3

2/2

3/6

1/8

l/4 = 1 apical meristem each visible on 4 shoot apices

examined and 2/l = 2 apical meristems on 1 shoot apex.

20

Photoperiodic

Treatment

24 Hour LD

TABLE 3

RELATIVE AREA OF SHOOT APEX

Area (cm )

Response

vegetative

No GA 1

2840ax

Plus lOjag GA-

4l l2bx

24 Hour LB

24 Hour SD

48 Hour SD

48 Hour LB

vegetative

floral

floral

floral

4348by

8l50cy

9112cy

3790bx

3212abx

4l90bx

2330ax

3900bx

The r e l a t i v e a rea was ca lcu la t ed from cu t t i ng out camera

l u c i d a drawings of shoot apices (50X) and weighing t o com

pare a g a i n s t a s t anda rd . Means followed by the same

l e t t e r i n each v e r t i c a l column ( a - c ) , or in each h o r i z o n t a l

row ( x , y ) , a re not s i g n i f i c a n t l y d i f fe ren t a t the 0.05

l e v e l acco rd ing t o Duncan's New Multiple Range Test

(Dixon, 1971) .

21

TABLE 4

AREA OF VEGETATIVE APICAL MERISTEMS PER SHOOT APEX

Photoperiodic

Treatment

24 Hour LD

24 Hour LB

Response

vegetative

vegetative

No GA-

{)xm )

324ax

352ax

Area

Plus lOjag GA-

(;am )

550ay

520ay

The area of vegetative apical meristems was calculated from

the height and width as shown in Figures 3a and 4a. Means

followed by the same letter in each vertical column (a),

or in each horizontal row (x,y), are not significantly

different at the 0.05 level according to Duncan's New

Multiple Range Test (Dixon, 1971).

22

TABLE 5

COMBINED WIDTH OF FLORAL APICAL MERISTEMS

PER SHOOT APEX

Photoperiodic

Treatment

24 Hour SD

Response

floral

No GA 1

796cy

Width (>im)

Plus iq^g GA-

540bx

48 Hour SD floral 535by 283ax

48 Hour LB floral 348ax 378ax

The width of floral meristems was calculated as shown in

Figure 3b. Means followed by the same letter in each

vertical column (a-c), or in each horizontal row (x,y),

are not significantly different at the 0.05 level according

to Duncan's New Multiple Range Test (Dixon, 1971).

Photoperiodic

Treatment

23

TABLE 6

WIDTH OF TERMINAL MERISTEM

Mean Width ( m)

Response No GA 1

Plus 10;ag GA-

24 Hour LD

24 Hour LB

24 Hour SD

48 Hour SD

48 Hour LB

vegetat

vegetat

floral

floral

floral

ive

ive

248ax

208ax

796 cy

265ay

348bx

228cx

190bx

360dx

153ax

378dx

Means followed by the same letter in each vertical column

(a-d), or in each horizontal row (x,y), are not signifi

cantly different at the 0.05 level according to Duncan's

New Multiple Range Test (Dixon, 1971).

24

in Table 5- Table 6 shows a comparison of width measure

ments for terminal apical meristems only of both vegetative

and flowering plants.

Discussion

Plants subjected to the 24 hour LD treatment remain

vegetative because the dark period was shorter than the

critical dark period (Takimoto, 1969). Lack of floral

primordia development results because phytochrome is pre

dominately P^^ due to insufficient time for dark reversion

to the innocuous P form (Vince-Prue, 1975). Also, the

light period of the 24 hour LD extends into a light

sensitive phase of the endogenous rhythm (Hamner and

Hoshizaki, 1974), resulting in inhibition of floral in

duction. Pharbitis exposed to the 24 hour LB treatment

remain vegetative because the light interruption shifts

phytochrome to a Pf '-Pj ratio which inhibits flowering in

SD plants, and the light break occurs during a light

sensitive phase of the endogenous rhythm, which interrupts

procession toward floral induction (Vince-Prue, 1975)-

The effect of phytochrome status has been reviewed

by Cummings et al. (1965) and Vince-Prue (1976). In the

24 hour LB treatment, the dark period following the light

interruption is not of sufficient length for reversion to

a P„ :P ratio which does not inhibit flowering in Pharbitis

25

(Evans and King, I969). The effect of the endogenous

rhythm has been reviewed by several workers (Hamner and

Takimoto, 1964; Vince-Prue, 1975; Palmer, 1976). Bunning

(1973) and Hamner and Hoshizaki (1974) have presented both

experimental evidence and hypotheses concerning endogenous

rhythms in a 24 hour cycle. Their perspective is that

phytochrome is the photoreceptive pigment, but cannot

account for all experimental results. It is apparent,

therefore, that both of the above photoperiodic components

inhibit floral induction of Pharbitis in a 24 hour LD or

LB treatment.

Even though plants from both the 24 hour LD and LB

treatments remain vegetative, the morphology of the shoot

apices of plants from these treatments is not the same .

All shoot apices of plants exposed to the 24 hour LB

treatment exhibited axillary bud development (Figure 3c);

however, this was not the case for plants maintained under

24 hour LD treatments (Figure 3a). Comparisons of the

number of apical meristems (Table 2) and the relative

area of the shoot apex (Table 3) show this difference

also.

The differences in effect of the 24 hour LD and LB

treatments may be accounted for by considering the

following: (a) time of occurrence of the inhibitory

Pj^:P ratio of phytochrome; or, (b) the possible effect

26

of the light break on endogenous GA levels within the

plant. The first consideration involves the progression

of P j, to P^ and the time of this progression in the photo

periodic cycle. In the 24 hour LD treatment, the period

of d^kness is eight hours, which is not a sufficient

length for phytochrome reversion in Pharbitis (Evans and

King, 1969). Also, the plant is exposed to a single period

of darkness for P^^ reversion to P . In the 24 hour LB

treatment, Pharbitis is exposed to two periods of darkness

in which the Pf^sPj, ratio begins to change, and neither

period is of adequate length for phytochrome conversion

to a ratio which is innocuous to floral primordia develop

ment .

The second consideration involves comparing the

shoot apices from plants of the 24 hour LB treatment

(Figure 3c and Table 2) to those of GA-treated plants from

the 24 hour LD and LB treatments (Compare Figure 3c to 4a

and 4c. See also Table 2). The light break may cause a

breaking of apical dominance in dwarf Pharbitis by

changing endogenous GA levels. The effect of a light

break in the middle of the dark period has been correlated

with an increase in GAs by Chailakhyan and Lozhnikora

(1966). Since dwarf Pharbitis is a GA deficeint mutant

(Ogawa, 1965), the photoperiodic effect is clearly much

more intricate than just an effect on internal GA levels.

27

Development of floral primordia in control plants

receiving the 24 hour SD treatment progresses more rapidly

than in other inductive treatments (Figure 3b vs. 3d and

3e). The shoot apex of these plants enlarges as flowering

occurs (Table 3), as does the terminal apical meristem

(Table 6). The length of darkness of the 24 hour SD is

adequate for phytochrome to change to a predominant status

of P^, and not interfere with flowering (Hendricks, 1963;

Evans and King, 1969). Also, no light is perceived during

a light sensitive phase of the endogenous rhythm (Hamner

and Hoshizaki, 1974; Heide, 1977), and the length of

darkness provides adequate time for synthesis of a floral

stimulus.

Pharbitis grown under 48 hour SD treatments exhibit

subapical elongation and developing axillary buds

(Figure 3d). Comparable subapical elongation is not seen

in any other Pharbitis plants when GA- is absent. The

developing axillary buds of dwarf Pharbitis from 48 hour

SDs are enlarged and distinction between the terminal and

axillary apical meristems is difficult (Figure 3d)' The

extended dark period of the 48 hour SD treatment affects

floral primordia development differently than the 24 hour

SD treatment but similarily to the 24 hour SD treatment

plus GA- (Figure 3d vs. 4b). It is possible that the sub

apical elongation which occurs in plants subjected to 48

28

hour SD treatments, and which occurs without applied GA

is due to: (1) the extended dark period which may favor

GA synthesis and/or utilization and the result would be

similar to exogenous GA application; or. (2) the extended

dark period which may favor synthesis and/or utilization

of another factor which affects apex development in a

manner similar to that of GA. Chemical feeding experi

ments with kaurene and other GA precursors caused no

growth response in Pharbitis nil Kidachi, but GA- changed

the dwarf habit to a pattern similar to normal Pharbitis

(Katsumi et al., 1964). Extracts of GA-like compounds

from dwarf Pharbitis show little or no GA activity in

bio-assays (Ogawa, 1965; Barendse and Lang, 1972). Also,

no GAs could be detected and confirmed in dwarf Pharbitis

by use of gas chromatography or mass spectrometry (Simmons,

1974). These facts seem to rule out the first interpre

tation.

The results obtained with Pharbitis grown under a

48 hour LB treatment should be compared to those of the

24 hour LB treatment. Plants exposed to the 48 hour LB

treatment produce floral primordia whereas those sub

jected to the 24 hour treatment remain vegetative. A

light interruption eight hours af-er the onset of dark

ness in a bidiurnal cycle (48 hour L3) re-establishes a

high P_..:P ratio but, in Fharbi-Js, the subsequent dark

29

period exceeds the eight to ten hours needed for phyto

chrome to revert to P^ (Evans and King, 1969) and the

P^^:Pj^ ratio is not inhibitory to flowering (Figure 2).

However, the development of floral primordia of Pharbitis

•receiving the 48 hour LB treatment is repressed to a level

below that of plants subjected to SDs. Since the dark

period is of sufficient length for phytochrome reversion,

the effec- on floral development is therefore due to light

perception during a light sensitive phase of the endogenous

rhythm. The developmental regulation effected by both

phytochrome and the endogenous rhythm and that effected

by the endogenous rhythm only should not be considered

the same for ?harbi~is, since responses to the 24 hour LB

and ^3 hour LB treatments are not the game.

Applied .:-AT did not induce flowering of dwarf

Fharbj-js expose! to 24 hour LD, or L3 treatments, in

dicating no direct effect on floral induction. The GA

effect on flowering, for dwarf Pharbitis (and possibly

for other SD plants), must be enhancement of growth and

development of floral buds after induction in "us. GA

accelerated flora- development cut did not affect in-

du:tion in the SD plants (Perilla ocimoides (Horavka

et ai., 19c2) and Cosmos bioinnatus (Molder and Owens,

19^^).

Apices of dwarf Pharbitis, gro^^. in 2'- hour LD or

L3 treatments, show subapical elongation in response to

30

applied GA^ (Figure 4a and 4c). Also, the area of the

apical meristems increases compared to control plants

(Table 4). This is a response which has been documented

as a general characteristic of a GA-treated plant (Sachs,

1965). . Comparison of apices from untreated and GA--treated

plants in 24 hour LDs reveals that, in addition to sub

apical elongation, GA- positively stimulated axillary bud

development. Reference to Figures 3 and 4 clearly shows

that applied GA- affects apical dominance in dwarf

Pharbitis except when grown under conditions of the 48

hour LB treatment. Jacobs and Case (I965) have also

shown that GA- stimulates axillary bud growth and may

play a major role in apical dominance. The floral

primordia of GA-treated plants exposed to the 24 hour SD

treatment are retarded in development compared to non-GA

plants (Figure 4b vs. 3b). For dwarf Pharbitis, GA- re

duces terminal and axillary apical meristem size (Tables

5 and 6 ) but apparently promotes differentiation of

axillary buds into flowers for plants exposed to the 24

hour SD. Dwarf Pharbitis responds to 48 hour SDs and

GA- by producing a floral apex which is greatly reduced

in size compared to those other induced plants (Figure

4d and Tables 5 and 6). No effects of applied GA^ on

the size of buds are noted during the 48 hour LB treat

ment (Tables 5 and 6).

31

These data show that floral inhibition effected by

both phytochrome and the endogenous rhythm (responses to

the 24 hour LB treatment) differs distinctly from the

floral repression evoked by the endogenous rhythm only

(responses to the 48 hour LB treatment). The effect of

GA- on apex development of dwarf Pharbitis also differs

from the above photoperiodic components. The influences

of GA-, the status of phytochrome, and/or the endogenous

rhythm apparently act separately in their effects on

development of the shoot apex.

CHAPTER III

A COMPARISON OF THE EFFECTS OF

PHOTOPERIOD AND GIBBERELLIN ON THREE DIFFERENT

PROCESSES OF GROWTH AND DEVELOPMENT IN PHARBITIS NIL

Pharbitis n U strain Violet (=normal) is a twining

vine with elongated internodes and contains many GA-like

compounds (Ogawa, 1965; Barendse and Lang, 1972).

Pharbitis nil Kidachi is a GA deficient dwarf variety

(Barendse and Lang, 1972). and the dwarf growth habit can

be overcome by GA- application (Takimoto, I969). Both

strains are qualitative SD plants; therefore, vegetative

and reproductive growth development can be analyzed

separately by manipulation of photoperiodic treatments.

In Chapter II. flower induction and development was

emphasized. In the following study, flowering, measured

as the number of floral buds per plant, was analyzed to

represent a change from one developmental mode to an

other; that is. from vegetative to reproductive. Stem

elongation was analyzed to represent a definitive process

of growth, as length, and leaf expansion was analyzed to

represent a process involving growth as a change in size.

These three responses were assayed following

exposure of plants to specific photoperiodic treatments.

The effect of GA- was analyzed by comparing the responses

32

33

of dwarf Pharbitis. normal Pharbitis. and the dwarf

supplemented with exogenous GA^, in combination with the

photoperiodic treatments used to distin^ish the develop

mental regulation of both phytochrome and the endogenous

rhythm from that of the endogenous rhythm only.

Materials and Methods

Procedures from Chapter II were followed for seed

germination and seedling establishment of normal and dwarf

Pharbitis • 'Nt.er. the seedlings were five days old, e;':peri-

aentation began.

To test both the effect of GA- and photoperiod,

twenty plants of dwarf Pharbitis and ten plants of the

normal strain were used for each photoperiodic treatment

shown in Figure 1. Ten of the dwarf plants from each

photoperiod were treated with 10 jag GA- each. This was

applied as 2 g in 10^1 of 0.05?5 Tween 20 to the

cotyledons during the main light period of each of the

first five photoperiodic cycles. The remaining ten dwarf

plants 01 each photoperiod were used as untreated controls.

In order to assay plants which were chronologically

the same age, the growth and development responses were

neasured ten days after seven repetitive cycles of 24

hour photoperiodic treatments (Takimoto, 1969). and three

days after seven repetitive cycles of 48 hour photoperiodic

treatments. Flowering was measured as the number of florae

34

buds per plant (Takimoto. 1969). Stem elongation was

measured as length of the main stem in centimeters. Leaf

expansion was estimated by measuring the length and width

of leaves, and the product of these measurements was ex

pressed as relative leaf area per plant. This estimation

of relative leaf area has been used for symmetrical leaves

by several workers (Manivel and Weaver, 1974; Wargo, 1978)

and has a direct functional relationship to actual leaf

area.

Results

The flowering responses of the dwarf and normal

strains, which result after seven cycles of photoperiodic

treatment, are presented in Table 7. The number of flowers

per plant was the same for plants exposed to either the 24

hour SD or 48 hour SD treatment. Plants subjected to 24

hour LD or 24 hour LB treatments remained vegetative,

while those receiving 48 hour LB treatments produced

about one-third the number of flowers as plants grown in

SDs.

Stem elongation responses, resulting from the

various photoperiodic treatments and/or GA availability,

are shown in Table 8. The photoperiodic treatments seem

to have little effect on stem elongation of dwarf plants

that are GA deficient. However, there is a detectable

35

TABLE 7

FLOWERING RESPONSES OF DWARF AND NORMAL PHARBITIS

FOLLOWING SEVEN CYCLES OF DESIGNATED

PHOTOPERIODIC TREATMENTS

Photoperiodic

Treatment Dwarf

24 Hour LD O.Oax

Flowers per plant

Dwarf + GA,

O.Oax

Normal

O.Oax

24 Hour SD

24 Hour LB

48 Hour SD

48 Hour LB

3.6cx

O.Oax

3.6cx

l.lbx

4.7cy

O.Oax

6.5dy

2.6by

6 .2cz

O.Oax

7.0dy

3.2by


(a-d), or in each horizontal row (x-z), are not signifi



36

TABLE 8

STEM ELONGATION OF DWARF AND NORMAL PHARBITIS



24 Hour SD

24 Hour LB

5•labx

5•labx

Photoperiodic

Treatment Dwarf

24 Hour LD 5.5bx

Length (cm)

Dwarf + GA 1

17.8ay

13.2ay

14. lay

Normal

9-5hz

7.9az

8.7bz

48 Hour SD 5.0ax 12.7ay 7.8az

48 Hour LB 5.4abx 13.7az 11.5CZ





37

TABLE 9

RELATIVE LEAF AREA OF DWARF AND NORMAL PHARBITIS



Photoperiodic

Treatment Dwarf

24 Hour LD 23.3bx

Relative Area (cm )

Dwarf + GA 1

32.Oby

Normal

33.2ay

24 Hour SD 20.6ax 28.lay 43.5bz

24 Hour LB

48 Hour SD

48 Hour LB

26.Obex

26.9CX

33.7dx

32.7by

30.7axy

43.0cy

34.5ay

35.8aby

52.8cz





38

difference between stem growth of plants of the 24 hour LD

and 48 hour SD treatments.

The relative leaf area for plants of both strains is

presented in Table 9- Dwarf plants subjected to the 24

hour SD, and not treated with GA-, have a smaller relative

leaf area than plants of the two other 24 hour cycles.

The results shown indicate that, in general,• plants from

bidiurnal, or 48 hour cycles, exhibit a larger relative

leaf area. Dwarf plants grown under the 48 hour LB treat

ment possess the largest relative leaf area of any group

not treated with GA.

Exogenous GA- applied to the dwarf and endogenous

GA of the normal strain caused an increase in flowering

of plants subjected to inductive photoperiods when com

pared to the untreated dwarf. Even though the availability

of GA increased flowering, only dwarf plants grown under

the 48 hour SD treatment, and treated with GA-, had a

floral response equal to that of the normal strain.

Both exogenous GA- applied to the dwarf and endogenous

GA of the normal caused increased stem length compared to

the untreated dwarf. The availability and effect of GA

on stem elongation is significant for every photoperiodic

treatment and the amount of growth effected by exogenous

application indicated that the level of GA^ treatment was

probably more than optimal and exceeded that of the normal

39

strain. Stem length of the normal strain was shorter for

plants subjected to the 24 hour or 48 hour SD treatments

than length of plants exposed to the 24 hour LD or LB '

treatments .

Application of GA3 to the dwarf, and endogenous GA

of the normal, increased leaf area under all photoperiodic

treatments . The leaf area of the GA-treated dwarf was

equal to that of the normal strain only under 24 hour LD,

24 hour LB, and 48 hour SD photoperiodic conditions. The

extended dark period of 48 hour SDs, in conjunction with

GA^, enhanced flowering, but had no such effect on leaf

expansion. Plants of both strains, if subjected to 48

hour LB treatments, developed a greater leaf area than

plants exposed to other photoperiodic treatments.

Discussion

The flowering of both normal and dwarf Pharbitis

follows the predictions made in Table 1. Floral inhibi

tion effected by both phytochrome and the endogenous

rhythm occurs in plants grown in 24 hour LD or 24 hour LB

treatments. Plants exposed to either 24 hour SD or 48

hour SD treatments flower in response to the lengths of

darkness in which a floral stimulus is present. The

flowering response of both strains subjected to the 48

hour LB treatment is repressed to a level below that of

40

plants exposed to SDs. The repression of flowering due to

light effect on the endogenous rhythm is seen as predicted

in Table 1.

Applied GA^ did not induce flowering of dwarf

Pharbitis exposed to 24 hour LD, or 24 hour LB treatments,

indicating no direct effect on floral induction. The GA

effect on flowering, for these two strains of Pharbitis

(and possibly for other SD plants), must be enhancement of

floral bud development after induction in SDs. GA also

accelerated floral development but did not affect induction

in the SD plants Perilla ocimoides (Horavka et al. , 1962) and

Cosmos bipinnatus (Molder and Owens, 1974). Lang and

Reinhard (196I) originally proposed that GA affected

flowering indirectly by altering stem growth. GA may also

affect mitotic activity of a meristem, rendering it more

responsive to a floral stimulus (Ogawa and Zeevaart, 1967).

Plants subjected to the extended dark period of the

48 hour SD treatment, and having available GA, show the

greatest increase in flowering. The exogenous GA- was

adequate in overcoming the growth deficiency of the dwarf;

however, both the extended dark period and the applied GA^

were required to increase flowering to that of the normal.

Imamura and Takimoto (1955). using normal Pharbitis, ob

served an increase in flowers per plant with an increase in

length of the dark period. An increase in flowering with

41

increased length of a dark period was also noted for Lemna

perpusilla (Shibata and Takimoto, 1975). Since plants sub

jected to 24 hour SD or 48 hour SD treatments received the

same amount of exogenous GA^, the concentration of GA^

should not be considered the limiting factor for plants

exposed to 24 hour SD treatments. Two possibilities may

be proposed: (a) the extended dark period may facilitate

GA enhancement of flowering; or (b) the extended dark

period may allow production of a factor which acts in

conjunction with GA to increase flowering. Dwarf Pharbitis

is a GA deficient mutant (Ogawa, 1965; Simmons, 1974);

therefore, the first possibility is unlikely. The ex

tended dark period must favor synthesis and/or utilization

of a factor which enhances flowering in dwarf Pharbitis.

Chailakhyan (1979) has proposed a bicomponent scheme,

comprised of gibberellins and anthesins, in which both are

required for flowering. If the above dark factor could be

considered anthesin, then Pharbitis nil grown under 48

hour SD treatments should prove to be a rich source of

this floral stimulant.

Both exogenous and endogenous GA increased stem

elongation and the amount of GAo applied to the dwarf

stain was more than adequate to overcome the dwarf growth

habit and stem length exceeded those of the normal. Stem

elongation of the normal strain follows a pattern linked

42

to floral induction. Normal plants exposed to the 24 hour

SD, and flowering, have the shortest stem lengths recorded

for 24 hour cycles. Stem elongation of plants subjected

to the 24 hour LD or 24 hour LB treatments and remaining

vegetative, is greater. Normal plants of 48 hour SD treat

ments produce a greater number of flowers but less stem

elongation than plants exposed to 48 hour LB treatments.

This indicates that when flowering is either not in progress

or repressed, GA may be utilized for stem growth. Okuda

(1959) also found that applied GA- resulted in accelerated

development of leaf primordia and increased stem elongation.

The effects of GAs on stem elongation, especially for

genetic dwarfs, have been well documented (Pelton, 1964;

Phinney and West, I960).

Leaf expansion was enhanced by applied GA-, and leaf

area measured for the GA-treated dwarf equaled that of

normal Pharbitis under photoperiodic conditions of the 24

hour LD and 24 hour LB treatments. This also supports the

above statement that GA is utilized in growth processes

when flowering is not in progress. iJ\/hen induced to flower

by 24 hour SD treatments, plants of dwarf Pharbitis ex

hibit less leaf expansion than vegetative plants, whereas

normal Pharbitis (with endogenously available GA) has a

greater leaf area. An increase in leaf expansion with

floral induction has also been reported for the SD plant

43

Chenopodium (Thomas, 196la), and the LD plants Spinacia

oleracea and Trifolium repens (Thomas, 196lb). Com

parisons of responses of plants to 48 hour cycles reveals

that leaf expansion is enhanced by the 48 hour LB treat

ment. This is in contrast to the decrease in flowering

when compared to the 48 hour SD treatment. Thus, available

GA may be utilized in leaf expansion when flowering is re

pressed.

A graphic summary of data from the three tables,

Figure 5. shows that flowering is affected more than

stem elongation or leaf expansion by available GA. When

flowering, leaf area, and stem elongation are charted

simultaneously, the shift of relationships due to GA is

as follows : dwarf control plants form the more closely

aligned cluster; the dwarf plants supplemented with GA-

show an intermediate cluster; and, the normal plants show

the least alignment of the clusters. It is important to

note that the shift of all three sets of clusters is in

the direction of increased flowering. Photoperiodic

treatments 1 and 3, the 24 hour LD and 24 hour LB treat

ments, respectively are adjacent. These results correlate

with the predictions in Table 1 concerning developmental

regulation effected by both phytochrome and the endogenous

rhythm. Photoperiods 2 and 4 are adjacent, which are the

24 hour SD and 48 hour SD treatments, respectively.

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46

Reference to Table 1 shows that neither phytochrome nor

the endogenous rhythm should inhibit flowering in plants

exposed to these photoperiods. Apparently, the effects of

these components on stem elongation and leaf expansion are

similar for plants grown under these photoperiods. Also,

the uniqueness of the 48 hour LB treatment is depicted in

Figure 5. Photoperiod 5, the 48 hour LB treatment, does

not align with any group. This also fits the prediction

in Table 1 that responses to this photoperiodic treatment

must be interpreted -differently than responses to those

above.

These data indicate that developmental regulation

effected by both phytochrome and the endogenous rhythm

differs from that effected by the endogenous rhythm only.

The effect of both exogenous GA on the dwarf and endogenous

GA in the normal also differs from the above photoperiodic

components. The status of the endogenous rhythm, and/or

phytochrome, and the influences of GA, act separately in

their effects on flowering, stem elongation, and leaf

expansion.

CHAPTER IV

SUMMARY

The i n f l u e n c e s of GA, phytochrome, and the endoge

nous rhythm on flower induction and development, stem

elongat ion, and leaf expansion of Pharbitis n i l have been

analyzed at the ce l lu l a r and organismal l eve l s . Data pre

sented support these main points : (a) developmental r e

gu la t ion due to the endogenous rhytrjr alone can c lear ly be

d is t inguished from tha t due to both phytochrome and the

endogenous rhythm (Table 7 and Figure 5); (b) combined

ef fec ts of both phytochrome and the endogenous rhythm

caused a breaking of apical dominance, and th i s i s a

response usua l ly observed for GA-treated plants (Figure

3c vs. 4a, 4b, and 4c) ; (c) if f loral induction occurs, GA

i s u t i l i z e d for flower development instead of stem and

leaf growth (Tables 7-9; Fig-ure 5); (d) the stem growth

deficiency of the dwarf i s overcome by applied GA , but

both GA and an extended dark period are required for

flowering of the dwarf to equal that of the normal (Table

7) , i nd ica t ing tha t an extended dark period f a c i l i t a t e s

synthesis and/or u t i l i z a t i o n of a factor which enhances

flowering; (e) the influences of Z-A, the s tatus of phy-^o-

chrome, and/or the endogenous rhythm are d i s t i nc t ly dif

ferent in these growth and developmental processes.

47

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Bernier, G. 1971. Structural and mxetabolic changes in the shoot apex in transition to floweri-g. Can. J. Bot. 49:303-819.

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48

49

'^"^^MacMilia^l;!- ' ' ' ' ' ^^^ Induction of Flowering.

Evans, L. T., and R_. W. King. 1969. Role of phytochrome m photoperiodic induction of Pharbitis nil Z. Pflanzenphysiol. 60:277-288"! '~

Gray, R. A. 1957. Alteration of leaf size and shape and other changes caused by gibberellins in plants. Am. J. Bot. 44:674-682.

Hamner, K. C , and T. Hoshizaki. 1974. Photoperiodism and circadian rhythms; an hypothesis. BioScience 24:407-4l4.

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Heide, 0. M. 1977. Photoperiodism in higher plants: an interaction of phytochrome and circadian rhythms. Physiol. Plant. 39=25-32.

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Jacobs, W. p., and D. B. Case. 1965- Auxin transport, gibberellin and apical dominance. Science 148:1729-1731.

Jensen. W. A. 1962. Botanical histochemistry. W. H. Freeman & Company, San Francisco.

50

Jones, R. L. 1973. Gibberellins: their physiological role. Ann. Rev. Plant Physiol. 24:571-598.

Katsumi, M., B. 0. Phinney, P. R. Jefferies, and C. A. Henrick. 1964. Growth response of the d-5 and an-1 mutants of maize to some kaurene derivatives. Science 144:849-850.

Lang, A. 1957 • The effect of gibberellin on flower formation. Proceedings National Academy of Sciences 43:709-717.

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