Cazebonne Et Al 1999

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Revista Chilena de Historia Natural72: 83-91, 1999

Salinity effects on germination and growthof Prosopis chilensis

Efectos de la salinidad en la germinaci6n y crecimiento de Prosopis chilensis

CONSTANCE CAZEBONNE, ANGELICA I VEG,DANIEL A VARELA2and LILIANA A CARDEMIL1Escuela de Ecologfa y Paisajismo, Facultad de Arquitectura y Bellas Artes, Universidad Central2Departamento de Biologfa, Facultad de Ciencias, Universidad de Chile,Universidad de Chile, Casilla 653, Santiago, Chile

ABSTRACTProsopis chilensis, also named Chilean algarrobo, is a leguminous tree, described as highly tolerant to heat shock, waterdeficit, salinity and injury stress. The objective of this research was to investigate the germination and growth responsesof seeds, seedlings and young plants of P chilensis under increasing concentrations of N aCl. Six groups of P chilensisseeds were germinated. One group was watered with distilled water (control group) and the other five were treated with0.3, 0.4, 0.5, 0.6, and 0.8 M NaCI. Control seeds germinated lOO% in 48 hours, while those imbibed in 0.3M and 0.4MNaCI reached lOO% after 21 days of imbibition. In 0.5M NaCl there was 66% germination after 28 days of imbibition.Above 0.5M NaCI there was no germination. Seedlings of 0.5 cm long germinated and maintained for 8 weeks in thesame NaCI concentration used for germination grew less than the control seedlings. Seedlings and young plants ofalgarrobo coming from seeds germinated and grown for 14 days in distilled water could grow a lot better in 0.3 to 0.8M NaCI. The seedlings reached a 69% growth of the control seedlings in 0.8M NaCl after 21 days of treatment, whileplants showed 58% growth of the control group after 8 weeks of treatment at this concentration. t was also observedthat the seedling cotyledonary expansion was delayed by salinity. Under salt treatment the number of leaves per plantwas reduced with a tendency to become significantly different to the control with 0.8M NaCI after 8 weeks of reatment.The conclusion of this research is that algarrobo can be consider as a Prosopis spp. highly tolerant to salinity becauseplants and seedlings can grow in 0.8M NaCI.

Key words: algarrobo, salinity, salt tolerance, imbibition, stress.

RESUMENProsopis chilensis, llamado algarrobo chileno, es un rbol leguminoso, considerado tolerante a estres termico, a deficithfdrico, a la salinidad, y a estres de heridas. El objetivo de este trabajo fue investigar la germinacion y crecimiento deplntulas y plantas jovenes de P chilensis a diferentes concentraciones de NaCI. De seis grupos de semillas, un grupofue regado con agua destilada (grupo control) y Ios otros 5 fueron tratados con 0,3, 0,4, 0,5, 0,6, y 0,8 M NaCI. Lassemillas control germinaron lOO% en 48 horas, mientras que as embebidas en 0,3M y 0,4M NaCI germinaron en un100% despues de 21 dfas de imbibicion. En 0,5M de NaCI hubo un 66% de germinacion a Ios 28 dfas de imbibicion.Sobre 0,5M de NaCl no hubo germinacion. Plntulas de 0,5 cm de longitud, provenientes de semillas germinadas endiferentes concentraciones de NaCl y mantenidas por 8 semanas en las concentraciones de NaCI usada en lagerminacion, crecieron mucho men os que las plntulas control. Plntulas y plantas jovenes de algarrobo provenientesde semillas germinadas y crecidas por 14 dfas en agua destilada pudieron crecer mejor en Ios tratamientos de 0,3 a 0,8M de NaCl. Las plntulas regadas con 0,8M de NaCI alcanzaron un 69% del crecimiento del control despues de 21 dfasde tratamiento, y un 58% del crecimiento del control alas 8 semanas de tratamiento. La expansion de Ios cotiledonesde las pliintulas tambien se retraso con el tratamiento salino. El numero de hojas por planta tiende a disminuir con lasalinidad existiendo una tendencia a ser significativamente diferente a control con 0,8M de NaCI despues de 8 semanas.La conclusion de esta investigacion es que algarrobo debe ser considerado como una especie de Prosopis altamentetolerante a la salinidad porque plantas y plntulas de algarrobo pueden crecer en 0,8M NaCI.

Palabras clave: algarrobo, salinidad, tolerancia a la sal, estres.

(Received March 7, 1998; accepted November 12, 1998; managed by Luis Corcuera)


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84 CAZEBONNE ET AL.INTRODUCTION

Trees of the leguminous genus Prosopisgrow in arid and semi arid regions of tropi-cal and subtropical areas of the world. Theyare considered underused trees, in spitethat the genus has potential for food, timber,fuel production and control of desertifi-cation (Felker et al. 1981, Torres 1985).Among Prosopis spp., Prosopis chilensis, aChilean mesquite named algarrobo, occursin the arid and semi arid region of Chile,from Copiap6 at the North (27 S) to Talca(37 S)(Roig et al. 1993). Algarrobo hasalso been planted in the Pampa delTamarugal as part of the reforestationprogram of Corporaci6n Nacional Forestal(Torres 1985).

Algarrobo trees are considered to betolerant to water stress, low and high tem-peratures (Felker et al. 1983, MedinaCardemil 1993), wound stress (Rodrfguez

Cardemil 1994, 1995) and high salinity(Arce et al. 1990).

Medina & Cardemil (1993) demonstratedthat P chilensis is highly tolerant to heatshock with a strong expression of heat shockproteins. Rodriguez Cardemil (1994,1995) found that seedlings responded tomechanical injury with a precocious devel-opment of secondary roots and increasingexpression of cell wall proteins.

Arce et al. (1990) have reported the ger-mination responses of three species ofProsopis: P chilensis, P tamarugo andP.alba when the seeds were imbibed indifferent concentrations of N a Cl. Felker etal. (1981), have also studied the growth ofdifferent Prosopis spp. at a maximum con-centration of 0.5 M NaCI with almost nogrowth of P chilensis at this concentration.However, studies on growth of seedlingsand plants of P chilensis in concentrationsof NaCI higher than 0.5 M have not beenperformed even when the seedlings have toestablish and plants have to grow in salinesoils such as that of the Pampa delTamarugal. Here the soil has a crust of saltwith a thickness ranging from 10 to 60 cmand containing sodium, magnesium, potas-sium and calcium (Briner 1985). Algarroboalong with other Prosopis spp. such as Ptamarugo y P alba, was used from 1965-

1970 by the Corporaci6n Nacional Forestal(CONAF) in a reforestation program torecover the ecology of salt flats of theAtacama Desert. In this program, nearly3136 ha of the Pampa del_ Tamarugal wasreforested with algarrobo (Aguirre Wrann1985).

The objective of this research was to testthe hypothesis that Prosopis chilensis ishighly tolerant to salt and, therefore, cangerminate and grow at salt concentrationshigher than that of sea water (0.5M NaCI)since is able to live over a crust of salt at thePampa of Tamarugal. This research is re-evaluating the germination and growth ofyoung plants and seedlings, and the leafnumber produced per plant at increasingconcentrations of NaCI from 0.3 to 0.8M.

MATERIAL AND METHODS

Germination experimentsSeeds of P chilensis were collected inPeldehue, Central Chile, in March 1993.For germination, the seeds were scarifiedfor 20 min in concentrated H2SO4 , and rinsedseveral times in distilled water. The seedswere germinated on trays covered withhumid paper (with distilled water ordifferent concentrations of N aCl) and keptunder continuous light during all thegermination period. Germination wasmonitored every 8 h during the first 48 hafter imbibition, and considered positivewhen the radicle appeared. For the seedsgerminated in saline solutions, the germi-nation was also monitored 14, 21 and 28days after the start of treatments.

For germination experiments, 150 seedswere scarified and germinated at 25C asdescribed above and divided into six groupsof 25 seeds each to be imbibed in differentconcentrations of N a Cl. The group I wasimbibed in distilled water; the group 2 wastreated with 0.3M NaCI; the group 3 with0.4M NaCI; the group 4 with 0.5 M NaCI;the group 5 with 0.6 M NaCI, and the group6 with 0.8 M NaCI. Each experiment wasrepeated four times and the percent of ger-mination was determined during 28 days.The significance of differences of germi-


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SALINITY EFFECTS ON PROSOPIS CHILENSIS 85nation among groups treated with differentN aCl concentrations was evaluated byKruskal-Wallis test after saline treatment.Seedling growth experimentsSeedlings of0.5 cm long derived from seedsgerminated at 25C in humid paper withdistilled water (control experiment) andfrom seeds germinated in the same substratebut irrigated with 0.3, 0.4, 0.5, 0.6 and 0.8M N a Cl, were grown for 8 weeks in thesame saline concentrations used forgermination. The length of the seedlingswere measured at the end of the 8 weekperiod. An ANOVA and Tukey Test wereperformed to evaluate the significance ofdifferences between lengths of seedlings atdifferent N aCl concentrations.

In another experiment, seeds were ger-minated at 25C in humid paper with dis-tilled water. After germination the seed-

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lings were grown for 14 days in the paperirrigated with distilled water, then dividedinto 5 groups, one for each saline treat-ment: distilled water (control); 0.4 M NaCl;0.5 M NaCl; 0.6 M NaCI; 0.8M NaCI. Theexperiments were run with three replicatesof each treatment and the growth was de-termined by measuring the length of theseedling axes. Growth determinations weremade after 7, 14 and 21 days of treatment.An ANOVA of repeated measurementswas performed to evaluate the significanceof differences between groups of seed-lings at different N a Cl concentrations.Plant growth experimentsTwo month old plants were used for growthexperiments under saline treatments. Theplants were obtained from seeds germinatedat 25C in humid paper with distilled waterand the seedlings were maintained in the

e o M 0.3 M

~ 0 . 4 M0.5 M--.-0.6 M--A-0.8 M

0 5 1 0 15 20 25 30Time of treatment days)

Fig. I: Germination of seeds of Prosopis chilensis during 28 days of treatment at different concentrationsof NaCI. The germination among treatments, after two days, showed significative differences (P


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86 CAZEBONNE ET AL.same substrate for 14 days into chambersat 25C with a photoperiod of 16 h. Afterthis period of time, the seedlings weremoved to a perforated Styrofoam platformwith the roots submerged into a recipientcontaining Hoagland II nutrient solution.After 4 weeks under this condition, theplants were transferred to pots with organicsoil and separated in 6 groups irrigatedwith: distilled water; 0.3 M NaCl; 0.4 MNaCl; 0.5 M NaCl; 0.6M NaCl and, 0.8MNaCl. During the treatments the plantswere kept in a greenhouse at a maximumtemperature of 30C in the day and aminimum of 18C at night. Plant growthwas monitored for two months by measuringthe length of the stem and determining thenumber of leaves of the main stem. Duringthe 8 week period of the experiment thebranches were not developed. Therefore,they were not considered as a growthparameter. An ANOVA of repeated measu-rements was performed to determine thesignificance of differences of the length

0 0.3 0.4 0.5 0.6 0.8Na Cl M)

Fig. 2: Growth of seedlings after 8 weeks of treatmentat different concentrations of NaCl. The seedlingswere 0.5 cm long at the beginning of the experimentand came from seeds germinated at the sameconcentration of NaCl of the growth treatment. Thesignificanceof differences (noted by different letters)was determined by the ANOVA and Tukey test(P


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SALINITY EFFECTS ON PROSOPIS CHILENSIS 8721 days of treatment. The high tolerance ofP chilensis to salinity was evident whenseedlings watered with 0.8 M NaCl wereable to grow one cm (from 8 to 9 cm) duringthe period going from 7 to 21 days of treat-ment.

The cotyledonary expansion was also af-fected by high N a Cl concentrations. Elevenout of 14 seedlings expanded their cotyle-dons when treated with water or 0.3, 0.4and 0.5 M NaCl; 8 out of 25 when treatedwith 0.6 M NaCl and, 6 out of 25 whentreated with 0.8 M NaCl.

When 6 weeks old algarrobo plants wereirrigated with different N aCl concentra-tions for 8 weeks, no significant differ-ences in the length of the stems of plantswere found during the first 4 weeks of salttreatment. The same was observed withrespect to the length of the stems betweensalt-treated plants and control plants (Fig-ure 4 ). After this time, the differences ingrowth between control plants and thosetreated with 0.8 M NaCl became signifi-cantly different (P


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88 CAZEBONNE ET AL.P chilensis (Felker et al. 1981) and, in Pchilensis, P tamarugo and P alba (Arce etal. 1990). However, none of these worksreport experiments performed in seedlingsand plants of Prosopis spp. withconcentrations of N aCl higher than in seawater, which is approximately 0.5 M NaCl,as done now in this work.

The results demonstrate that P chilensisis a tree highly tolerant to salt stress be-cause:a) Although salinity in general delays theonset of germination there is 68% germina-tion in 0.5 M NaCl after 28 days of treat-ment.

b)The seedlings can grow in 0.8 M NaCl,being more salt tolerant than germinatingseeds.

60 0 M0 0.3 M50 0.4 M0 0.5 M

- A 0.6 ME 40 0.8 M1:::-i30Q

E20

10

0 0 1 2 l 4

c) Plants can grow in 0.8 M NaCl for 8weeks with not significant differences ingrowth respect to the control plants forthe first 4 weeks of treatment.d) Similarly, the number of leaves of theplant stem increased in all N aCl concentra-tions during the 8 weeks of treatment withnot statistical differences in relation to thecontrol plants. However, there is a ten-dency in the plants to increase the differ-ences of the leaf number among the groupsover time of treatment and, probably, asignificative difference will be found be-tween the control group and that treatedwith 0.8 M NaCl ifthe period of the experi-ment is extended beyond 8 weeks.e) The plants can reassume the rate ofgrowth of the control group and the size of

5 7 8 9Time o treatment weeks)

Fig. 4: Stem growth of plants during 8 weeks of treatment at different concentrations of NaCl. TheSignificance of Differences for growth af the stems among groups treated with different salt concentrations,were evaluated by an ANOVA of repeated measurements. A P


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SALINITY EFFECTS ON PROSOPIS CHILENSIS 89

40

UJ 30I)-

11I) 20cEsz

10

0::.

-

1

0 M0.3 M0.4 M0.5 M0.6 M0.8 M T

~ = ~: - . -------.------- -

2 3 4 5 6 7 8 9Time of treatment weeks)

Fig. 5: Effect ofNaCl treatment on the number of leaves of the main stem of Prosopis chilensis plants. Thenumber of leaves was determined during 8 weeks for each treatment. The plants are the same used in theexperiment of Figure 4. The significance of differences were also evaluated by an ANOVA of repeatedmeasurements. A P=0.094 was found between the control group and tha t treated with 0.8 M NaCl at the eigthweek of treatment. Vertical bars = standard deviations.Efecto del NaCI en el numero de hojas del tallo principal de plantas de Prosopis chilensis. El numero de hojas fue determinadodurante 8 se man as para cada tratamiento. Las plantas fueron as mismas usadas en el experimeto de la Figura 4. Las diferenciassignificativas fueron eval uadas por una ANOV A de mediciones repetidas encontrandose un P=0,094 entre el grupo control yel grupo tratado con 0.8 M de NaCI a as 8 semanas de tratamiento. Barras verticale s =desviaciones estandard.the leaves recovers if they are returned tobe irrigated with tap water.

Prosopis chilensis seems to increase itsthermotolerance when the seeds are germi-nated at 35C (Medina & Cardemil 1993).In contrast, the tolerance of the seedlingsto salinity is decreased when the seedlingscame from seeds germinated in 0.3, 0.4 and0.5 M NaCl. Indeed, seedlings coming fromthese seeds grew less than those treatedwith salt but coming from seeds germi-nated in distilled water (see Fig. 2 and 3).From these results we can conclude thatduring growth and development of the seed-lings of algarrobo acquires tolerance tosalinity. However, more research needs tobe done in Prosopis chilensis to find outthe molecular mechanisms of this acquiredsalt tolerance throughout seedling devel-

opment (Winicov & Bastola 1997, Flowerset al. 1997).

The length of the stem is affected earlierand by a lower concentration of NaCl (0.6M) than the leaf number. In addition, thesize of the leaf area is also affected byNaCl becoming smaller under treatmentsfrom 0.5 to 0.8 M. Thus, the effect ofNaClon the length of the stems and number ofleaves of plants seems to indicate that thesalt treatment affects more the growth oforgans than the differentiation of them inProsopis chilensis.

We expected to have a large statisticaldispersion of the data among indivictuals ofthe same group, since there is a high ge-netic variability in Prosopis chilensis asoccurs in all Prosopis spp. due to theobligate cross pollination for seed produc-


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90 CAZEBONNE ET AL.tion (Felker et al. 1981, Rodhes & Felker1988). This was true for the experimentsof seedling coming from seeds germinatedin distilled water and for those with plantsgrown in Hoagland's solution II. However,this statistical dispersion was rather low inthe germination experiments and in thosewhere the growth of seedlings was evalu-ated after irrigation with the same N a Clconcentrations used for germination (Figs.1 and 2). Probably this is due to the fact thatseeds acquire a synchronized growth givenby germination at different salt concentra-tions, as it does other stress conditions.

Felker et al.(l981) observed less growthof P chilensis plants than those of Particulata, P pallida and P tamarugo, withalmost full losses of leaves, using 0.25 and0.5 M NaCl added to a nitrogen-free nutri-ent solution. From these experiments theyconcluded that P chilensis is less salttolerant than the other Prosopis spp. inspite that P chilensis is able to grow onsoils covered with salt.

In spite of the difference between theirdata and ours, our results seems to favorthat P chilensis is a tree highly tolerantto salinity. One might speculate thatalgarrobo is more salt tolerant than most ofthe salt tolerant terrestrial plants reportedso far, such as the genera, Chenopodium,Suaeda, Salicornia, Atriplex (Somers1979), Phillyrea latifolia (Gucci et al. 1997)and Mesembryanthemum crystallinum(Yen et al. 1997). However, without analy-ses and quantification of the salt contentsof the algarrobo plants grown in salineconditions and without knowing the physio-logical and molecular mechanisms of thesalt tolerance of algarrobo, it is not pos-sible to compare, in this respect, Prosopischilensis with other species (WinicovBastola 1997, Yen et al. 1997).

In any case, the high tolerance ofalgarrobo can explain why it has been pos-sible for CONAF(Corporaci6n NacionalForestal) to reforest part of the Pampa delTamarugal with P chilensis, where theseeds of Prosopis spp. such asP. tamarugogerminate and seedlings have to grow andsettle in a soil covered with a crust of salt.t will be possible then to consider P

chilensis as a tree that can provide food,

fuel, timber and to halt desertification inthe coastal deserts of the world using seawater for irrigation. Because salinity insoil affects 7% of the land's surface andabout 5% of the cultivated land with a 20%of irrigated land suffering from secondarysalinization, (Flowers et al. 1997), Prosopischilensis can be considered as well as agenetic resource for salt tolerance.

ACKNOWLEDGMENTS

We are grateful of Dr. Ernes to Medina andJose Gregorio Rodrfguez, both from Insti-tuto Venezolano de Investigaci6n Cientffi-ca, Caracas Venezuela, of Dr. OrlandoBalboa, Facultad de Ciencias Universidadde Chile (deceased in 1996) for review andcomments on this manuscript. We thankMr. Hector Sanchez for growing Prosopischilensis plants in Hoagland's nutrientsolution. Support is acknowledged fromDID Universidad de Chile and fromFONDECYT grant N 1950401 granted toLiliana Cardemil.

LITERATURE CITEDARCE P, C MEDINA 0 BALBOA (1990) Toleran cia a

la salinidad en la germinacion de 3 especies deProsopis P. alba, P chilensis y P. tamarugo . Cien-cia e Investigacion Agraria 17:1-75

AGUIRRE J J J WRANN (1985) The genus Prosopisand its management at the Tamarugal Pampa. In:Habit M (ed) The Current State of Knowledge ofProsopis tamarugo : 3-31. Food and AgricultureOrganization of The United Nations Publisher.

BAZZAS FA (1973) Seed germination in relation to salttolerance in three population of Prosopis farcta.Oecologia (Berlin) 13:73-80.

BRINER C (1985) Phenotypic characterization of hetamarugo biotypes at the Tamarugal Pampa. In: MHabit (ed) The Current State of Knowledge of Prosopistamarugo: 221-225. Food and Agriculture Organi-zation of The United Nations Publisher.

DAFNI A M NEGBI (1978) Variability in Prosopisfarcta in seed germination as affected by temperatureand salinity. Israel Journal of Botany 27: 147-159.

FELKER P, P CLARK, AE LAAG PF PRATT (1981)Salinity tolerance of tree legumes: mesquite Prosopisglandulosa var. Torreyana, P. velutina and P.articulata algarrobo (P. chilensis , Kiawe P pallidaland tamarugo P. tamarugo grown in sand culture onnitrogen-free media. Plant and Soil 61: 311-317.


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SALINITY EFFECTS ON PROSOPIS CHILENSIS 91FELKER P, GH CANNELL, PR CLARK, JF OSBORNP NASH (1983) Biomass production of Prosopis

species (mesquite), Leucaena and other leguminoustrees grown under heat drought stress. Forestry Science29: 592-606.FLOWERS TJ, A GARCIA, M KOY AMA AR YEO

(1997) Breeding for salt tolerance in crop plants. Therole of molecular biology. Acta PhysiologiaePlantarum 19: 427-433.

GUCCI R, G ARONNE, L LOMBARDINI M TATTINI(1997) Salinity tolerance in Phillyrea species. NewPhytologist 135: 227-234.

LEONCINI RM J ZAMORA ( 1969) Pampa delTamarugal (Recopilaci6n Bibliografica). Tesis: 68.Pontificia Universidad Cat6lica de Chile.

MEDINA C L CARDEMIL (1993) Prosopis chilensis isplant highly tolerant to heat shock. Plant CellEnvironment 16: 305-310.

RODHES D P FELKER ( 1988) Mass screening Prosopis(Mesquite) seedlings for growth at sea water salinityconcentrations. Forest Ecology and Management 24:169-176.

RODRIGUEZ JG L CARDEMIL (1994) Cell wallproteins in seedlings cotyledons of Prosopis chilensis.Phytochemistry 35: 281-286.

RODRIGUEZ JG L CARDEMIL (1995) Tissue specificexpression of cell wall proteins of seedlings ofProsopis chilensis during development and woundstress. Physiologia Plantarum 93: 457-463.

ROIG FA, JB CAVAGNARO SO TRIONE (1993) Con-servaci6n y mejoramiento de as especies del generoProsopis. En: Contribuciones Mendocinas a la Quin-ta Reunion Regional para America Latina y el Caribede la Red de Forestaci6n del CIID: 10-15. EditorialIADIZA-CRICIT -CIID.

SOMERS GF (1979) Natural halophytes as a potentialresource for new salt-tolerant crops. In: A. Hollaender(ed) The Biosaline Concept: 101-115. Plenum Press,New York.

TORRES H ( 1985) Planning the conservation anddevelopment of the Tamarugal Pampa. In: Habit M(ed) The Current State of knowledge of Prosopistamarugo :65-70. Food and Agriculture Organizationof the United Nations Publisher.

WINICOV I OR BASTOLA (1997) Salt tolerance incrop plants: New approaches trough tissue cultureand gene regulation. Acta Physiologiae Plantarum19: 435-449.YEN HE, D ZHANG, J LIN, G E EDW ARDS MSB KU(1997) Salt-induced changes in protein compositionin light-grown callus of Mesembryanthemumcrystallinum. Physiologia Plantarum 101: 526-532.

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