Annals of Arid Zone 30 (3) : 247-258, 199]

PROPAGA TION OF PROSOPIS SPECIES: PROBLEMPERSEVERANCE AND PERSPECTIVES

K. G. RAMAWAT AND DILIP NANDWANI

Department of Botany, University of Jodhpur, Jodhpur-342 001

ABSTRACT

Prosopis species are predominant components of arid-land vegetationbut they are degenerated as biotic pressure has caused large scaledeforesta tion. Plant tissue culture has been proved successful inregeneration of a large number of tree species. However, I'rosopisspecies are recalcitrant to regenerate and efforts made hy variousworkers to regenerate and propagate P;osopis are discussed in thelight of achievements made in the laboratory.

INTRODUCTION

The Indian desert (22°-32°N and 68°-76°E) covering an area of 28600 km2

represents several specific characteristics. The climate and soil conditions arecharacterised by high temperature, low and erratic rainfall, evaporation, far exceedingprecipitation, low organic matter in the soil and strong desiccating winds. It is alsothe most densely populated (61 persons/km2) of the desert areas of the world(Khoshoo and Subrahmanyam 1989). Due to obvious pressure on land, marginallands are cultivated and available vegetation cover is being over exploited leadingto desertification (Paroda 1979). It is in this context that there is an urgent needfor development of biotechnology for rapid multiplication of trees for afforestation.

Prosopis cineraria is now available only in protected land areas while P. ju/ifiorathrives well in all soils including saline areas of Luni river and Sambhar lake. Theother important plants are Acacia ni/otica, Tecome/la undulata, Ziziphus mal/ritiana,Sa/vadora persica. S. oleoides, Balanites aegyptiaca. All these species can withstandthe long dry and hot periods. In case of monsoon failure they survive without adrop of water for years. It may be due to the fact (Breazeale et al. 1950) thatcertain plants during high moisture regime (e. g. coul night) absorb atmosphericmoisture. In case of P. tamarugo (recently introduced in India) the unusualepicuticular configuration could be an important system which enhances the foliarabsorption of atmospheric moisture (Hull and Bleckmann 1977). It has been pointedout that large amount of mucilage contained in its leaves helps the process of waterabsorption from atmosphere. Recently (Serrato-Valenti et al. 1989) it has beenshown that the stomata together with the anticlinal walls of the epidermal cells arevery important sites of water absorption. The histo-chemical tests of leaf sectionrevealed the presence of poly ions at these sites (Serrato-Valenti et al. 1989). These

248: RAMAWAT& NANDWANI

polyions are themselves able to link water in amounts directly related to the intensityof the available negative charges (Modensi and Vanzo 1980).

Origin and importance of Prosopis species: All Prosopis species are xerophyticand occur in arid regions of the world. Burkart (1976) described the 44 species ofProsopfs distributed in tropics and subtropics of both hemispheres. The three speciesin the section Prosopis are natives of Asia and North Africa. Argentina is consideredto be the largest centre of diversity for Prosopis. The details of botanical description,distribution and biology has been described by Burkart (1976) and Leakey andLast (!980).

Uses: According to Burkart (1976) at least 14 Prosopis species are potentiallyimportant for afforestation and fodder productions, whereas a few are consideredas weed. P. cineraria is a multipurpose tree of western Rajasthan. It will not beexaggerated ifit is termed as back-bone of rural economy. Prosopis juliftora is mainlyused as fuel wood and living fence.

Conventional practices and improvement

Prosopis species are propagated through seeds. Being leguminous seeds, theypossess hard seed coat and in some cases hard endocarp of fruit wall. Therefore,seeds require pretreatment to enhance success in germination up to 70-80 percentMost species produce abundant seeds (e. g. 1 kg/plant in P. cineraria).

Vegetative propagation is possible by suckers in P. cineraria and by rooting ofstem cuttings in P. juliftora (Leakey and Last 1980) and P. alba (Felker and Clark1981). However, the percentage of plantlets obtained through these methods was notvery high but they have demonstrated the possibilities of application of the method.Considerable improvement in percentage rooting of P. alba stem cutting was obtainedby fertilizer application to the stock plants (De-Souza and Felker 1986). Air layeringhas been used successfully to obtain large number of plants of P. cineraria (Solankiet al. 1984). Therefore it is desirable to evolve further these methods before utility atlarge scale can be established. Simultaneously more species should be tested for theseconditions.

Most species of Prosopis are diploid (2n - 28) except P. juliftora in whichtetraploid plants are also known. No serious efforts have been made for hybridisationof P. cineraria with other species of Prosopis. Little information is available abouthybridisation work in Prosopis species pertaining to section Algarrobia (See Leakeyand Last 1980).

Prosopis species exhibit a wide range of characters due to heterozygosity.Therefore it is esscntiaU to select the plants for their desirable characters viz. leafprotein, absence of protease inhibitors, thorniness, leaves yield, timber quality and

PROPAGATION OF PROSOP1S SPECIES: 249

quantity etc. These species show moderate to high salinity resistence. These plantsare infested with a variety of insects. After careful selection improvement programmecan be implemented by conventional breeding methods or through plant tissue, cellIlnd protoplast culture methodology.

In vitro approllches : Plant tissue culture provides all excellent system to stud y thefactors involved in vegetative multiplication and the possibilities of obtaining improvedplants by genetic engineering. Vegetative multiplication through tissue culture hasemerged as major applied aspect of plant tissue culture research (Murashige 1974;Bajaj 1986). A large number of research groups are.working to develop technologyfor their regeneration through plant tissue culture as an alternative mean to provide.large scale plants for afforestation' of arid lands. The results obtained with Prosopisspecies are summarised in Table 1 and discussed in the light of results obtainedby other workers on Prosopis species.

Table 1. Tissue culture and regeneration studies in various species of Prosopis.

Shoot develop- Batchelor etment al. 1989

MS medium + 1AA(0.25 mg/I) Kinetin(4.5 mg/l)MS medium 1AA

(3.0 mg/I) Kinetin(0.05 mg/!)

3-5 shootsformation bygrowth regul-ator1-3 shoots, Goyal & Arya

medium factors 1984aShoot formation CAZR1 (Per.

Comm.)Shoot formation Nandwani &

Ramawat 1989

S. No.

1.

2.

Plant species

2

Prosopis cineraria

Prosop is tamarl/go

Explant

3

Hypocotyl

stem

Bud explant

Stem, Root

Stem

Stem

Stem

HypocotylNodal section

Shoot tip

Hypocotyl

Medium

4

-do-

MS medium

MS medium +Kin. (1.5 mg/I) +BAP (1.5 mg/I)MS medium +BAP (10.0 mg/I)NAA (5.0 mg/I)MS medium +BAP (5.0 mg/I)MS medium +MS medium

MS medium

MS medium +BAP (5.0 mg/I)

Result

5

Shoot Bud

Shoot regenera-tionPlantlets forma-tion Shoot

regenerationShoot-like struc-turePlantlets forma-tion

Reference

6

Goyal & Arya1981

Goyal & Arya1984

Nandwani 1990

Nandwani, 1990Jordan

et al. 1985Jordan, et al.1987Nandwani &Ramawat 1989,Nandwani 1990

----------------- ------------

250: RAMAWAT& NANDWANI

2 3 4 5 6

Stem MS medium + Shoot buds for- Nandwani 1990NAA (0.2 mg/I)+mationBAP (5.0 mg/I)

3. P. alba Shoot tip MS medium Contamination, Anno. 1987somatic embryo

La teral bud MS medium + Shoot multiplica- Tabone et al.explant BAP (4.4 x 10-5 tion 1986

M) tIAA (2.9 x10-5M)

4. P. chilensis Node MS medium Callus Jordan et a1.1985

Nodal segme- B5 medi um + Embryoid for- Jordan et al.nt, callus 2,4-D mation 1987

(2.0 mg/I) +cysteine (60 mg/I)

Shoot tip MS medium + Multiple shoots Jordan 1988NAA (0.3 mg/I), formationBAP (0.1 mg/I),GAa (0.01 mg/I)

stem MS medium + Shoot prolifera- Batchelor et a!.BA (15.0 mg/?) , tion 1989NAA (5.0mgfl)

5. P. juliflora Stem MS medium Not known TERI, Delhi (per.comm.)

Meristem/ Shoots Annon. 1987shoot tipNodal s( g-men!Nodal MS medium, Shoots develop- Wainright and Eng-cutting kinetin BAP ment land 1987

(0.25 uM)Stem MS medium Multiple shoots Nandwani & Rama-

IAA (0.1 mg/I) wat 1989BAP (5.0 mg/I)

Stem MS medium Shoot develop- Batchelor et al.ment 1989

Stem MS medium + Plant:ets forma- Nandwani & Rama-IAA (0.1 mg/I), tion wat, 1991BAP (5.0 mg/!)

6. P. tamarugo Meristem, Microbial conta- Annon. 1987node, callus mination, Embryo

like structure

7. Prosopis sp Meristem/ Delgado (Per.shoot cotyle- Annon., comm.)don 1987

PROPAGATION OF PROSOPIS SPECIES: 251

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Callus cullure: Callus induction was observed in all the three species (P. cineraria(PC), P. juliflora (PJ) and P. tamarugo (PT) on several treatments of medium compon-ents including growth regulators (Table 2) incorporated in the MS medium usingvarious explants Nandwani and Ramawat 1989).

Except P. juliflora, it was possible to maintain callus culture from P. cinerariaand P. tamarugo (Nandwani and Ramawat 1989) and callus and cell suspension cult-ures from p. chilensis and P. tamarugo (Jordan et al. 1987; Jordan 1988). However,all attempts to regenerate the isolated and subcultured callus failed. It may beinteresting to note that Jordan et al. (I987) observed a single embryo/shoot apex likestructure in the cultures grown on B5 medium supplemented with 2, 4-D.

Bud Break: Bud break from nodal explants in different species of Prosopis can beobtained on diverse media (Goyal and Arya 1984; Jordan et a!. 1987; Wainright andEngland 1987; Yao et al. 1989; Nandwani 1990). MS medium containing high cytok-inin (Nandwani 1990) or high auxin (Goyal and Arya 1984) or high concentration of acytokinin and an auxin (Batchelo et al. 1989) support shoot regeneration from stemsexplants of P. cineraria. These findings suggest that response is not precisely contro-lled by concentration and quality of exogenous growth regulators. Perhaps, therequirement is quite different than that provided exogenously. But subsequent changesin metabolic level and endogenous level of growth regulators pr0mote shoot forma-tion. This conclusion is supported by the fact that in most cases only single shootformation has been achieved. The complete stimulation of explant was lacking, there-by production of adventitious shoots remained suppressed (Table 3).

In P. cineraria, we have produced evidence that explants obtained from differentgenotypes as well as explants of same genotype differ in their regenerative potentialon same medium. These explants showed considerable variatioE in phenotypic expre-ssion in relation to delayed regeneration, size, length and vigour of regenerated shootsand leaf expansion (Nandwani 1990). Thus, results obtained by various workersare mostly due to different material (genotype) used, making the reproducibility ofresults very diffIcult. Therefore these studies require a detailed account of explantsource used in the investigation. Some of these speeies are very recalcitrant to regene-rate. In case of p.jllliflora several hundred permutation and combinations of mediumfactors could not produce subculturable callus and shoot number could not be enhan-ced more than seven. Similar results with low shoot number were also obtained withp. chilensis, P. cineraria and P. jllliflora nodal explants by Yao et a1. (1989).

Adventitious shoot formation

Adventitious shoot formation: In P. tamarugo various growth hormonesincorporated in MS medium induce single shoot from hypocotyl explants. In one case

PROPAGATION OF PROSOPIS SPECIES: 253

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it was observed that when a few hypocotyls grown on high cytokinin (HC) contain-ing medium are transferred to MS medium devoid of growth hormone, it producedmultiple shoot buds at the base of developing single shoot. Therefore, a sequence ofexperiments were designed to evaluate the precise role of growth hormones, particula-rly the cytokinin. This has resulted in formation of multiple shoot buds from hypoco-tyles in successive passage on hormone free (HF) and cytokinin supplemented media.Embryonic explants grown on HF or HC medium for a passage and then transferredvice versa, produced multiple shoots. It was observed that number and proliferationin shoot buds increased when tissues grown for second passage on HC medium.Reduction in cytokinin concentration in the medium resulted in elongation of a fewshoots from the clump of organogenetic mass. Elongated shoots produced roots onrooting medium containing an auxin.

It has also been observed that if both cotyledons are allowed to remain attachedwith the embryonic axis, the presence of cotyledons enhanced the multiple shootformation in the explants. In absence of attached cotyledons not only the number ofshoot buds per explant remained low, but the process was also delayed with the

Pt.

ptantlet

Pc.

Plantlet

Fig. I. Differential morphogenetic response of various explants from embryonic axis ofProsopis cineraria (Pc) and P. tamarugo (PI) (AP : with (+) or without (-) apicalmeristem' H: hypocoty,1 R : root, C : cotyledon, S:stem).

PROPAGATION OF PROSOPIS SPECIES ::255

growth of explant, the number of shoot buds increased by formation of new adven-titious buds. It appears that these buds at a later stage develop adventitiously andfrom newly proliferating callus cells, while earlier were produced from axillary buds.In certain cases, apical meristems in axillary position proliferate to produce a phylodylike structure.

As far as our information goes, adventitious shoot formation from mature orjuvenile explants bf Prosopis species has not been reported. Or; the basis of resultsobtained with p. tamarl/go successful regeneration of adventitious shoots from juve-nile explants of P. cineraria has also been obtained. However, the number of shootsper explant remained low in P. cineraria as compared to P. tamarugo. Goyal andArya (J 981) obtained single shoot from juvenile explant, whose exact nature' wasuncertain.

Protoplast culture

The single report (Shekhawat and Kackar 1987) described the method forisolation of protoplast from leaves of P. cineraria.

RootingIt has been observed that rooting can be induced in P. taml/rl/go and P.juliflora

with relative ease as compared to P. cineraria (NandwaniI990; Goyal and Arya 1984).These system require more refinem~nt and increased frequency and subsequentviability in field conditions. However, in case of P. cineraria the age of explant,culture medium, genotype and age of shoots markedly affect the rooting behaviour ofin vitro regenerated shoots. Therefore, to develop a reproducible rooting system, allfactors require consideration. Once rooted, establishment of these shoots does notpose difficulties. Howeve, low watering is required.

Evidence for callus morphogenesis

It is mentioned earlier that callus once isolated from explants and maintainedseparately than it never regenerated. But there were cases when highly regenerativetissues were obtained from juvenile explants of P. famarl/go. It was suspected thatsuch a high regeneration cannot be obtained without callus morphogenesis.Histological observations of regenerative mass obtained in successive passages fromjuvenile explants grown on high cytokinin containing medium showed shoot budsdevelopment from callus produced by proliferating tissues. This is first evidence ofcallus regeneration in any of the Prosopis .species (.Table 4). Increased nitrogencontent enh'lI1ced shoot bud proliferation while added inhibitors of auxin· synthesisand auxin transport enhanced callus formation in the regenerative tissues(Nandwani 1990).

Conclusion and ProspectsThe Prosopis species are most valued trees of arid .regions. In case of tree

species, which are primarily required for biomass production, regeneration throughcallus culture is an ideal system to produce large number of plants though they may

256: RAMAWAT & NANDWANI

Table 4 : Effect of various factors on regeneration of multiple shoot buds ofP. tamarugo.

S. No. MS Medium +BAP (5.0 mg/I)Medium factors(mg/I)

Shoot buds Growth ofproduced shoot buds

Remarks

Healthy growth ofshoot buds, elongationin 2 shootsVigour increased.Reduced growth ofproliferating mass

Relative value of shoot buds formation:- -Nil; + low, ++ Moderate, + ++ Higll

1.

i.ii.

lll.

iv.v.

2.i.

ii.

3.i.ii.

iii.4.

i.

ii.iii.

NitrogenKN03 (NH4h S04500 4000300 1000

4000 500NH4 NOs

1900 16503800

Adenine sulphate25.0

100.0

Gibberellic acid0.11.05.0Calcium chloride220

440880

ex:

0<:

oc:

oc

oc

++++

++

+++

+

++

++++

+

+

+++

Fleshy thick leaves,Green, proliferatingshoot buds

Poor growth, No callusHealthy shoot buds,Shoots 2.0 em elonga-ted

Shoot buds and growthof inoculumGreen, fleshy shoots.Elongation in 3 shoots.

differ slightly from each other. Clonal propagation leading to monoclonal populationis not required in such cases.

The present state of knowledge about regeneration in Prosopis suggested thatstilI there is wide gap betw~en laboratory feasibility of regeneration and practicalfield level application of technology. For afforestation programmes hundred thousand

PROPAGATION OF PROSOPIS SPECIES: 257

plants are required regularly. Considering mortality at all steps and frequency ofregeneration this cannot be achieved with nodal explants. In case of P. tamarugocallus regeneration have been achieved but frequency of shoot elongation is still low.Therefore, still the conventional methods of propagation (seeds) have to be used tillthese barriers are removed.

After obtaining a highly regenerative system in P. tamarugo the work onfollowing lines will be useful for large scale multiplication of this and related Prosopisspecies.

1. Precise control of elongation of shoot buds.2. Details of factors affecting transplantation.3. Isolation, culture and fusion of protoplasts of P. tamarugo with other species

of Prosopis. Since one system is regenerative, chances of obtaining regenera-tive hybrids are good.

4. Development of regenerative system in other species of Prosopis on thesame lines.

REFERENCES

Bajaj, Y. P. S. 1986. Bioteehnology of tree improvement for rapid propagationand biomass energy production. In: Biotechnology in Agriculture and ForestyTrees: Vol. 1. Y. P. S. Baj3j (ed.) Springer-Verlag, Heidelberg, Berlin pp. 1-23.

Batchelor, C. A., Yao, D., Koechler, M. J. and Harris, P. J. C. 1989. In vitropropagation of Prosopis species (P. chi lensis, P. cineraria and p. julitfora). Annalsdes Sciences Forests 46 : 110-112.

Breazeale, E. L., McGeorg, W. T. and Breazeale, J. F. 1950. Moisture absorptionby plants from an atmosphere of high humidity. Plant Physiology 25: 413-419.

Burkart, A. 1976. A monograph of the genus Prosopis (Leguminosae sub. fam.Mimosoideae). Journal of Arnold Arboretum, Harvard University 57 : 219-249.and 450-525.

De Souza, S. M. and Felker, P. 1986. The influence of stock plant fertilization ontissue concentrations of N, P and carbohydrates and the rooting of Prosopis albacuttings. Forest Ecology and Management 16: 181-190.

Felker, P. and Clark, P. R. 1981. Rooting of mesquite (Prosopis) cuttings. Journalof Range Management 34 : 446-448.

Goyal, Y. and Arya, H. C. 1981. Differentiation in cultures of Prosopis cinerariaLinn. Current Science 50 : 468-469.

Goyal, Y. and Arya, H. C. 1984. Tissue culture of desert trees: I. Clonal multipli-cation of Prosopis cineraria by bud culture. Journal of Plant Physiology 115 :183-189.

Hull, H. M. and Bleckmann, C. A. 1977. An unusual epicuticular wax ultrastructureon leaves of P.osopis tamarugo (Leguminosae). American Journal of Botany 64 :1083-1091.

258: RAMAWAT& NANDWANI

Jordan, M., Cortes, I. and Goreux, A. 1987. Potentialities of cell and callus tissueculture to regenerate two mesquite species (Prosopis tamarugo and p. chilensis).Gartenbauwi ssenschaft 52 (4): 166-169.

Jordan, M. 1988. In vitro culture of Prosopis species. In : Cell and tissue culture inforestry. Vol. 3. Bonga, J. M. and Durzan, D. J. (eds.) Martinus Nijhoff Pub.Boston. pp. 370-384.

Khoshoo, T. N. and Subrahmanyam, G. V. 1989. Ecodevek)pment of arid lands inIndia with non-agricultural economic plants - a holistic approach. In: Plantsfor arid lands. Wickens, G. E. Goodin, J. R. and Field, D. V. (eds.) UnwinHyman. Boston, pp. 243-265.

Leakey, R. R. B. and Last, F. T. 1980. Biology and potential of Prosopis speciesin arid environments, with particular reference to p. cineraria. Journal of AridEnvironment 3 : 9-24.

Modensi, P. and Vanzo, C. 1980. The cortical surfaces in Parmelia saxatilis andp. caperata, a histochemical approach. Lichenologist 18 : 320-338.

Murashige, T. 1974. Plant propagation through tissue culture. Annual Review ofPlant Physiology 25 : 135-166.

Nandwani, D. and Ramawat, K.G. 1989. In vitro growth of three species of Prosopis.in Proc. Tissue Culture Conference VII. Tandon, P. (ed.) NEHU, shillong.

Nandwani, D. 1990. Tissue culture of forest plants of Rajasthan 1. Prosopis sppPh. D. thesis, University of Jodhpur, Jodhpur, pp. 101.

Nandwani, D. and Ramawat, K.G. 1991. Callus cultures and plantlets formation inProsopis juliftora (swartz.) D.C. Indian Journal of Experimental Biology 29(6).253-257.

Paroda. R. S. 1979. Plant resource of Indian arid zones for industrial uses. In : Aridland plant resources. Goodin J. R. and Northington, K. D. (eds.) Inter .

. C.A.S.L.A.S., Texas pp. 261-281.Serrato-Valenti, G., Melone, L., Modenosi, P. and Bozzini, A. 1989. Leaflet structure

of Prosopis tamarugo Phil. (Leguminosae) and its water uptake. Phytomorphology29: 181-188.

Shekhawat, N.S. and Kackar; A. 1987. Protoplasts of Prosopis cineraria: Isolationand culture. Nitrogen fixing tree. Research Reports, 5 : 51-53.Solanki, K. R., Kackar, N.L. and Jindal, S.K. 1984. Propagation in Prosopis

cineraria (L.) Mac Bride by air layering. Current Science 53 : 1166-1187.Wainright, H. and England, N. 1987. The micropropagation of Prosopis juliftora

(Swartz) D.C. : Establishment in vitro. Acta Horticulturae. 212 : 49-53.Yao, D., Batchelor, C.A., Koechler, M.J. and Harris, P.J.C. 1989. In I'itro regenera-

tion of Prosopis species (P. chilensis P. cineraria and P. juliftora) from nodalexplants). Chinese JOurnal of Botany 1 : 89-97.