Short communication

Factors affecting in vitro development

of embryonic axes of cashewnut

Sudripta Das, Timir B. Jha, Sumita Jha*

Centre of Advanced Study, Department of Botany, University of Calcutta,

35, Ballygunge Circular Road, Calcutta 700019, India

Accepted 17 March 1999

Abstract

Cashewnut embryos as small as 1±2 mm were successfully cultured to obtain complete plantlets.

The best results for both radicle emergence (99.7%) and seedling development (95.5%) were

obtained in embryos 11±14 mm in size, cultured in MS basal media with BA (2 mg/l) and GA3

(2 mg/l). Plants obtained were successfully transferred to the field with a 90% survival rate. The

system may serve as an alternative efficient propagation methodology in cashewnut. # 1999

Elsevier Science B.V. All rights reserved.

Keywords: Anacardium occidentale; Cashew; Embryo culture; Embryo rescue

1. Introduction

Cashewnut (Anacardium occidentale L., Anacardiaceae) is an importantplantation crop of great commercial value. Though there has been considerableimprovement in the crop through conventional breeding techniques, progress hasbeen slow because the tree is heterozygous and takes 10±12 years to reach fullmaturity. Efforts have been made to select high yielding types and propagatethem by asexual methods (Samson, 1986) but the rate of multiplication has notbeen adequate to meet the growing demand. There have been no reports on in

Scientia Horticulturae 82 (1999) 135±144

Abbreviations: BA, 6-benzylaminopurine; GA3, gibberellic acid; Kn, kinetin; PVP, polyvinylpyr-

rolidone

* Corresponding author. Tel.: +91-33-475-3681; fax: +91-33-474-6619

E-mail address: s.jha@cubmb.ernet.in (S. Jha)

0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 4 6 - 1

vitro response from tissues of mature trees of members of Anacardiaceae due tothe recalcitrant nature of the trees. In cashewnut, very limited success has beenachieved even from seedling explants. Phillip (1984) reported direct organogen-esis in cotyledon sections in cashew. Jha (1988) reported the induction ofmorphogenesis from callus derived from zygotic embryos. Multiple shoot budinduction from cotyledonary nodes of cashewnut has been reported by D'silvaand D'souza (1992) and Das et al. (1996).

One of the problems of cashew breeding is embryo abortion. Low percentagesof fruit set (3±4%) have been reported in cashewnuts from South India (Nambiarand Thankamma Pillai, 1980). Embryo abortion is caused by incomplete embryodevelopment, mutations in embryo covering structures or a recalcitrant type ofdormancy for which no method of release has been developed (Nambiar andThankamma Pillai, 1980). Culturing excised embryos has long been known tobypass dormancy in species in which seed generation frequency is low ornegligible (Hu and Wang, 1986). Excised immature zygotic embryos grown onnutrient media often bypass the stages of dormancy, the embryos develop intoweak seedlings, displaying only those structures that are already present at thetime of embryo excision. Such precocious germination is of regular occurrence incashewnut. The objective of the present work is to study the factors that regulatethe development of cashewnut embryos in vitro.

2. Materials and methods

2.1. Plant materials

Immature nuts of A. occidentale var. KV-26 were collected during March±Mayfrom 5±10-year-old grafted trees grown at Arabari Forest Range, Midnapore,West Bengal, India. The size of the nuts and embryos were measured before andafter cultivation on different nutrient media.

2.2. Sterilization

The nuts, from which the embryos were excised, were dipped in 70% ethanolfor 10 min, flamed and surface sterilized with 0.1% mercuric chloride for 20 min.Nuts were then washed with sterile distilled water five times, cut open asepticallyand the embryos dissected out using forceps. Intact ovules were sterilized thesame way as the nuts and were cultured in tubes.

2.3. Cultural conditions and media

Excised embryos and intact ovules were incubated in darkness or under 16 hphotoperiod at 25 � 18C day and night temperature. Illumination was provided

136 S. Das et al. / Scientia Horticulturae 82 (1999) 135±144

by cool-white fluorescent lamps at ca. 25 mE mÿ2 sÿ1. Embryos were grown ondifferent basal media solidified with agar (0.7%) and supplemented with activatedcharcoal (0.1±1.0%), sucrose (1.0±6.0%) and polyvinylpyrrolidone (PVP, 0.5%).Several basal media were tried including B5 (Gamborg et al., 1968), MS(Murashige and Skoog, 1962), SH (Schenk and Hildebrandt, 1972), LV (Litvay etal., 1985) and MS-1 (MS inorganics and B5 vitamins). Various concentrationsand combinations of growth regulators such as BA (1.0±10.0 mg/l), Kn (1.0±10.0 mg/l), GA3 (1.0±5.0 mg/l), L-proline (50±100 mg/l) and L-glutamine (100±500 mg/l) were investigated for germination and growth of zygotic embryos. Allmedia were adjusted to pH 5.6 prior to addition of charcoal and autoclaving at1218C and 103.4 Kpa for 15 min. Embryos and ovules were cultured in tubescontaining 20 ml media and transferred to new tubes every 21 days unlessotherwise stated. Each experiment had 40 embryos/ovules per treatment and wasrepeated thrice.

2.4. Developmental stages of nuts and embryos

Nuts and embryos were classified into five developmental stages according tolength. A1, nut size � 6±8 mm, embryo size � 1±2 mm; A2, nut size � 9±12 mm,embryo size � 3±4 mm; A3, nut size � 14±18 mm, embryo size � 5±6 mm; A4,nut size � 20±24 mm, embryo size � 7±10 mm; A5, nut size � 28±30 mm,embryo size � 11±14 mm.

2.5. Transfer of plants to soil

Plantlets were cultured in MS basal media for 2±3 months during which time theroot system usually reached the bottom of the tube and five to six leaves were formed.Plantlets were transferred to a peat-perlite (1 : 1) soil mix in planting containers in ahumidity chamber at 90% humidity, fertilized weekly with commercial plant food(Phostrogen) and the humidity was gradually lowered to ambient conditions over thenext two months. Following acclimatization, plants were moved to a greenhousewith a minimum and maximum achieved temperatures of 288 and 328C, respectivelyand 70±80% humidity. They were grown in the greenhouse for 2±4 months, beforethey were successfully transferred to the field.

3. Results and discussion

3.1. Effect of cultural conditions and size of cashew embryos on radicle

emergence and seedling development

Cultures of young ovules, with embryos inside, turned brown and died in thefirst two weeks of culture initiation, even when incubated in the dark and in the

S. Das et al. / Scientia Horticulturae 82 (1999) 135±144 137

presence of activated charcoal (0.3%) and PVP (0.5%). When embryos werecultured within intact seed coat in the presence of charcoal and PVP, the seedcoats turned brown in the first week of culture and the embryos were unable toburst through the seed coat and died. Cultures of excised embryos of differentsizes showed phenolic exudation within two days of culture initiation, even in thepresence of activated charcoal and PVP. Maximum exudation was noticed whenlarger embryos (A4±A5 size) were cultured, although the browning of the embryoswas more noticeable in smaller embryos (A1±A2 size).

The presence or absence of light affected the viability and germination ofzygotic embryos in cashew (Table 1). Dark incubation was necessary for the firstfour weeks until the emergence of radicle. These cultures were then transferred tolight and kept under such conditions for seedling growth (Fig. 1(c)) anddevelopment until the seedlings attained a stage to be transferred to theglasshouse for acclimatization. There was evidently a direct correlation betweenthe size of the embryos and the frequencies of radicle emergence (Table 1):35.0% for A1 embryos and 98.0% for A5 embryos. On the other hand, initialincubation under light conditions for four weeks resulted in a low frequency ofradicle emergence for all sizes of embryos sampled (0.0% for A1 to 20.0% for A5

embryos). A direct relationship also existed between the size of embryos and thefrequencies of seedling development (21.0±89.5%).

3.2. Effect of different basal media and growth regulator combinations on radicle

emergence and seedling development of zygotic embryos

The frequencies of both radicle emergence and seedling development werelower when embryos were cultured on basal media without growth regulators(BM), as compared to cultures on basal media with growth regulators (BMGR,Table 2). When growth regulator supplemented media were compared, the bestresults of radicle emergence (96.5%) and seedling development (71.25%) wereobtained with MS media. When explants were cultured on basal media (BM), thebest frequencies were noticed in LV media (20.0%) for radicle emergence,although further development of seedlings on this media was at a lower frequency(2.22%). Since the composition of inorganics is similar in MS and MS-1 media,the use of B5-vitamins in place of MS-vitamins in MS-1 may have caused thedifference in the frequencies of radicle emergence and seedling developmentrecorded (Table 2).

Optimal levels of Ba (2 mg/l), Kn (3 mg/l), GA3 (2 mg/l) and L-glutamine(100 mg/l) were combined variously, to determine the best combination of growthregulators to be used along with MS basal media. For this experiment, only A3

size embryos were cultured on MS basal with different growth regulatorcombinations, initially for four weeks under dark and subsequently six weeksunder 16 h photoperiod. The highest frequencies of radicle emergence (96.5%)

138 S. Das et al. / Scientia Horticulturae 82 (1999) 135±144

Table 1

Effect of size of zygotic embryos of cashewnut in frequencies (%) of radicle emergence (RE) and seedling development (SD)

Cultural conditions Embryo size

A1 A2 A3 A4 A5

RE SD RE SD RE SD RE SD RE SD

Dark incubation 35.0 21.0 58.0 39.7 96.5 71.2 96.8 80.2 98.0 89.5

Light incubation 0.0 0.0 2.0 1.0 5.5 1.5 15.5 2.0 20.0 3.25

ANOVA-significances

Radical emergence

(RE)

Seedling development

(SD)

Embryo size (A) **a **a

Cultural conditions (B) **a **a

A � B **a **a

Results were obtained after 10 weeks of incubation (4 weeks under dark conditions and 6 weeks under 16 h photoperiod in case of dark incubation;

10 weeks under 16 h photoperiod in case of light incubation). Media: MS basal � Ba (2.0 mg/l) � GA3 (2.0 mg/l) � activated charcoal (0.3%, w/v),

PVP (0.5%, w/v) and agar (0.7%).a Significant at p > 0.01.

S.

Da

set

al./S

cientia

Ho

rticultu

rae

82

(1999)

135±144

139

and seedling development (71.25%) were obtained when BA and GA3 werecombined (Table 3). The combinations of BA and Kn and Kn and GA3 producedsimilar frequencies of radicle emergence (89.0% and 87.5%, respectively), butthe frequencies of seedling development were lower (50.5% and 47.0%,respectively). When L-glutamine was used in combination with the growthregulators, the frequencies of radicle emergence (40.0%, 21.25% and 35.8%)were reduced. When BA and GA3 were used in combination with L-glutamine,

Fig. 1. (a±b) Excised embryos of different sizes in culture: aÐA1 embryo, 1±2 mm in size; bÐA2

embryo, 3±4 mm in size. Bar � 0.5 mm. (c) Stages of development if excised zygotic embryos

leading to plantlet formation. Bar � 1.8 cm. (d) Acclimatized zygotic embryo derived seedling

before transfer to the field. Bar � 2.5 cm.

140 S. Das et al. / Scientia Horticulturae 82 (1999) 135±144

Table 2

Effect of different basal media (BM) or basal medium with growth regulators (BMGR) on frequencies of radicle emergence and seedling development

in zygotic embryo cultures of cashewnut

Germination stages Media

B5 MS MS-1 SH LV

BM BMGR BM BMGR BM BMGR BM BMGR BM BMGR

Radicle emergence (%) 3.33 15.0 23.7 96.5 13.75 48.75 15.0 60.0 20.0 68.8

Seedling development (%) 0 8.76 3.33 71.25 3.33 23.75 5.0 38.8 2.22 55.5

ANOVA-significances

Basal media (BM) BM with growth

regulators (BMGR)

Germination stages (A) **a **a

Basal media types (B) **a **a

A � B **a **a

Media: BM, basal media � sucrose (3%) � activated charcoal (0.3%) � PVP (0.5%); BMGR, basal media � BA (2 mg/l) � GA3 (2 mg/l) � sucrose

(3%) � activated charcoal (0.3%) � PVP (0.5%); Embryo size sampled: A3. All results were obtained after four weeks incubation in dark followed by

six weeks under 16 h photoperiod.a Significant at p > 0.01.

S.

Da

set

al./S

cientia

Ho

rticultu

rae

82

(1999)

135±144

141

decrease of both radicle emergence (96.5% to 52.5%) and seedling development(71.25% to 30.25%) were recorded.

The most important aspect in culturing immature embryos is to define a culturemedia that can sustain their growth and development. In less than optimum, theimmature embryos may fail to survive, turn into undifferentiated callus orgerminate prematurely (precocious germination). To our knowledge, there has notbeen any report on embryo culture in cashewnut so far. In this species, the lowpercentages of germination (Nambiar and Thankamma Pillai, 1980) and the highabortion rate in vivo, is associated with the inability of embryos to burst throughthe seed coat, the high degrees of recalcitrant dormancy, and the precociousgermination nature of cashew embryos, which was also noticed in vitro. In thepresent study, immature cashew embryos, as small as 1 mm could be excisedfrom the surrounding tissues, cultured and grown into complete healthy plants invitro. The problem of embryo ability and culture establishment was accentuatedby browning of embryos and exudation of phenolics in culture media, which wasovercome to some extent by the use of activated charcoal along with PVP.Activated charcoal not only absorbs toxic exudates, but also growth regulatorsand nutrients (Weatherhead et al., 1978). But, in cashew, the effect of activatedcharcoal was not inhibitory to the growth of embryos. Absence of charcoal causedreduction of radicle emergence in embryos, suggesting a direct influence of

Table 3

Effect of different combinations of growth regulators on frequencies (%) of radicle emergence and

seedling development in cashew

Growth regulator combinations Radicle

emergence

(%)

Seedling

development

(%)

1. Ba (2 mg/l) � L-glutamine (100 mg/l) 40.0 18.88

2. Ba (2 mg/l) � Kn (3 mg/l) 89.0 50.5

3. Ba (2 mg/l) � GA3 (2 mg/l) 96.5 71.25

4. Kn (3 mg/l) � L-glutamine (100 mg/l) 21.25 10.0

5. Kn (3 mg/l) � GA3 (2 mg/l) 87.5 47.0

6. GA3 (2 mg/l) � L-glutamine (100 mg/l) 35.8 17.5

7. BA (2 mg/l) � GA3 (2 mg/l) � L � glutamine (100 mg/l) 52.25 30.25

Anova-significances

Germination stages (A) **a

Growth regulators (B) **a

A � B **a

Media: MS basal � sucrose (3%) � activated charcoal (0.3%) � PVP (0.5%); Embryos sampled

were of A3 size. Results were obtained after four weeks of dark incubation and six weeks under

16 h photoperiod.a Significant at p > 0.01.

142 S. Das et al. / Scientia Horticulturae 82 (1999) 135±144

charcoal in rooting of embryos, which has been previously reported in Litchichinensis (Kantharajah et al., 1992).

The presence or absence of light affected the viability and radicle emergence ofcashew embryo cultures in vitro. It is obvious when incubating embryos that acertain light regime is needed to induce normal germination. One arbitrary way todetermine light versus dark incubation is to examine the nature of the in situcovering structure surrounding the developing embryo. It should be safe to adoptdark incubation if the covering tissue is thick enough for little light penetration,which was the case in cashew. Since light may stimulate precocious germination,it is advisable to incubate immature embryos in darkness during the period ofradicle emergence, before transferring them to light for further development(Dolezel et al., 1980), as was done in cashew.

The best results of radicle emergence and seedling development were obtainedin cashew embryos using BA (2 mg/l) and GA3 (2 mg/l) in combination. In manycases reported, exogenously supplied growth regulators are not required forembryo cultures. Monnier (1978) suggested that embryos can be considered asplants with their own endogenous hormones. However, there are several cases inwhich addition of either cytokinins, auxins or gibberellins has greatly facilitatedembryo cultures, as in cashew. The induction of precocious germination by GA3

has been observed in cultured embryos of other species (Norstog, 1961; Skene,1969; Custers and Kruit, 1982), no such observations were made in cashew. Theeffect of L-glutamine in reducing the frequencies of radicle emergence andseedling development was not clear because L-glutamine is known to benefitembryo development (Raghavan, 1976).

Acknowledgements

Authors gratefully acknowledge financial assistance for UGC, Government ofIndia and Mr Tapas Roychowdhury, Range Officer, Arabari Research Range,Midnapore, India for plant materials.

References

Custers, J.B.M., Kruit, S., 1982. Embryo culture with Cucumis species. In: Plant tissue culture

1982, Proc. Vth Intl. Congress in Plant Tissue Culture, Japanese Association of Plant Tissue

Culture, Tokyo, pp. 777±778.

Das, S., Jha, T.B., Jha, S., 1996. In vitro propagation of cashewnut. Plant Cell Rep. 15, 615±619.

Dolezel, J., Novak, F.J., Luzny, J., 1980. Embryo development and in vitro culture of Allium cepa

and its interspecific hybrids. Zeitschrift Pflanzenzuecht 85, 177±184.

D'silva, I., D'souza, L., 1992. In vitro propagation of Anacardium occidentale L. Plant Cell Tiss.

Org. Cult. 29, 1±6.

S. Das et al. / Scientia Horticulturae 82 (1999) 135±144 143

Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Nutrient requirements of suspension cultures of

soybean root cells. Exp. Cell Res. 50, 151±158.

Hu, C.Y., Wang, P.J., 1986. Embryo culture: Technique and application. In: Evans, D.A., Sharp,

W.R., Ammirato, P.V. (Eds.), Handbook of Plant Cell Culture, Vol. 4. Macmillan Publishing Co.,

New York, pp. 43±96.

Jha, T.B., 1988. In vitro morphogenesis in cashewnut Anacardium occidentale L. Ind. J. Exp. Biol.

26, 505±507.

Kantharajah, A.S., McConchie, C.A., Dodd, S.A., 1992. In vitro embryo culture and induction of

multiple shoots in lychee (Litchi chinensis Sonn.). Ann. Bot. 70, 153±156.

Litvay, J.D., Verma, D.C., Johnson, M.A., 1985. Influence of loblolly pine (Pinus taeda L.) culture

medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus

carota L.). Plant Cell Rep. 4, 325±328.

Monnier, M., 1978. Culture of zygotic embryos. In: Thorpe, T.A. (Ed.), Frontiers of plant tissue

culture. University of Calgary Press, Calgary, Canada, pp. 277.

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco

tissue cultures. Physiol Plant 15, 473±497.

Nambiar, M.C., Thankamma Pillai, P.K., 1980. Cashew. In: Bose, T.K. (Ed.), Fruits of IndiaÐ

Tropical and Subtropical, Naya Prokash, Calcutta, India, pp. 409±438.

Norstog, K.J., 1961. The growth and differentiation of cultured barley embryos. Am. J. Bot. 48,

876±884.

Phillip, V.J., 1984. In vitro organogenesis and plantlet formation in cashew (Anacardium

occidentale L.) Ann. Bot. 54, 449±452.

Raghavan, V., 1986. Experimental embryogenesis in vascular plants. Academic Press, New York,

pp. 567.

Samson, J.A., 1986. Tropical fruits. Longmann Inc., Essex, UK, pp. 124.

Schenk, R.U., Hilderbandt, A.C., 1972. Medium and techniques for induction and growth of

monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50, 199±204.

Skene, K.G.M., 1969. Stimulation of germination of immature bean embryos by gibberellin acid.

Planta 87, 181±192.

Weatherhead, M.A., Burden, L., Henshaw, G.G., 1978. Some effects of activated charcoal as an

additive to plant tissue culture media. Z. Planzenphysiol. 89, 141±147.

144 S. Das et al. / Scientia Horticulturae 82 (1999) 135±144