Proe. Indian Acad. Sci. (Plaat Sci.), Vol. 91, No. 1, Fobruary 1982, pp. 37-41. ~~) Printr in .

Regeneration of plantlets from callus of cardamomum Maton

N K SKINIVASA RAO, S NARAYANASWAMu E K CHACKO and R. DORE SWAMY Division of Plaut Physiology aad Bioeh Iudiat~ Instituto of Horticultuml P-~~oareh (iCAR.), Bartgalore 560080, India *Emeritu~ Seienti~t, Couneil of Seieatific and Industrial Roaearch, Ncw Ddhi

MS roceivcd 3 November 1980 ;rovised 10~tobor 1981

Abstract. Embryo caltus aud caUus of rootstoekg of in vitro-raigcd seedling~ of Elettaria eardamomum wero growa oa MS mcdium ~uppleme~ted with CW + 2, 4-13 + BAP. Difforentiationof Skoot buds, roots and le,aves lr to tho dovelop- ment of plantlots could be induced in callus by withdrawing 2, 4-I) of substituting it by IAA or NAA in low cortcer~tratiorts.

Keywords. CaUus cutturo ; eardamom ; rogeneration.

1. Introduction

Reports on induction of shoot buds and whole from tissue cl:Itures of both monocotyledonous and dicotylecionovs plants have been numerous in recent years as evident from the spate of publications op the subject (see rexir by Murashige 1974 ; Narayanaswamy 1977). Clonal proFagation through tisst, e cuIture has been successful witb many spice and condiment plants such as Foeniadum vulgare (Maheshwari and Gupta 1965), Anetht.m graveolens (RatnambZ and Chorra 1974), Carum corvi (Ammirato 1974) and Capsicum annuum (Gunay and Rao 1978). Speetacular rate of mu[tiplication of tl~rmeric (Curr longa) plants llave been reported in cu[tures of young veg~tative buds isolated from the root stock (Nada- guada et al 1978). This prompted us to investigate the potenfial for organogenesis in tissue cultures of the eardamom (Elettaria cardamomzim Maton of ) widely used as a condiment. This paper reports the successful regeneration of shootbuds and plantlets from seedling calIus of the herbaceous perennial ,

2. Methodology and results

2.1. Seed get'mination

Dry seeds ofcardamom were surface sterilised by 0" lPA mercurio chloride solution to which a few drops of the detr Teepol had been added. After ~ashing

Contribution nurabor 944 of Indian Instituto of ttorticulturai Rose~rch. 37 38 N K $Irinivasa Rao et al

thoroughly in sterilized water, the seeds were sown on White's (1963) nutrient agar. $1ender seedlings were obtained in three weeks on incubation at 26 ~ C. Whole seedlings bearing the tirst sheathing lr and plumule were transfr to Murashige and Skoog's (19627 medium (M$) to which auxins, cytokinins and coconut water (CW) as specitied (table 1) and sucrose (2%,) had been addr l~e IKDTA was used as the iron source. Each treatment eomprised 12 replicates. l~mbryo caUtts was also obtained direetly from seeds sown on the medium (figure 1) eontaining ah auxin such as 2, 4-D (2,4-diehlorophyenoxy aeetie atid).

2"2. Callus induction Five-woek-old 3 cm long seedlings bearing the first sheathing leaf and plumtde were transferred to MS medium to whieh CW (18% v/v)+ 2, 4-,D(2mg/l)or indole-3-butyrie acid (IBA 2mg/l) of naphthalr acetie acid (NA& 2mg/l) and benzylaminopurine (BAP 2mg/l) had been added in eombinations as needed. ProIiferation of ceUs from the rootstock was observed in 75% of the cvAtures resu l- ting in the formation of exttberant callus in 6 weeks aftr incubation (figme 2). MS supplemented with CW (18~ v/v) + 2, 4-]3 (2mg/l) + BAP (0.5mg/I) was conducive for 6allus initiation and growth which could be augmentedby the addi- tiorr of casein hydroIysate (CH, lgm/l) in the medium. Neither yeast extract 250mg/I nor mar extraer (250mg/l) proved favourable for callus growth~ 2, 4-I) could, however, be repIaced by IBA for callusing. Propionocarmine squashes of the proliferating caUus showr cells of ddverse sizes and shapes (figure 3). Traeheidat differentiation of cells was marked.

2-3. Regeneration of sltoot buds Calhts grov, ing on MS + CW + 2, 4-D was subcultured on medium devoid of 2, 4-D but containing 1AA (2mg/l) or NAA (lmg/l). Three weeks after transfer green nodular strue tutes developed in the eallus indicatin g the initiation of organised grov, th centres. Callus grown on medium with higher eoneentrations of the auxin beea~me friable and was not conduciv*, for shoot bud induetion. But induetion

Table 1. R~~ponscof ealli to growth regulators in vitro on sequentia transfer.

SI. Media composition Naturr of No. (Hormone eonc~trations in mffl) response

1. MS + CW(18 v/v} +2,4-D(2) CaUusing good 2. MS + CW (18 ~ v/v) + 2, 4-D (2) + CH (1 g/! ) + BAP Cailusing exuberartt (o-5) 3. MS + CW(18~/o v/v) + IAAorlBA(I) q-NAA.(2) Callust grcw as vascular nodules 4. MS +CW(18 V/v) +IAA(i) -~BAP(2) Shoot bud initiation in eallus (80%) 5. MS +CW(10~,{o v]v)+-BAP(2-5) +IAA (I) 4-6 shoot buds per subculture Regenerathgn in 39

Figures 1~5. Callas irtdaotio~ arLd ghoot bad regeaeration m card~mom. 1. Callasirtg of soodlirtg~ in vitro ort MS + CW (18 %v/v) + 2,4-D (2 mg/l) d-BAP (0"5 mg/l) 6 wookg aftor incubatio~. • 1 "5. 2. Nodular callu~ dorived from root stock of scr transfr to MS mcdium + IAA (1 mg/l)+NAA (2mg/l) x 1"5. 3. Propioaocarmiae gquash preparatiort from root-stock cal[tts pieco showirtg freo c,~,llg aad coll aggreg~te~ • 800. 4. Roge.neration of a sttoot bud la primaxy ca[[ttg growa ort bis + BAP (2 mg/l) -I- 1AA (mg/l), x 1.5. 5. Claster of ghoot bttds regertorated from callas s,xbealtures ort MS + CW (10 ~v/v) q-- BAP (2mg/l) +IAA (l mg/I) x 1"5. Regeneration in E,lettaria cardamomum 41

occurred if MS medium was supplemented with CW (10~ v/v)+ BA1s (2-5 mg/l) with or without the addition of 1AA and incubated for 6 weeks under 500 lux (figures 4,5). Fottr to six shoot buds could be obtained fruto eaeh callus subculture of lmiform size. Rooting occurred at the base of individual shoot buds on prolonged ineubation in the same medium (aged eultures) or when indivi- dual sttootlets were isolated and grown on White's medium to whieh NAA (2mg/l) and sucrose (1~) had been added.

3. Discussion

R.egeneration of plantlets in caUus cu[ture is an alternate means of propagation in cardamom. Calltts subeulturr eoul4 develop 4-6 regenerants, each of which was capable of rooting, when isolated and grown, forming a whole . Plant- lets propagated thus might not conforto to parent genotype, having been obtainr fruto seeci[ingcaUi,/qevertheless, tissue culture provides a methott by which a large number of strains could be obtaine6 for selection of desirable variants. Also, it offers a method of rapid multiplication of elite varieties through multiple shoot induction. Preliminary studies havr shown that test tv.be plants could be success- fully transferred to soil.

Acknowledgements

The authors thank Dr G S Randhawa, the then Director, Indian Institt, te of Hortictdtural Researeh, Bangalore, for his keen interest and for providing faei- lities for tissue calture work. Tbanks are also due to Dr D Gm~du Rao, Senior Plant Pathologist, for the supply of cardamom seeds.

Referencos

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