Indian Journal of Experimental Biology Vol. 49, September 2011, pp. 711-716

In vitro propagation and mass scale multiplication of a epiphytic orchid, calceolaris (Buch.-Ham ex J.E.Sm.) D.Don. using immature seeds

Promila Pathak*, Hossein Piri, S P Vij, K C Mahant & Shaveta Chauhan Orchid Laboratory, Department of Botany, Panjab University, Chandigarh 160 014, India

Received 24 May 2010; revised 20 May 2011

In vitro asymbiotic seed germination potential of its immature seeds (36 weeks after pollination) of G. calceolaris was successfully tested on three different agar gelled nutrient media i.e. Murashige and Skoog (MS), Mitra et al. (M) and potato dextrose agar (PDA). Seeds germinated within 15.75±0.75 to 35.75±0.75 days in the three different media. The protocorms developed therefrom subsequently differentiated into first leaf and root primordia, and complete seedlings were obtained within 111.25±1.25 to 141.25±1.25 days on MS and M media. The protocorms, though failed to differentiate further on basal PDA medium, despite repeated subculturings, incorporation of peptone (P; 1gl -1), yeast extract (YE; 2 gl -1) and coconut water (CW; 20%) in the medium proved beneficial in inducing differentiation, in these germinating entities. Additional use of growth additives (P/YE/CW), in general, favoured better germination, protocorm formation and seedling development. The optimal nutritional combination during seed germination, protocorm growth and multiplication and seedling development was found to be CW (10%) enriched MS medium.

Keywords: , Immature seeds, Nutrient media, Seed germination, Seedling development.

Orchid seeds lack endosperm, and an appropriate Sikkim, Arunachal Pradesh, Tripura and Meghalaya machinery to directly utilize their own lipid food (330-660 m). The species is ornamentally significant reserves. They require a suitable fungal stimulus for for its beautiful flowers and has an immense potential germination and further development; on contacting a to serve as a putative parent in hybridization suitable substratum, only 0.2-0.3% of them are able to programmes aimed at producing elite genotypes. Its germinate in nature. Moreover, their propagation natural populations are declining rapidly under the through conventional means has lost its significance duress of progressive and in commercial ventures, because vegetative extensive commercial collections; the species has multiplication through division and back bulb culture recently been listed as critically endangered 1. Though is rather slow and yields only a meager number of the morphogenetic response of Gastrochilus plantlets even after 5-6 years. On the other hand, calceolaris inflorescence axes has been tested in tissue culture techniques which offer a great potential vitro 2, in the present study, an attempt has been made in maintaining the genetic fidelity of clones and to develop an appropriate propagation system for its conservation of genetic resources have opened mass multiplication , by assessing its in vitro new possibilities in conservation and asymbiotic seed germination potential, using commercialization of these . immature seeds from unripened, green and Gastrochilus calceolaris (=Saccolabium calceolaris undehisced capsules. Lindl .) bearing yellowish-green, fragrant, and long- lasting flowers is distributed in , India, , Materials and Methods , and . In India, it is distributed Source and sterilization of the explants —The along the Himalayas from Garhwal Eastwards to cultures were initiated using immature seeds procured from unripe, green and undehisced capsules —————— [36 weeks after pollination (wap)]. The capsules *Correspondent author (‘pods’) were scrubbed with ‘teepol’ (1%), washed Phone: +91-172-2534018; Mobile: 09876701963 E-mail: [email protected]; thoroughly under running tap water for 15-20 min and [email protected] surface sterilized for 7 min with HgCl ₂ solution 712 INDIAN J EXP BIOL, SEPTEMBER 2011

(0.1%), with 1-2 drops of ‘teepol’ as a wetting agent the non-significant difference of additives at 5% level prior to washing with sterilized distilled water. The of significance, various groups of additives showing pods were also treated with streptomycin (0.03%) for identical/similar response were formed statistically. 5 min and repeatedly washed with sterilized double To this end, Tukey test was performed at 5% level distilled water so as to remove all the traces of the with respect to each response. sterilant, and subsequently, dipped in 70% ethyl alcohol for 30 sec, flame sterilized, and were split Results and Discussion open longitudinally, with a sterilized blade. The seeds In G. calceolaris, immature seeds (Fig. 1 a), were scooped out and inoculated on the nutrient successfully germinated asymbiotically, in vitro (Fig. media, in a laminar flow cabinet, under aseptic 1 b), in all the three tested nutrient media (MS, M and conditions. PDA). The possibility of bypassing fungal Culture media —Three different nutrient media, requirement of orchid seeds during germination 5 viz. Murashige and Skoog (MS) 3, Mitra et al. (M) 4 in vitro has added new dimensions to orchid and (PDA) containing 2% sucrose and gelled with propagation and a large number of orchid species 0.9% agar were used. The culture media were also and hybrids representing diverse habits and habitats 6-13 supplemented with peptone (P; 1gl -1), yeast extract have responded to asymbiotic germination in vitro . (YE; 1-2gl -1) and coconut water (CW; 10-20%), as The technique often known as ‘Green Pod Culture’ growth additives. The pH of the media was adjusted facilitates easy sterilization, and ensures better to 5.5 using 0.1 N HCl or NaOH. About 25ml l -1 of the frequency of germination, and reduces the time 14 medium was dispensed in each test tube and the lapse between pollination and sowing of seeds . culture tubes were plugged with cotton buns before However, as the technique requires the use of all pressure and at 121°C for seeds/embryos in a single sowing, the importance of ²־ autoclaving at 1.1 kg cm 20 min. a proper stage at which the fruit has to be harvested Incubation of cultures and data recording —The assumed a great significance. Further, harvesting cultures were maintained at 25°±2°C and 12 h time was considered specific not only to genera and 15 photoperiod provided by cool white fluorescent light hybrids but also to species . During the present (40 µmol m -² s -¹). The cultures were examined studies, the immature seeds procured 36 weeks after regularly and the data were recorded on the pollination from green capsules showed the swelling germination frequency and time taken (in days) for of embryos within 25.75±0.75 to 35.75±0.75 days in germination, chlorophyll synthesis, protocorm the three nutrient media . The easy germinability of formation, emergence of leaf and root and subsequent immature seeds can be attributed to their distended seedling development. Subculturing was done at testa cells, metabolically awakened embryos, and 16 4 weeks intervals or as and when required. For every absence of dormancy factors . The technique has treatment, 16 replicates were maintained. also proved advantageous for hybrid rescue, cloning Hardening, deflasking and establishment of of apomictic genotypes, exploiting the seedlings in greenhouse —The procedure involved polyembryonate potential of the seeds and 17 gradual removal of growth additives, sucrose, facilitating genetic transformations . vitamins, and inorganic salts from the nutrient The spherules emerged out, produced chlorophyll medium. The selected healthy seedlings with 2-3 and developed into protocorms. The protocorms leaves and 1-2 roots were washed with lukewarm subsequently differentiated into first leaf and root water to remove the traces of agar followed by primordia and complete seedlings were obtained treatment with a mild fungicide (Bavistin; 0.01%) within 130.00±0.00 and 136.25±1.25 days solution and streptomycin (0.03%) for 4-5 min. These respectively in MS and M media. In basal PDA were, then, transferred to greenhouse and potted in 6 medium, the chlorophyllous protocorms were formed, cm diameter clay pots, filled with potting mixture of but failed to differentiate further, despite repeated charcoal pieces, pine bark, brick pieces and subculturing. Sphagnum moss (1:1:1:1). In the present study, M and MS media supported Statistical analysis —Multivariate analyses of the best germination (68.75±1.25% within 25.75±0.75 variance was performed with respect to each response days) followed by PDA (48.75±1.25% in 35.75±0.75 (mean ± SE). As multivariate ANOVA results showed days. A variety of culture media including some PATHAK et al. : IN VITRO PROPAGATION AND MASS SCALE MULTIPLICATION 713

Fig. 1—In vitro propagation and mass scale multiplication of G. calceolaris using immature seeds [a, Immature seeds at the time of inoculation (x20); b, Spherule emerging out by rupturing seed coat (x20); c-d, Protocorm multiplication on M+CW (10%); and M+YE (1gl -¹); e, Seedling development on MS+CW(10%); f-g, Protocorm differentiation and multiplication on PDA+CW (20%); h, Seedlings ready for hardening; i, seedlings transferred to pot]. species specific ones have been formulated for could also multiply at an average rate in CW at 10 and in vitro culture of orchid seeds/tissues 3-4,11 . The use 20% of its concentration in M and PDA media of M medium has been well-documented in respectively, the protocorms multiplied rapidly in CW literature for a number of orchid species 9. (10%) enriched MS medium. Addition of CW (10%), Additional use of growth additives (P/YE/CW), in in general, proved beneficial during seed germination general, favoured better germination, early protocorm and protocorm growth in M and MS media (Fig. 1 c) formation and seedling development (Fig. 1 c and 1 d). though its effect was more pronounced in MS The per cent and onset of germination response and medium, as seedlings were obtained within the time taken for morphogenetic stages leading to 111.25±1.25 days (Fig. 1e). CW has been used seedling development, varied with quality and favourably for germination in Cymbidium 18 , concentration of growth additives used (Table 1). Rhynchostylis retusa 19 , Dendrobium species 20 , and Amongst the three nutrient media used multiplication Paphiopedilum purpuratum 16 and for seedling growth of protocorms was observed only on basal MS in Dendrobium species 20 . Protocorms on CW free medium; incorporation of growth additives in this medium hardly grew and turned brown as compared medium, however, in general improved the rate of to those in nutrient media supplemented with CW 19 . It multiplication of these entities. Though protocorms was, however, inhibitory to germination in 714 INDIAN J EXP BIOL, SEPTEMBER 2011

Table 1—Effect of different nutrient media with and without growth additives during asymbiotic seed germination and seedling development in G. calceolaris [Values are mean ± SE] Nutrient media Growth additives MS M PDA Germination frequency (%) Basal medium a68.75±1.250 x a68.75±1.250 y a48.75±1.250 z P (1 gl -¹) b88.75±1.250 x b78.75±1.250 y b78.75±1.250 z YE (1 gl -¹) c99.50±0.500 x c88.75±1.250 y c48.75±1.250 z YE (2 gl -¹) d68.75±1.250 x d58.75±1.250 y d68.75±1.250 z CW (10%) e99.50±0.500 x e99.50±0.500 y e58.75±1.250 z CW (20%) fe 78.75±1.250 x fe 88.75±1.250 y fe 88.75±1.250 z Protocorm development (days) Basal medium a45.50±0.50 x a45.75±0.75 x a58.50±0.50 z P (1 gl -¹) be 40.75±0.75 x b40.75±0.75 x b50.75±0.75 z YE(1 gl -¹) ce 30.75±0.75 x ce 35.50±0.96 x ce 60.75±0.75 z YE(2 gl -¹) d50.75±0.75 x d51.00±0.58 x d55.75±0.75 z CW(10%) e30.75±0.75 x e35.75±0.75 x e60.75±0.75 z CW(20%) f45.50±0.50 x f45.75±0.75 x f50.75±0.75 z Development of 1 st leaf primordium (days) Basal medium a71.50±0.86 x a74.00±2.31 y a0.00±0.00 z P(1 gl -¹) bf 61.50±0.87 x bf 74.00±2.31 y bf 81.50±0.87 z YE(1 gl -¹) ce46.50±0.87 x ce 61.50±0.87 y ce 0.00±0.00 z YE(2 gl -¹) d72.50±1.44 x d76.50±0.87 y d86.50±0.87 z CW(10%) e52.00±1.22 x e61.50±0.87 y e0.00±0.00 z CW(20%) f66.50±0.87 x f74.00±2.3 y f81.50±0.87 z Development of 1 st root primordium (days) Basal medium a86.50±0.87 x a86.50±0.87 y a 0.00±0.00 z P(1 gl -¹) b74.00±2.31 x b81.50±0.87 y b101.50±0.87 z YE(1 gl -¹) ce 61.50±0.87 x ce 71.50±0.87 y ce 0.00±0.00 z YE(2 gl -¹) d86.50±0.87 x d91.50±0.87 y d111.50±0.87 z CW(10%) e61.50±0.87 x e76.50±0.87 y e0.00±0.00 z CW(20%) f84.00±2.3 x f86.50±0.87 y f101.50±0.87 z Seedling development (days) Basal medium a130.00±0.00 x a136.25±1.25 y a0.00±0.00 z P(1 gl -¹) bf 125.00±0.00 x bf 126.25±1.25 y bf 150.00±0.00 z YE(1 gl -¹) ce 111.25±1.25 x ce 126.25±1.25 y ce 0.00±0.00 z YE(2 gl -¹) d131.25±1.25 x d141.25±1.25 y d162.50±1.44 z CW(10%) e111.25±1.25 x e121.25±1.25 y e0.00±0.00 z CW(20%) f121.25±1.25 x f131.25±1.25 y f150.00±0.00 z P: Peptone; YE :Yeast extract; CW: Coconut water . Same superscript alphabetical letters denotes that t he corresponding means are in the same group using Tukey’s multiple range test at 5%; a, b, c, d, e, f written as superscripts on left side of the data represent significant difference between the treatments whereas x, y, z written as superscripts on right side of the data represent significant difference between the nutrient media Dendrobium 21 , and root differentiation in Dendrobium development; the additive also proved useful in moschatum 20 . Almost similar results were obtained inducing differentiation in the protocorms on PDA during early stages of seed germination, rhizogenesis medium. This complex additive has been successfully and seedling development when YE (1gl-1) was used to culture embryos of several orchid species 11 . Its supplemented in MS medium. Peptone, whenever growth promotory nature has been correlated with its used in any of the three nutrient media not only peptides and other unknown contents 22 whereas amino enhanced the frequency and advanced onset of acids, amides and vitamin contents of this additive germination, but also favoured the healthy protocorm are considered responsible for its benign nature 23 . PATHAK et al. : IN VITRO PROPAGATION AND MASS SCALE MULTIPLICATION 715

Additional use of growth additives in PDA References medium, though, in general, enhanced the frequency 1 Agoo E M G, Cootes J, Golamco A, Jr de Vogel, E F & Tiu and advanced onset of germination, YE at 1 gl -1 and D. Gastrochilus calceolaris , in IUCN 2009 . IUCN red list of CW at 10% could not induce the differentiation in the threatened species . Version (2009) 1. 2 Vij S P, Sood A & Plaha K K , In vitro breakdown of apical protocorms. These, however, when used at higher dormancy and development of vegetative shoots from -1 concentration [YE (2gl ); CW (20%)] successfully inflorescence segments in Saccolabium calceolare Lindl., in induced differentiation in these germinating entities Biology, conservation and culture of orchids , edited by Vij S P (Fig. 1 f and g), and seedlings with 2-3 leaves and (Affiliated East-West Press Pvt. Ltd., New Delhi) 1986. 473. 3 Murashige T & Skoog F, A revised medium for rapid growth 1-2 roots were obtained in 162.50±1.44 and and bioassays with tobacco tissue cultures, Physiol 150.00±0.00 days respectively. YE favoured Plantarum , 15 (1962) 473. 24 enhanced germination in Goodyera repens , and 4 Mitra G C, Prasad R N & Roy Chowdhury A, Inorganic salts Orchis laxiflora 25 , but proved inhibitory during and differentiation of protocorms in seed callus of an orchid germination in Dendrobium 20 . The protocorms and correlated changes in its free amino acid content, Indian J Exp Biol , 14 (1976) 350. multiplied rapidly on YE supplemented media in 26 27 5 Knudson, L, Non-symbiotic germination of orchid seeds, Bot Coelogyne barbata , Spiranthes sinensis , and Gaz , 73 (1922) 1. 28 Satyrium nepalense and Vanda cristata . 6 Arditti J, Clements M A, Fast G, Hadley G, Nishimura G & Based on the present observations, the optimal Ernst R, Orchid seed germination and seedling culture—A nutrient medium for seed germination, protocorm Manual, in Orchid biology , reviews, and perspectives , Vol. II edited by Arditti J (Cornell University Press, Ithaca, New growth and rapid multiplication, and seedling York) 1982. 243. development in G . calceolaris is MS+CW (10%). 7 Hossain M M, Sharma M & Pathak Promila, In vitro mass The seedlings with 2-3 leaves and 1-2 roots were propagation of an economically important orchid, subjected to hardening procedure (Fig. 1 h). The Cymbidium aloifolium (L.) Sw, J Orchid Soc India , 22 hardened plants were subsequently transferred to (2008) 91. 8 Hossain M M, Sharma M & Pathak Promila, Cost-effective pots containing potting mix (Fig. 1 i). The potted protocol for in vitro mass propagation of Cymbidium plants were kept in the greenhouse conditions and aloifolium (L.) Sw-A medicinally important orchid, Eng-Life the data were recorded regularly on their Sci, 9 (2009) 1. performance to evaluate their survival frequency. 9 Pathak Promila, Vij S P & N Gautam, Effects of alternate About 70-80% of survival was observed after gelling agents on in vitro asymbiotic germination and seedling development in Aerides multiflora Roxb.: An 3 months of their transfer. attempt toward developing a cost effective protocol, in Proc While assessing the in vitro morphogenetic 18 th WOC Dijon France (Actes Proceedings 2005 France- response of young inflorescence axes of G. 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