Promila Pathak*, Hossein Piri, S P Vij, K C Mahant & Shaveta Chauhan

Transcription

1 Indian Journal of Experimental Biology Vol. 49, September 2011, pp In vitro propagation and mass scale multiplication of a critically endangered epiphytic orchid, Gastrochilus 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 , 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 ±1.25 to ±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: Gastrochilus calceolaris, Immature seeds, Nutrient media, Seed germination, Seedling development. Orchid seeds lack endosperm, and an appropriate machinery to directly utilize their own lipid food reserves. They require a suitable fungal stimulus for germination and further development; on contacting a suitable substratum, only % of them are able to germinate in nature. Moreover, their propagation through conventional means has lost its significance in commercial ventures, because vegetative multiplication through division and back bulb culture is rather slow and yields only a meager number of plantlets even after 5-6 years. On the other hand, tissue culture techniques which offer a great potential in maintaining the genetic fidelity of clones and conservation of plant genetic resources have opened new possibilities in conservation and commercialization of these plants. Gastrochilus calceolaris (=Saccolabium calceolaris Lindl.) bearing yellowish-green, fragrant, and longlasting flowers is distributed in China, India, Nepal, Thailand, and Malaysia. In India, it is distributed along the Himalayas from Garhwal Eastwards to *Correspondent author Phone: ; Mobile: ppathak_2000@yahoo.com; ppathak_2008@rediffmail.com Sikkim, Arunachal Pradesh, Tripura and Meghalaya ( m). The species is ornamentally significant for its beautiful flowers and has an immense potential to serve as a putative parent in hybridization programmes aimed at producing elite genotypes. Its natural populations are declining rapidly under the duress of progressive habitat destruction and extensive commercial collections; the species has recently been listed as critically endangered 1. Though the morphogenetic response of Gastrochilus calceolaris inflorescence axes has been tested in vitro 2, in the present study, an attempt has been made to develop an appropriate propagation system for its mass multiplication, by assessing its in vitro asymbiotic seed germination potential, using immature seeds from unripened, green and undehisced capsules. Materials and Methods Source and sterilization of the explants The cultures were initiated using immature seeds procured from unripe, green and undehisced capsules [36 weeks after pollination (wap)]. The capsules ( pods ) were scrubbed with teepol (1%), washed thoroughly under running tap water for min and surface sterilized for 7 min with HgCl₂ solution

2 712 INDIAN J EXP BIOL, SEPTEMBER 2011 (0.1%), with 1-2 drops of teepol as a wetting agent prior to washing with sterilized distilled water. The pods were also treated with streptomycin (0.03%) for 5 min and repeatedly washed with sterilized double distilled water so as to remove all the traces of the sterilant, and subsequently, dipped in 70% ethyl alcohol for 30 sec, flame sterilized, and were split open longitudinally, with a sterilized blade. The seeds were scooped out and inoculated on the nutrient media, in a laminar flow cabinet, under aseptic conditions. Culture media Three different nutrient media, viz. Murashige and Skoog (MS) 3, Mitra et al. (M) 4 and (PDA) containing 2% sucrose and gelled with 0.9% agar were used. The culture media were also supplemented with peptone (P; 1gl -1 ), yeast extract (YE; 1-2gl -1 ) and coconut water (CW; 10-20%), as growth additives. The ph of the media was adjusted to 5.5 using 0.1N HCl or NaOH. About 25ml l -1 of the medium was dispensed in each test tube and the culture tubes were plugged with cotton buns before autoclaving at 1.1 kg ² cm pressure and at 121 C for 20 min. Incubation of cultures and data recording The cultures were maintained at 25 ±2 C and 12 h photoperiod provided by cool white fluorescent light (40 µmol m - ² s - ¹). The cultures were examined regularly and the data were recorded on the germination frequency and time taken (in days) for germination, chlorophyll synthesis, protocorm formation, emergence of leaf and root and subsequent seedling development. Subculturing was done at 4 weeks intervals or as and when required. For every treatment, 16 replicates were maintained. Hardening, deflasking and establishment of seedlings in greenhouse The procedure involved gradual removal of growth additives, sucrose, vitamins, and inorganic salts from the nutrient medium. The selected healthy seedlings with 2-3 leaves and 1-2 roots were washed with lukewarm water to remove the traces of agar followed by treatment with a mild fungicide (Bavistin; 0.01%) solution and streptomycin (0.03%) for 4-5 min. These were, then, transferred to greenhouse and potted in 6 cm diameter clay pots, filled with potting mixture of charcoal pieces, pine bark, brick pieces and Sphagnum moss (1:1:1:1). Statistical analysis Multivariate analyses of variance was performed with respect to each response (mean ± SE). As multivariate ANOVA results showed the non-significant difference of additives at 5% level of significance, various groups of additives showing identical/similar response were formed statistically. To this end, Tukey test was performed at 5% level with respect to each response. Results and Discussion In G. calceolaris, immature seeds (Fig. 1 a), successfully germinated asymbiotically, in vitro (Fig. 1 b), in all the three tested nutrient media (MS, M and PDA). The possibility of bypassing fungal requirement of orchid seeds during germination in vitro 5 has added new dimensions to orchid propagation and a large number of orchid species and hybrids representing diverse habits and habitats have responded to asymbiotic germination in vitro The technique often known as Green Pod Culture facilitates easy sterilization, and ensures better frequency of germination, and reduces the time lapse between pollination and sowing of seeds 14. However, as the technique requires the use of all seeds/embryos in a single sowing, the importance of a proper stage at which the fruit has to be harvested assumed a great significance. Further, harvesting time was considered specific not only to genera and hybrids but also to species 15. During the present studies, the immature seeds procured 36 weeks after pollination from green capsules showed the swelling of embryos within 25.75±0.75 to 35.75±0.75 days in the three nutrient media. The easy germinability of immature seeds can be attributed to their distended testa cells, metabolically awakened embryos, and absence of dormancy factors 16. The technique has also proved advantageous for hybrid rescue, cloning of apomictic genotypes, exploiting the polyembryonate potential of the seeds and facilitating genetic transformations 17. The spherules emerged out, produced chlorophyll and developed into protocorms. The protocorms subsequently differentiated into first leaf and root primordia and complete seedlings were obtained within ±0.00 and ±1.25 days respectively in MS and M media. In basal PDA medium, the chlorophyllous protocorms were formed, but failed to differentiate further, despite repeated subculturing. In the present study, M and MS media supported the best germination (68.75±1.25% within 25.75±0.75 days) followed by PDA (48.75±1.25% in 35.75±0.75 days. A variety of culture media including some

3 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+; 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 in vitro culture of orchid seeds/tissues 3-4,11. The use of M medium has been well-documented in literature for a number of orchid species 9. Additional use of growth additives (P/YE/CW), in general, favoured better germination, early protocorm formation and seedling development (Fig. 1 c and 1 d). The per cent and onset of germination response and the time taken for morphogenetic stages leading to seedling development, varied with quality and concentration of growth additives used (Table 1). Amongst the three nutrient media used multiplication of protocorms was observed only on basal MS medium; incorporation of growth additives in this medium, however, in general improved the rate of multiplication of these entities. Though protocorms could also multiply at an average rate in CW at 10 and 20% of its concentration in M and PDA media respectively, the protocorms multiplied rapidly in CW (10%) enriched MS medium. Addition of CW (10%), in general, proved beneficial during seed germination and protocorm growth in M and MS media (Fig. 1 c) though its effect was more pronounced in MS medium, as seedlings were obtained within ±1.25 days (Fig. 1e). CW has been used favourably for germination in Cymbidium 18, Rhynchostylis retusa 19, Dendrobium species 20, and Paphiopedilum purpuratum 16 and for seedling growth in Dendrobium species 20. Protocorms on CW free medium hardly grew and turned brown as compared to those in nutrient media supplemented with CW 19. It was, however, inhibitory to germination in

4 714 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] Growth additives Germination frequency (%) P (1 gl - ¹) YE (1 gl - ¹) YE (2 gl - ¹) CW (10%) CW (20%) Protocorm development (days) P (1 gl - ¹) Development of 1 st leaf primordium (days) P(1 gl - ¹) Development of 1 st root primordium (days) P(1 gl - ¹) Seedling development (days) P(1 gl - ¹) Dendrobium 21, and root differentiation in Dendrobium moschatum 20. Almost similar results were obtained during early stages of seed germination, rhizogenesis and seedling development when YE (1gl- 1 ) was supplemented in MS medium. Peptone, whenever used in any of the three nutrient media not only enhanced the frequency and advanced onset of germination, but also favoured the healthy protocorm Nutrient media MS M PDA a 68.75±1.250 x a 68.75±1.250 y a 48.75±1.250 z b 88.75±1.250 x b 78.75±1.250 y b 78.75±1.250 z c 99.50±0.500 x c 88.75±1.250 y c 48.75±1.250 z d 68.75±1.250 x d 58.75±1.250 y d 68.75±1.250 z e 99.50±0.500 x e 99.50±0.500 y e 58.75±1.250 z fe 78.75±1.250 x fe 88.75±1.250 y fe 88.75±1.250 z a 45.50±0.50 x a 45.75±0.75 x a 58.50±0.50 z be 40.75±0.75 x b 40.75±0.75 x b 50.75±0.75 z ce 30.75±0.75 x ce 35.50±0.96 x ce 60.75±0.75 z d 50.75±0.75 x d 51.00±0.58 x d 55.75±0.75 z e 30.75±0.75 x e 35.75±0.75 x e 60.75±0.75 z f 45.50±0.50 x f 45.75±0.75 x f 50.75±0.75 z a 71.50±0.86 x a 74.00±2.31 y a 0.00±0.00 z bf 61.50±0.87 x bf 74.00±2.31 y bf 81.50±0.87 z ce 46.50±0.87 x ce 61.50±0.87 y ce 0.00±0.00 z d 72.50±1.44 x d 76.50±0.87 y d 86.50±0.87 z e 52.00±1.22 x e 61.50±0.87 y e 0.00±0.00 z f 66.50±0.87 x f 74.00±2.3 y f 81.50±0.87 z a 86.50±0.87 x a 86.50±0.87 y a 0.00±0.00 z b 74.00±2.31 x b 81.50±0.87 y b ±0.87 z ce 61.50±0.87 x ce 71.50±0.87 y ce 0.00±0.00 z d 86.50±0.87 x d 91.50±0.87 y d ±0.87 z e 61.50±0.87 x e 76.50±0.87 y e 0.00±0.00 z f 84.00±2.3 x f 86.50±0.87 y f ±0.87 z a ±0.00 x a ±1.25 y a 0.00±0.00 z bf ±0.00 x bf ±1.25 y bf ±0.00 z ce ±1.25 x ce ±1.25 y ce 0.00±0.00 z d ±1.25 x d ±1.25 y d ±1.44 z e ±1.25 x e ±1.25 y e 0.00±0.00 z f ±1.25 x f ±1.25 y f ±0.00 z P: Peptone; YE :Yeast extract; CW: Coconut water. Same superscript alphabetical letters denotes that the 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 development; the additive also proved useful in inducing differentiation in the protocorms on PDA medium. This complex additive has been successfully used to culture embryos of several orchid species 11. Its growth promotory nature has been correlated with its peptides and other unknown contents 22 whereas amino acids, amides and vitamin contents of this additive are considered responsible for its benign nature 23.

5 PATHAK et al.: IN VITRO PROPAGATION AND MASS SCALE MULTIPLICATION 715 Additional use of growth additives in PDA medium, though, in general, enhanced the frequency and advanced onset of germination, YE at 1 gl -1 and CW at 10% could not induce the differentiation in the protocorms. These, however, when used at higher concentration [YE (2gl -1 ); CW (20%)] successfully induced differentiation in these germinating entities (Fig. 1 f and g), and seedlings with 2-3 leaves and 1-2 roots were obtained in ±1.44 and ±0.00 days respectively. YE favoured enhanced germination in Goodyera repens 24, and Orchis laxiflora 25, but proved inhibitory during germination in Dendrobium 20. The protocorms multiplied rapidly on YE supplemented media in Coelogyne barbata 26, Spiranthes sinensis 27, and Satyrium nepalense and Vanda cristata 28. Based on the present observations, the optimal nutrient medium for seed germination, protocorm growth and rapid multiplication, and seedling development in G. calceolaris is MS+CW (10%). The seedlings with 2-3 leaves and 1-2 roots were subjected to hardening procedure (Fig. 1 h). The hardened plants were subsequently transferred to pots containing potting mix (Fig. 1 i). The potted plants were kept in the greenhouse conditions and the data were recorded regularly on their performance to evaluate their survival frequency. About 70-80% of survival was observed after 3 months of their transfer. While assessing the in vitro morphogenetic response of young inflorescence axes of G. calceolaris 2, it was reported that lower floral buds aborted, whereas the ones near the apex (subterminal) variously matured into flowers or leafy shoots depending upon their physiological age. Explants, however, regenerated positively in M medium containing casein hydrolysate (CH; 2 gl -1 )/ urea (25 mgl -1 ) and IAA (2 mgl -1 ). To summarise, the propagation protocol was successfully developed in vitro, for mass scale multiplication of a medicinally important and critically endangered epiphytic orchid, Gastrochilus calceolaris, using immature seeds, suggesting thereby that such studies, may be extended to other related medicinally important orchid taxa, as well. Acknowledgement Financial assistance from Department of Biotechnology, Ministry of Science and Technology, Government of India is gratefully acknowledged. References 1 Agoo E M G, Cootes J, Golamco A, Jr de Vogel, E F & Tiu D. Gastrochilus calceolaris, in IUCN IUCN red list of threatened species. 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