State Forest Research Institute, Post Box No. 159, Itanagar , India 1 Department of Botany, Rajiv Gandhi University, Itanagar , India
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1 Indian Journal of Biotechnology Vol 6, April 2007, pp Effects of different culture media on seed germination and subsequent in vitro development of protocorms of Hygrochilus parishii (Veith & Rchb.f.) Pfitz (Orchidaceae) R Shadang, Padmanabh Dwivedi 1*, S N Hegde and N Ahmed State Forest Research Institute, Post Box No. 159, Itanagar , India 1 Department of Botany, Rajiv Gandhi University, Itanagar , India Received 19 September 2005; revised 24 May 2006; accepted 20 July 2006 Seeds of Hygrochilus parishii (Veith & Rchb.f.) Pfitz were inoculated on eight different culture media (basal) MS, MKC, V&W, I&Y and half strength of respective media. Seeds germinated within d almost on all media with varying percentage and developed into greenish protocorm after d. Best seed germination was observed in ½ strength MKC medium. Healthy protocorms were observed within 100 d in V&W media. Sub-cultured protocorms in all the above media supplemented with NAA, BAP, 2,4-D and IAA in a range of mg L -1, both individually and in combination, and additives like banana pulp (BP, 10%) and coconut milk (CM, 15%) produced well developed callus. Multiplication of protocorms was best on ½ strength MS + NAA (2.0 mg L -1 ). Formation of root and leaf was early in V&W medium having CM (15%) and BP (10%) but without sucrose. Keywords: culture media, Hygrochilus parishii, orchid, protocorms, seed germination IPC Code: Int. Cl. 8 A01H4/00 Introduction North-Eastern part of India is home to a number of rare and endemic orchid species. Some of these species are disappearing due to extensive collection and habitat destruction by anthropogenic activities. Hygrochilus parishii (Veith & Rchb.f.) Pfitz, an epiphytic plant is one of those species that is endemic and has become rare 1. Due to its handsome multicoloured and long lasting flower quality, the species has received wide attention of floriculturists and orchid lovers. As measure to conserve this species, experiments on seed germination and subsequent protocorms and seedling growth have been carried out in vitro. Though most of the orchid species produce millions of seeds in a single capsule 2,3, in natural conditions less than 5% germinate due to specific nutritional requirement 4,5. Asymbiotic in vitro germination of orchid seeds has been reported by several workers 6-8. In the present communication, an attempt has been made for in vitro seed germination and subsequent development of *Author for correspondence: Tel: ; Fax: pdwivedi25@rediffmail.com protocorms and seedling growth from green undihisced mature capsules of H. parishii to produce a large number of plants. Materials and Methods Eight different basal culture media - MKC (Modified Knudson C ) 9, MS (Murashige & Skoog) 10, V&W (Vacin & Went) 11 and I&Y (Ichihashi & Yamashita) 12 and their half strengths were used for seed germination and protocorm development. ph of the media were adjusted to 5.6 before autoclaving. The growth and development of protocorms and subsequent seedlings were tested on all the above media supplemented with filtersterilized NAA, BAP, IAA, 2,4-D ( mg L -1 ), either singly or in combination. The media were also enriched with additives like coconut milk (CM) and banana pulp (BP). Undihisced 8-month-old green capsules were washed thrice in running tap water followed by application of a detergent solution with brush. The capsules were then dipped in 70% ethanol for 5 min and washed thoroughly with sterilized distilled water. The capsules were flamed before opening by a sterile blade. Seeds were then distributed directly onto the
2 SHADANG et al: PROTOCORM DEVELOPMENT OF H. PARISHII IN VITRO culture media. The inoculated flasks were kept at room temperature (22'-2OC) under lux light with 14 h photoperiod. The number of days required for germination and protocorm development were recorded. Protocorms thus obtained were inoculated onto basal media used for seed germination with different combinations of NAA, BAP, IAA, 2,4-D and additives like CM and BP. Growth response in all the cultures was recorded. Results and Discussion The seeds of H. parishii inoculated in eight basal I nutrient media germinated after d and developed into greenish protocorms within 100 d of culture. Seed germination was 90% in MKC, followed by 80% in MS medium. On V&W medium though only 60% germination occurred, the protocorms showed better growth with early leaf (1~2 in numbers) and roots (1-2 in numbers) formation 4-5 months after culture % germination was recorded in I&Y, 34 MKC and?h MS, and 20-40% in?h V&W and 34 I and Y (Table 1). The promotory effect of growth regulators in in vitro culture has been worked out by a number of worker^'^-'^. In the present study, BAP (2.0 mg L-') in MS media showed significant response in shoot and root development (Table 2). Higher concentration of NAA (2.0 mg L-I) in 34 MS, V&W media favoured protocorm multiplication (Figs 1 & 2) but 2,4-D (2.0 mg L-') in WKC initiated callus in protocorms (Fig. 3). IAA (1.0 mg L-') in MS initiated shoot bud formation after 60 d of sub-culture (Fig. 4). All protocorms developed shoot buds in both full and?h strength MS media supplemented with BAP (2.0 mg L-') and NAA (2.0 mg L-I) (Figs 5&6). With BAP (1.0 mg L-') + NAA (2.0 mg L-') in 95 strength MKC Figs &2. Multiplication of protocorms on Yz strength MS protocorm was observed' md V&W supplemented with NAA (2.0 mg ~1); 3. Callus in H Excellent callus was observed in MKC, MS or %MS MKC+ 2,4-D (2.0 mg L-1); 4. shoot bud initiation in MS+IAA media in combination of BAP (1.0 mg L-') + 2,4-D (1.0 mg L-j); 5 & 6. Protocorms developed shoot & buds in MS & (2.0 mg L-') + IAA (1.0 mg L-I). BP, CM and potato extract have been successfull used in place of 17 sucrose in Sarcanthine orchids. In our experiments, incorporation of CM (15%) or BP (10%) instead of sucrose in MKC, MS, 34 strength MS and V&W media initiated direct differentiation of shoots and roots from protocorms. In other media the response was very poor (Table 2). Seed germination frequency was lower in 34 strength than in full strength of culture media. In case of protocorms, higher concentration of BAP Yz strength MS supplemented with BAP (2.0 mg L-I) and NAA (1.0 mg L-I); and 7. Seedling growth in V&W +CM (15%)+BP (10%). (2.0 mg LA') initiated bud in majority of culture media. 2,4-D (2.0 mg L-') caused callusing of protocorms and NAA (2.0 mg L-') proved effective for protocorm multiplication. Combination of BAP (1.0 mg L-I), 2,4-D (2.0 mg L-I) and IAA (1.0 mg L-') favoured formation of shoot buds. However, CM '' (15%) and BP (10%) when added in place of sucrose
3 258 INDIAN J BIOTECHNOL, APRIL 2007 S..No Media Time taken for seed germination (d) Table 1 Response of seed germination in Hygrochilus parishii. Number of days taken for formation of protocormlike bodies (Plbs) % Germination Observation 1 MKC Inoculated seeds swell after 16 d, followed by germination, Plbs found after 80 d, Plbs small in size. 2 ½ MKC Inoculated seeds swell after 16 d, followed by germination, 40% of Plbs turned brownish and died after 90 d, Plbs were small in size. 3 MS Inoculated seeds swell after 16 d, followed by germination, Plbs found after 90 d, Plbs large in size. 4 ½ MS Inoculated seeds swell after 16 d, followed by germination, few of the protocorms turned brownish and died after 90 d. 5 V&W Inoculated seeds swell after 20 d, followed by germination, protocorms formed with 1-2 leaf, 2-5 mm long, 1-2 root 5-10 mm long, after 5 months. 6 ½ V&W Inoculated seeds swell after 20 d, followed by germination, protocorms formed with 1-2 leaf, 2-5 mm long, 1-2 root 5-10 mm long, after 4 months. 7 I&Y Inoculated seeds swell after 16 d, followed by germination, Plbs found after 80 d. 8 ½ I &Y Inoculated seeds swell after 16 d, followed by germination, white protocorms formed after 90 d only, few turned green. Time taken for Plbs: n = 120, df = 7, F = , p< [p = 0.000] Time taken for germination: n = 120, df = 7, F = 3.702, p< [0.001] Result: n = 120, df = 7, F = , p < [p = 0.000] Culture Media Table 2 Response of protocorms grown on various culture media supplemented with different growth regulators Growth regulators (mg L -1 ) BAP NAA 2,4-D IAA CM BP Callus % Response Plbs multiplication Shoot buds after 2 month of Plbs inoculation MKC % 10%
4 SHADANG et al: PROTOCORM DEVELOPMENT OF H. PARISHII IN VITRO 259 Culture Media Table 2 Response of protocorms grown on various culture media supplemented with different growth regulators Contd. Growth regulators (mg L -1 ) BAP NAA 2,4-D IAA CM BP Callus % Response Plbs multiplication Shoot buds after 2 month of Plbs inoculation ½ MKC % 10% MS % 10% ½ MS % 10%
5 260 INDIAN J BIOTECHNOL, APRIL 2007 Culture Media Table 2 Response of protocorms grown on various culture media supplemented with different growth regulators Contd. Growth regulators (mg L -1 ) BAP NAA 2,4-D IAA CM BP Callus % Response Plbs multiplication Shoot buds after 2 month of Plbs inoculation V&W % 10% ½ V&W % 10% I&Y % 10% ½ I&Y % 10% (-) Nil, BAP (6-Benzyl amino purine), NAA (Naphthalene acetic acid), 2,4-D (2,4-Dichlorophenoxy acetic acid), IAA (Indole-3-acetic acid), CM (Coconut Milk), BP (Banana Pulp). Each concentration of hormone had 10 replicates, and the experiment was conducted 3 times.
6 SHADANG et al: PROTOCORM DEVELOPMENT OF H. PARISHII IN VITRO 261 promoted better response (Fig. 7). The survival rate of these seedlings after in vivo transplantation in the potting mixture (Brick:Charcoal:Tree fern::1:1:1) was 70%. The method thus suggested in this paper can be used for large-scale propagation of this orchid species. Acknowledgement Thanks are due to the Department of Biotechnology, Govt. of India, New Delhi for the financial assistance. We also thank the Director, State Forest Research Institute, Itanagar for facilities. References 1 Singh D K, Wadhwa M & Singh K P, A conspectus of orchids of Mizoram, their studies and conservation, J Orchid Soc India, 4 (1990) Shushan S, Developmental anatomy of an orchid Cattleya x Trimos, in The orchids: A scientific survey, edited by C L Withner (Ronald Press, New York) Withner C L, Orchids physiology, in The orchids: A scientific survey, edited by C L Withner (Ronald Press, New York) Rao A N, Tissue culture in orchid industry, in Applied and fundamental aspects of plant cell tissue and organ culture, edited by J Reinert & Y P S Bajaj (Springer Verlag, Berlin) 1977, Hazarika R B & Sarma C M, In vitro germination and regeneration of Dendrobium transparens Lindl, J Orchid Soc India, 9 (1995) Knudson L, Non-symbiotic germination of orchid seeds, Bot Gaz, 73 (1922) Hegarty C P, Observation on the germination of orchid seed, Am Orchid Soc Bull, 28 (1955) Arditii J, Factor affecting the germination of orchids seeds, Bot Rev, 33 (1967) Knudson L, A new nutrient solution for the germination of orchid seeds, Am Orchid Soc Bull, 15 (1946) Murashige T & Skoog F, A revised medium for rapid growth and bioassay with tobacco tissue culture, Physiol Plant, 15 (1962) Vacin E F & Went F W, Some ph changes in nutrient solutions, Bot Gaz, 110 (1949) Ichihashi S & Yamashita M, Studies on the media for orchids seed germination, J JPN Soc Hort Sci, 45 (1977) Kano K, Studies on the media for orchid seed germination, Mem Fac Agric Kagawa Univ, 20 (1965) Methews V H & Rao P S, In vitro culture of Vanda hybrid (Vanda TMA x Vanda Miss Joaquim): II Studies on seedling explants, Proc Indian Natl Sci Acad, 51 (1980) Kumaria S, Chrungoo N & Tandon P, Activities of some oxidative enzymes in axenic cultures of protocorms of Cymbidium giganteum Wall as influenced by different growth regulators, J Orchid Soc India, 4 (1990) Sharma S K & Tandon P, Influence of growth regulators on asymbiotic germination and early seedling development of Coelogyne punctulata Lindl, in Biology, conservation and culture of orchids, edited by S P Vij (Affiliated East West Press, New Delhi) 1986, Intuwong S & Sagawa Y, Clonal propagation of sarcanthine orchids by aseptic culture of inflorescences, Am Orchid Soc Bull, 46 (1973)
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