Asymbiotic Germination of Immature Seeds in an Ornamentally Important Fox-tail Orchid, Aerides multiflora Roxb.

Transcription

1 VEGETOS Vol. 28 (4) : (2015) / Asymbiotic Germination of Immature Seeds in an Ornamentally Important Fox-tail Orchid, Aerides multiflora Roxb. Jagdeep Verma*, Sayeeda K. Bhatti 1 and Jaspreet K. Sembi 2 Received: / Revised: / Accepted: / Published online: This article is published in open access at Abstract Present paper deals with asymbiotic germination potential of immature seeds in Aerides multiflora Roxb. for its rapid mass propagation in vitro. Seeds were inoculated on Potato Dextrose Agar (PDA) medium with and without growth adjuncts (CH, Casein Hydrolysate; P, Peptone, YE, Yeast Extract; each 0.1%) and activated charcoal (AC; 0.2%). Seed germination initiated after 1.20±0.42 weeks (wks) on CH supplemented medium. The percent germination response showed significant differences in majority of combinations used; it was lowest (48.00±2.16) in basal medium and highest (99.50±0.85) in medium supplemented with CH and AC. Protocorm formation started after 4.15±0.48 wks of culturing on CH and AC containing medium. First leaf and first root differentiated after 7.30±0.82 and 14.00±0.67 wks of culturing respectively on medium supplemented with P and AC. Seedlings with 3-4 leaves and 2-3 roots were obtained after 18.30±0.67 wks. The medium supplemented with CH and AC proved to be the best for seed germination, protocorm formation and multiplication, and that containing P and AC for early leaf and root differentiation, and seedling development. The seedlings were subjected to hardening and 72.56±3.18% of them survived under greenhouse conditions. Keywords: Asymbiotic germination, Aerides multiflora, immature seeds, orchid, protocorm. Introduction Orchid seeds are the smallest in the plant kingdom. They are produced in very large numbers but their embryos lack access to nutrient reserves. In nature, their germination is exceptionally low (less than 1%) because of their total dependence upon appropriate fungal stimulus (mycotrophy) for this. However, ever since Knudson (1922) suggested that the fungal requirement of orchid seeds can be bypassed by supplying an appropriate nutrition in vitro, the technique of asymbiotic seed germination has been successfully employed in a variety of orchid species and hybrids (Arditti et al. 1982, Lin et al. 1994, Pyati and Murthy 1995, Faletra et al. 1997, Kondo et al. 1997, Gangaparsad et al. 1999, Jamir et al. 2002, Pant et al. 2002, 2011, Dutra et al. 2008, Pathak et al. 2011, Roy et al. 2011, Piri et al. 2013, Sathiyadash et al. 2013, Zhang et al. 2013, Nenekar et al. 2014). The application of tissue culture techniques which help in achieving round the year rapid mass propagation of desired genotypes have opened up new possibilities of their conservation as well as commercialization. Aerides Lour. is a beautiful genus of foxtail orchids known for attractive flowers and foliage of its species. Many of its species and hybrids have been propagated in vitro through seed and organ culture (Murthy and Pyati 2001, Sheelavanthmath et al. 2005, Das et al. 2008, Basu et al. 2010, Chalaparmal et al. 2011, Sembi et al. 2011, Parab and Krishnan 2012, Srivastava et al. 2013, Hongthongkham and Bunnag, 2014). Aerides multiflora Roxb. is one of its species with immense potential to serve as a putative parent in hybridization programmes for producing elite quality genotypes (Vij et al. 2013). However, the size and frequency of its natural populations are on gradual decline because of habitat destruction, and unscrupulous collections for commercial and scientific purposes. Therefore, the asymbiotic germination potential was presently tested for rapid in vitro mass propagation of Aerides multiflora Roxb. by using immature seeds procured from unripe green fruits. Materials and Methods Unripe green fruits of Aerides multiflora were collected 12 weeks after pollination (WAP) Department of Botany, Shoolini Institute of Life Sciences & Business Management, Solan , Himachal Pradesh, India, 1 Department of Botany, Shoolini University of Biotechnology & Management Sciences, Solan , Himachal Pradesh, India 2 Department of Botany, Panjab University, Chandigarh , Chandigarh, India *Corresponding author verma.jd@gmail.com 162

2 Asymbiotic Germination of Immature Seeds in Fox-tail Orchid, Fig 1 (A-I). Morphogenetic changes during in vitro seed to seedling development in Aerides multiflora. A, immature seeds just inoculated on basal PDA medium. B, Swelled embryo and broken seed coat. C-D, Protocorms (PDA+CH). E, Differentiation of first leaf (PDA+CH). F, Differentiation of first root (PDA+P). G, Seedlings with 2-3 leaves and 1-2 roots (PDA+P+AC). H, Seedlings in hardening process. I, Hardened seedlings in clay pots. from Tihra town (31º46 N longitude, 76º40 E latitude, 1057 m altitude) of Sarkaghat subdivision (district Mandi, Himachal Pradesh, Northwest Himalaya). The fruits were scrubbed with liquid detergent (teepol; 1%) with a soft brush and washed under running tap water for min. Further sterilization was done under aseptic conditions inside the laminar air flow cabinet. Fruits were surface disinfected by dipping in mercuric chloride (HgCl 2 ; 0.2% with 2-3 drops teepol as a wetting agent) for 6-7 min followed by washing with sterilized distilled water for 4-5 times to remove the traces of mercuric chloride. Finally they were flamed after dipping in 70% ethyl alcohol. The sterilized fruits were then split longitudinally with a sterilized surgical blade to scoop out the immature seeds. The powdery seeds were inoculated on the surface of Potato Dextrose Agar (PDA) medium with and without three growth adjuncts (CH, Casein Hydrolysate; P, Peptone, YE, Yeast Extract; each 0.1%). Effect of activated charcoal (AC; 0.2%) was also studied separately in all combinations. The cultures were incubated at 25±2 o C temperature and 12 hr photoperiod. They were observed at 1 wk time interval for recording various morphogenetic changes (swelling of embryos, emergence of spherules, protocorm formation, leaf and root 163

3 Table 1. In vitro germination response of Aerides multiflora immature seeds on PDA medium with and without growth adjuncts Growth adjunct (s) Germination Response (%) Initiation of germination (wks) Jagdeep Verma et al. Time taken (wks) for the development of Spherule Protocorm 1 st leaf 1 st root 1 st seedling ±2.16 a 1.40±0.52 a 3.40±0.70 a 5.30±0.48 a 12.20±0.42 e 17.20±0.42 c 22.90±0.57 e AC 75.80±2.15 c 1.50±0.53 a 3.20±0.79 a 5.20±0.63 a 10.50±0.71 c 16.90±0.57 c 20.60±0.70 b P 60.20±2.20 b 1.70±0.48 a 4.50±0.71 b 5.20±0.42 b 11.40±0.70 de 17.20±0.63 c 22.20±0.79 d P+AC 79.90±1.37 d 1.60±0.79 a 3.40±0.52 a 5.10±0.57 b 7.30±0.82 a 14.00±0.67 a 18.30±0.67 a YE 59.80±2.10 b 1.20±0.52 a 3.45±0.55 a 4.50±0.53 b 10.40±0.52 c 17.40±0.70 c 21.30±0.48 c YE+AC 80.00±1.15 d 1.50±0.71 a 3.20±0.67 a 4.25±0.70 b 8.20±0.79 b 15.00±0.82 b 19.50±0.85 b CH 75.00±1.41 c 1.40±0.52 a 3.40±1.13 a 4.20±0.79 a 11.00±0.82 cd 17.90±0.88 c 21.60±0.52 cd CH+AC 99.50±0.85 e 1.20±0.42 a 3.20±0.52 a 4.15±0.48 a 8.20±0.79 b 15.20±0.63 b 19.20±0.42 b Data are shown as mean ± standard deviation. Values in a column with the same superscripts are not significantly different at p e differentiation, seedling development). Subculturing was done at 6 wks interval or when required. Seedlings obtained were hardened in vitro by sequential removal of growth adjuncts and dextrose from the nutrient mix at 2 wks intervals. These were then removed from flasks, washed thoroughly with luke warm water to free them of agar and potted in 6 cm diameter clay pots filled with potting mixture (Charcoal, Brick pieces, Bark; 1:1:1) topped with moss. The potted plants were mist irrigated and hardened for 4 wks in humidity chamber. These were then shifted to the greenhouse where they were mist irrigated and covered with porous polybags for initial 2 wks. Emergence of new roots and leaves was taken as an indication for successful acclimatization of the seedlings. The data for each combination were collected in 10 replicates and the values expressed in Table 1 are means of these. Results were analyzed using a completely random design. These were subjected to one-way analysis of variance and post hoc tests to detect the significant differences (p 0.05) in various nutritional combinations using SPSS 17.0 (SPSS Inc., USA). Results and Discussion The immature seeds of Aerides multiflora (Fig. 1A) successfully germinated asymbiotically on basal PDA medium as well as cultures supplemented with various growth adjuncts in vitro. Various morphogenetic changes observed from seed to seedling development have been summarised in Table 1. These are now presented in detail and discussed in the light of relevant literature. Germination of immature seeds initiated after 1.20±0.42 wks of culturing. The swelling of embryo and breaking of seed coat (Fig. 1B) was the first sign of successful germination process. The ability of orchid seeds to germinate prior to reaching maturity is well documented in literature (Withner 1943, Pathak et al. 2001, Piri et al. 2013), and such a culture of immature seeds is often referred to as embryo culture, green-pod culture, green-fruit culture or green-capsule culture. This technique is very useful as it follows an easy sterilization procedure and helps in reducing the time lapse between flower pollination and seed sowing (Rao and Avdhani 1964, Pyati and Murthy 1995, Alouffa et al. 1998) Moreover, as seeds are saved from direct exposure to sterilizing agents, the germination frequency has also been found to be better (Tsuchiya 1954, Sagawa 1963). Immature seeds exhibit better germination because of their distended testa cells, metabolically awakened embryos and absence of dormancy factors (Linden 1980, Arditti et al. 1981, Yam and Weatherhead 1988). Yasugi (1983) successfully achieved germination even immediately after fertilization in Dortis pulcherrima. During present study, differences were observed in time taken for initiation of germination response in various nutritional combinations (1.20± ±0.48 wks) but these were not significant. This suggests that orchid seeds can germinate in a wider variety of nutritional regimes but the germination frequency varies with respect to the actual chemical stimulus present in the culture medium. Presently, basal medium invoked germination response in 48.00±2.16% seeds and various growth adjuncts helped improving germination percentage. It was maximum (99.50±0.85) in medium containing CH and AC. The results showed that AC not only improved germination percentage but also 164

4 Asymbiotic Germination of Immature Seeds in Fox-tail Orchid, proved effective in reducing time period between seed sowing and seedling development in various nutritional combinations (Table 1). Activated charcoal has widely been used to darken the medium used for seed and organ (leaf/ root) culture. According to Van Waes (1987), the addition of AC to the agar substrate improves seedling survival, which tends to exude phenolics. The seedlings grown in AC supplemented medium are reported to have more fresh weight because of its better light absorbing ability and enhanced availability of energy quantum per unit of plant material (Werckmeister 1970). The swelled embryos (spherules) became green with the development of chlorophyll and acquired polarity to develop into pear shaped structures known as protocorms (Fig. 1C). The medium supplemented with CH and AC proved better for early protocorm development as well as their multiplication (Fig. 1D). Morphologically, a protocorm is considered as a state between an undifferentiated tissue and shoot primordium (Kanase et al. 1995). Functionally, it is believed to behave like a cotyledon for supplying nutrition to developing embryo as well as its subsequent growth into seedling (Lee and Lin 1987, Batygina et al. 2003, Stewart and Kane 2006, Johnson et al. 2007). The protocorm differentiation started first of all in medium containing P and AC; they developed first leaf (Fig. 1E) and root (Fig. 1F) after 7.30±0.82 and 14.00±0.67 wks respectively. Same combination resulted in early seedling development. Seedlings with 2-3 leaves and 1-2 roots (Figs 1G-H) were obtained in 18.30±0.67 wks old cultures. Several complex organic growth supplements such as Peptone (P), Yeast Extract (YE), Casein Hydrolysate (CH), Coconut Water (CW), Urea (U), etc. are routinely employed to enrich the culture media for orchid seed germination and subsequent seedling development. The growth promotory effect of these additives has been attributed to their organic nitrogenous compounds, minor elements and vitamin constituents (Raghavan, 1976). Presently the effect of P, YE and CH was assessed during germination and seedling development, and all of them proved beneficial during various morphogenetic stages. Ever since Lami (1927) observed that P improved seed germination in Vanda and Phalaenopsis, it has added in culture media used for culturing of many other orchid taxa as well. Its growth promotory nature has been attributed to presence of various peptides, amino acids, amides, vitamins and some other unknown contents (Pathak et al. 2001). Peptone has proved beneficial for early organogenesis of protocorms during present study. Literature studies also reveal that P favour germination, protocorm multiplication, differentiation and seedling growth in various orchids including Acampe praemorsa, Aerides multiflora, Cymbidium macrorhizon, Gastrochilus calceolaris, Rhynchostylis retusa and Vanda testacea (Vij et al. 1981, Oliva and Ariditti 1984, Pathak et al. 2001, 2011). Kaur and Bhutani (2012, 2014) reported the effectiveness of P in PLB multiplication and early plantlet development. The importance of YE as a source of reduced nitrogen is well documented in literature. It also proved beneficial in improving germination frequency in presently conducted experiments. Ichihashi (1990), Pathak et al. (2011) and Piri et al. (2013) reported usefulness of YE in seed germination and protocorm development in different orchid species. However, its inhibitory effects have also been suggested by some workers in a few taxa (Weatherhead et al. 1986, Devi et al. 1990, Pathak et al. 2001). In our study, CH proved to be the best for seed germination, and protocorm development and multiplication. It is a complex mixture of casein, amino acids and some other relatively simple substances, and its ability to improve orchid seed germination has been demonstrated in many studies (Harvis 1972, Vij et al. 1981, Lakshamanan et al. 1995, Ng and Saleh 2011, Srivastava et al. 2013). Its composition, which varies with the extent of hydrolysis of nitrogen substances in the casein, seems to account for its variable effects during orchid germination (Pathak et al. 2001). Hardening (acclimatization) is of important concern during lab to land transfer of micropropagated plants as it helps reducing their mortality rate (Pospisilova et al. 1999). Therefore seedlings with 2-3 leaves and 1-2 roots (Figs. 1G-H) were subjected to successive in vitro and ex vitro hardening. They were hardened in vitro by sequential removal of growth adjuncts and dextrose at 2 wks interval for initial 6 wks. These were then removed from flasks, washed thoroughly with luke warm water to free them of agar and potted in 6 cm diameter clay pots filled with potting mixture (Charcoal, Brick pieces, Bark; 1:1:1) topped with moss (Fig. 1I). The potted plants were mist irrigated and hardened for 4 wks in humidity chamber. This is important because plantlets produced in vitro under high humidity conditions are much susceptible to desiccation when transferred to greenhouse (Wetzstein and Sommer 1983). The seedlings were then shifted to the greenhouse where they were again mist irrigated and covered with porous polybags for initial 2 wks. Emergence of new roots and leaves was taken as an indication for successful acclimatization of the seedlings. 165

5 Jagdeep Verma et al. After 8 wks of deflasking, 72.56±3.18% seedlings survived under greenhouse conditions. References Alloufa M A I, Araujo K C L and Dutra M D (1998). Clonal propagation of Oncidium cebolata using tissue culture. Proc 9 th Intl Conf on Plant Tissue and Organ Cult (Abst.), Jerusalem, Israel, Pp. 41. Arditti J, Michaud J D and Oliva A P (1981). Seed germination of North American orchids. Native California and related species of Calypso, Epipactis, Goodyera, Piperia, Platanthera. Bot Gaz : ( h t t p : / / dx.doi.org/ /337245). Gangaprasad A, Decruse S W, Seeni S and Menon V S (1999). Micropropagation and Restoration of the endangered Malabar daffodil orchid Ipsea malabarica. Lindleyana 14: Harvais G (1972). The development and growth requirements of Dactylorhiza purpurella in asymbiotic cultures. Can J Bot 52: ( Hongthongkham J and Bunnag S (2014). In vitro propagation and cryopreservation of Aerides odorata Lour. (Orchidaceae). Pak J Biol Sci 17: Arditti J, Clements M A, Fast G, Hadley G, Nishimura G and Ernst R (1982). Orchid seed germination and seedling culture- A manual. In: Orchid Biology, Reviews and Perspectives, J Arditti (ed.). Cornell University Press, Ithaca, New York, Basu A K, Samanta S K and Sing N K (2010). Response pattern of three orchid species for asymbiotic germination, seedling growth and hardening. Proc 6 th Intl Plant Tissue Cult and Biotech Conf, Dhaka, Bangladesh, Batygina B T, Bragina E A and Vasilyeva V E (2003). The reproductive system and germination in orchids. Acta Biologica Cracoviensia Series Bot 45: Chalaparmal S, Thohirah L A, Fadelah A A and Abdullah N A P (2011). Hybridization of several Aerides species and in vitro germination of its hybrid. Afr J Biotechnol 10: Das A K, Das J, Gogoi H K and Srivastava R B (2008). Mass propagation of orchids through in vitro seed culture technology. J Cell Tissue Res 8: Devi J, Nath M, Devi M, Deka P C (1990). Effect of different media on germination and growth of some North East Indian species of Dendrobium. J Orchid Soc India 4: Ichihashi S (1990). Effect of light on root formation of Blettila striata seedlings. Lindleyana 5: Jamir C, Devi J and Deka P C (2002). In vitro propagation of Cymbidium iridioides and Cymbidium lowianumn. J Orchid Soc India 16: Johnson T R, Stewart S L, Dutra D, Kane M E, Richardson L (2007). Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae) preliminary evidence for the symbiotic culture advantage. Plant Cell Tissue Organ Cult 90: ( -z). Kanase A and Takano T (1995). Comparative morphology on the histo and organogenesis in protocorm like body of four species in orchids. Proc Nagoya Intl orchid show, S Ichihashi and H Inoue (eds). Nagoya, Japan, pp Kaur S and Bhutani K K (2012). Organic growth supplement stimulants for in vitro multiplication of Cymbidium pendulum (Roxb.) Sw. Hort Sci (Prague) 39: Kaur S and Bhutani K K (2014). In vitro conservation and asymbiotic propagation of Coelogyne flaccida (Lindl.): A threatened orchid. Plant Biosystems 148: ( dx.doi.org/ / ). Dutra D, Johnson T R, Kauth P J, Stewart S L, Kane M E and Richardson L (2008). Asymbiotic seed germination, in vitro seedling development and greenhouse acclimatization of the threatened terrestrial, orchid Bletia purpurea. Plant Cell Tiss Organ Cult 94: ( dx.doi.org/ /s ). Faletra P, Dovholuk A, King T and Sokoloski K (1997). Saving Cypripedium reginae. Orchids 66: Knudson L (1922). Non-symbiotic germination of orchid seeds. Bot Gaz 73: ( dx.doi.org/ /332956). Kondo K, Tanaka C, Shimada T and Dhtani M (1997). Developmental morphology of seeds and micropropagation of Orchis aristata (Orchidaceae) in axenic culture. Ann Tsuk Bot Gard 16: Lakshmanan P, Loh C S and Goh C J (1995). An in 166

6 Asymbiotic Germination of Immature Seeds in Fox-tail Orchid, vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture. Plant Cell Rep 14: ( Lami R (1927). Influence D,une peptone sur la germination de quel-ques vandees. Compt Rend Acad Sci Paris 184: 151. Lee N and Lin M G (1987). Controlling the flowering of Phalaenopsis. Proc symposium on forcing culture of Hortic crops, LR Chang (ed.). Taichung District Agric Improv Sta, Taichung, Taiwan, China, Lin Y J, Chen C C, Yeh F T, Chiu N Y and Tsay H S (1994). Tissue culture of Bletilla formosana. The influence of seed maturity and pre treatement on seed germination and seedling development. J Agri Res China 43: Linden B (1980). Aseptic germination of seeds of northern terrestrial orchids. Ann Bot Fennici 17: Murthy H N and Pyati A N (2001). Micropropagation of Aerides maculosum Lindl. (Orchidaceae). In Vitro Cell. Dev. Biol. - Plant 37: ( Nenekar V, Shriram V, Kumar V and Kishor P B K (2014). Asymbiotic in vitro seed germination and seed development of Eulophia nuda Lindl., an endangered medicinal orchid. Proc National Acad of Sci, India, 84: ( dx.doi.org/ /s ). Ng, C and Saleh, N. M. (2011). In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies. Plant Cell Tissue Organ Cult 105: ( dx.doi.org/ /s ). Oliva A P and Ariditti J (1984). Seed germination of North American orchids, II native Calfornia and related species of Aplectrum, Cypripedium and Spiranthes. Bot Gaz 145: ( dx.doi.org/ /337484). Pant B, Chaudhary R P, Subedi A and Shakya L R (2002). Nepalese Orchids. Proc of the Intl Orchid wave in Simanami, An Intl orchid conf on conservation and propagation of endangered wild orchids of the world, Simanami, Japan, Pant B, Shrestha S and Pradhan S (2011). In vitro seed germination and seed development of Phaius Tancarvilleae Blume. Sci World 9: Parab G V and Krishnan S (2012). Rapid in vitro mass multiplication of orchids Aerides maculosa Lindl. and Rhynchostylis retusa (L.) BI. From immature seeds. Indian J Biotech 11: Pathak P, Mahant K C, Gupta A (2001). In vitro propagation as an aid to conservation and commercialization of Indian orchids: Seed culture. P Pathak, R N Sehgal, N Shekhar, M Sharma, A Sood (eds.). Orchids: Science and Commerce, Bishen Singh Mahendra Pal Singh, Dehradun, India, pp Pathak P, Piri H, Vij S P, Mahant K C and Chauhan S (2011). In vitro propagation and mass scale multiplication of a medicinally important and critically endangered epiphytic orchid, Gastrochilus calceolaris (Buch.-Ham ex J.E.Smith) D. Don using immature seeds. Ind J Exp Biol 49: Piri H, Promila P and Bhanwra R K (2013). Asymbiotic germination of immature embryos of a medicinally important epiphytic orchid Acampe papillosa (Lindl.) Lindl. Afric J Biotechnol 12: Pospisilova J, Ticha I, Kadlecek P, Haisel D and Plzakova S (1999). Acclimatization of micropropagated plants to ex vitro conditions. Biol Plant 42: ( A: ). Pyati A N and Murthy H N (1995). In vitro seed germination and seedling development of Dendrobium ovata (Willd.) Krunzl. J Orchid Soc India 9: Raghavan V (1976). Experimental embryogenesis in Vascular Plants. Academic Press, New York, pp Rao A N and Avdhani P N (1964). Some aspects of in vitro Culture of Vanda seeds. Proc 4 th world Orchid Conf, Singapore, Malaysia, pp Roy A R, Patel R S, Patel V V, Sanjeev S and Deka B C (2011). Asymbiotic seed germination, mass propagation and seedling development of Vanda coerulea Griff ex.lindl. (Blue Vanda): An in vitro protocol for an endangered orchid. Sci Horticult 128: ( dx.doi.org/ /j.scienta ). Sagawa Y (1963). Green pod cultures. Florida Orchidist 6: Sathiyadash K, Muthukumar T, Murugan S B, Sathishkumar R, Uma1 E, Jaison S and Priyadharsini P (2013). In vitro asymbiotic seed germination, mycorrhization and seedling development 167

7 Jagdeep Verma et al. of Acampae praemorsa (Roxb.) a common south Indian orchid. Asian Pacific J Rep 2: Sembi J K, Verma J, Pathak P and Vij S P (2011). Regeneration competence of Aerides multiflora root segments: a study in vitro. J Orchid Soc India 25: 5-8. Sheelavanthmath S S, Murthy H N, Hema B P, Hahn E J and Paek Y (2005). High frequency of protocorm like bodies (PLBs) induction and plant regeneration from protocorm and leaf sections of Aerides crispum. Sci Horticult 106: (h ttp: // dx.d oi.o rg / / j.scienta ). Srivastava D, Gayatri M C and Sarangi S (2013). In vitro seed germination as an aid to conserve Aerides maculosum Lindl., an endemic and endangered orchid. Int J Pharm Bio Sci 4: Stewart S L and Kane M E (2006). Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue Organ Cult 86: ( dx.doi.org/ /s y). Tsuchiya I (1954). Possibility of germination of orchid seeds from immature fruits. Hawaii Orchid J 4: Van Waes J (1987). Effect of activated charcoal on in vitro propagation of Western European Orchids. Acta Hort 212: Vij S P, Sood A and Plaha K K (1981). In vitro seed germination of some epiphytic orchids. In: Contemporary trends in plant sciences, SC Verma (ed.). Kalyani Publishers, New Delhi, pp Vij S P, Verma J and Sathish Kumar C (2013). Orchids of Himachal Pradesh. Bishen Singh Mahendra Pal Singh, Dehradun. Weatherhead M A, Zee S Y and Bairelto G (1986).Some observation on early stages of development of Eulophia yushuiana. Mem Hong Kong Natl Soc 7: Werckmeister P (1970). Die steuerung von Vermehrung (Proliferation) und Wachstum in der Merstemkultur von Cymbidium und die Verwendung eines Kohlen-Nahrmediums. Die Orchidee 21: Wetzstein, H Y and Sommer H E (1983). Scanning electron microscopy of in vitro cultured Liquidambar styraciflua plantlets during acclimatization. J Amer Soc Hort Sci 108: Withner C L (1943). Ovule culture: A new method for starting orchid seedlings. Amer Orchid Soc Bull 11: Yam T W and Weatherhead M A (1988). Germination and seedling development of some Hongkong orchids. Lindleyana 3: Yasugi S (1983). Ovule and embryo development in Dortis pulcherrima (Orchidaceae). Amer J Bot 70: Zhang Y, Lee Y, Deng L and Zhao S (2013). Asymbiotic germination of immature seeds and the seedling development of Cypripedium macranthos Sw., an endangered lady's slipper orchid. Sci Horticult 164: