OF CYMBIDIUM IRIDIOIDES D. DON. AND THE ROLE OF DIFFERENT FACTORS Aolemla Pongener and Chitta Ranjan Deb* Department of, Nagaland University, Headquarters: Lumami, Mokokchung 798 601 (University Branch Office), Nagaland, India *Correspondent Author: debchitta@rediffmail.com / dedeb@bsnl.in ABSTRACT Immature embryos of Cymbidium iridioides were successfully cultured on five different media with various supplements and maintained in three different light conditions. Within three week of culture, embryos formed nodular structures. Cultures under full light condition formed green protocorm-like bodies while, other light conditions failed to support optimum germination. Immature embryos of 10 months after pollination registered optimum germination (~95%) on MS medium containing sucrose (2%), CW (5%) and NAA + BA (3 +3 µm in combination). The germinated embryos formed PLBs and differentiated within two passages on germination media. The advanced protocorm-like bodies were converted into rooted plantlets on MS medium containing sucrose (3%), NAA (3 µm) and BA (6 µm) where as many as 20 shoot buds/plbs developed. The rooted plantlets were hardened on ½MS medium with sucrose (1%) and charcoal, brick pieces and chopped mosses (at 1:1 ratio) as substratum for 4-6 wk before transferring to community potting mix. About 80% survival rate was registered after two months of potting. KEYWORDS Cymbidium iridioides, effect of organic carbon sources, field establishment of regenerates, immature embryo culture, mass multiplication. INTRODUCTION Amongst the flowering plants, orchids represent the most evolved and one of the largest groups. The flowers exhibit a highly colorful, attractive and long shelf life with varied shapes and 1 sizes and have a great value in floriculture industry as cut flower and potted plants. The regeneration and multiplication of orchids through seeds in nature is limited due to suppressed endosperm and requirements of fungal stimulus 1-3. In vitro culture of
OF CYMBIDIUM IRIDIOIDES D. DON. AND THE ROLE OF DIFFERENT FACTORS orchid seeds/embryos plays an important role in commercial production of orchids and conservation programme. Since Knudson 4 showed the possibility of in vitro germination of orchid seeds bypassing the fungal requirements, the technique has been accepted as an important tool for propagating orchids and has been applied to several commercially viable and or threatened orchid s 2-3,5-10. However, in vitro seed/embryo germination of orchids is greatly influenced by several factors, like seed age, nutrient media, organic carbon sources, different adjuncts and quality and quantity of plant growth regulators (PGRs) 2-3,5-8,11. However none of these basal media with different adjuncts and supplements fulfills the requirements of the entire orchidaceous group. Cymbidium iridioides is an epiphytic/lithophytic orchid that produces attractive flowers during October-November which are 7-8 cm across, brown with red streaks (Figure 1a) and has immense horticultural importance. In this communication, we describe the interplay between different factors on successful in vitro culture of immature embryo, plant regeneration and mass multiplication of C. iridioides. MATERIALS AND METHODS Explants source and sterilization of the explants The green pods of different developmental stages (6 to 16 months after pollination [MAP]) were harvested at two months interval and were used for initiation of culture. The green pods were thoroughly scrubbed with liquid laboratory detergent (1:100 ratio, v/v) before washing under running tap water. The green pods were sterilized by dipping in 0.5 % (w/v) aqueous solution of HgCl 2 for 5 min and subsequently rinsed repeatedly 4 or 5 times with sterilized distilled water. Finally, the green pods were flamed off before scooping out the embryos in a laminar flow cabinet. The immature embryos/seeds of various developmental stages were scoped out from the sterilized pods and cultured on different media. Culture media and initiation of culture Five different basal media were tested for the present study viz. MS 12, Mitra et al 13 and Knudson C 14, B 15 5 and SH 16. All the media were fortified with different levels (0-4%) (w/v) of organic carbon sources like dextrose, glucose and sucrose, coconut water (CW) (0-20%) (v/v). For seed germination all the media were further supplemented with different quality and quantity of plant growth regulators (PGRs) like α- naphthaleneacetic acid (NAA) and N 6 benzyl adenine (BA) (0-12 µm) either singly or in combination. Difco-bacto agar (0.8%) was used as gelling agent and the ph of the media was adjusted to 5.6 using 0.1 N NaOH and 0.1 N HCl. About 15 ml of medium was dispensed in each test tube (size: 25x150 mm) and plugged before autoclaving at 1.05 kg cm -2 pressure and at 121 o C for 20 min. The immature embryos at different MAP scooped out from the sterilized green pods and inoculated on different culture media. All the cultures were maintained in different light conditions (dark, diffused light [20 µmol cm -2 s -1 ] and full light [40 µmol cm -2 s -1 ) under cool white fluorescent light with 12 h photoperiod at 25±2 o C. The cultures were monitored regularly and the data were scored at one wk intervals. The seeds were allowed to germinate and differentiate into 2
OF CYMBIDIUM IRIDIOIDES D. DON. AND THE ROLE OF DIFFERENT FACTORS 3
OF CYMBIDIUM IRIDIOIDES D. DON. AND THE ROLE OF DIFFERENT FACTORS protocorm like bodies (PLBs) on the same initiation medium. The germinated embryos and or PLBs were maintained for another two passages on optimum culture conditions for further differentiation. Protocorm like bodies were sub-cultured at four wk interval until mentioned otherwise. For each treatment 20 culture vials were used and all the experiments were repeated at least thrice. The experimental design was completely randomized. Differentiation, plantlet regeneration and mass multiplication The PLBs developed from the cultured immature embryos were maintained on the optimum germination medium for further differentiation. The PLBs with the first set of leaflets (advanced stage PLBs) were separated from the germination medium and cultured on regeneration media. For regeneration of plantlet and mass multiplication different media like MS, Mitra et al, and Knudson C were tested and all the media were enriched with 0-4% sucrose (w/v), CW (0-20%) (v/v) and different PGRs like NAA, IAA, BA, Kn, TDZ either singly or in combination. The resulted multiple shoot buds/plantlets were separated from the regeneration and cultured on fresh regeneration medium for further multiplication. The well rooted plantlets were maintained for 2-3 passages on the regeneration medium before they were transferred to hardening condition. Hardening of plantlets and transferring to community potting mix About 6-7 cm long rooted plantlets (with 2-3 roots) were taken out from the regeneration medium and washed off the traces of agar with a soft brush and transferred on culture vials containing ½MS medium supplemented with 1% sucrose freed from any PGRs. In the culture vials charcoal pieces, brick pieces and chopped mosses (at 1:1 ratio) was used as substratum. The cultures were maintained for 4-6 wk in normal laboratory condition before transferring to community potting mix (CPM) containing sand: brick pieces: coconut husk: charcoal pieces: decayed wood at 1:1 ratio with a moss topping and covered with holed transparent poly bag. The potted plants were watered weekly. The potted plants were exposed to normal day light for about 1 h in a day for initial one wk and subsequently increased the exposure period by 2 h from the second week and finally after one month the plantlets were left in the normal full day light condition. RESULTS Initiation of culture The cultured immature embryos started swelling within three wk of culture as the first sign of germination. After three wk of culture in the dark, the first sign of germination was observed as yellowish nodular swelling of the seeds (Fig 1 b) while, the cultures in diffused light and full light conditions delayed initial response. The embryos/seeds cultured in the dark condition formed hairy like structure followed by PLBs formation when they are transferred to full laboratory light condition. Though the light cultured seeds delayed germination but subsequently healthy PLBs formation resulted. About 95% germination was registered from the full light grown culture followed by diffused light (90%) and dark condition (80%) (Figure 2). 4
100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 Dark Time taken for nodulation (days) Time taken for PLBs formation (days) % germination Diffused light (20 µmol m -2 s -1 ) Figure-2 Full light (40 µmol m -2 s -1 ) Figure 2: Effects of different light conditions on in vitro embryo culture of C. iridioides. The age of the green pod/immature embryos was found to be the key factor for successful asymbiotic germination of seeds. Seeds age up to 8 MAP delayed germination while, the embryos from the green pod age >14 MAP did not germinate at all. The optimum germination was recorded with green pod age of 10 MAP (Table 1). Amongst the different nutrient media studied during the present investigation, not all the media found to be equally suitable for the successful germination of embryos. Of the five media tested in the present study, optimum germination was occurred on MS medium (95%). Besides MS medium, other media supported moderate germination but in most of the cases either delayed differentiation or failed to differentiate into either PLBs or plantlets (Table 2). 5
Table 1 Effect of green pod age on in vitro germination of immature embryos of C. iridioides * Green pod Days taken for Germination Types of response age (MAP) Nodulation PLBs formation 1 st leaflets rate (%) (±SE) # 6 - - - - No response 8 35 65-35 (±1.5) Slight nodular swelling of embryos but fewer PLBs formed 10 20 58 74 95 (±2.0) Most of the germinated embryos converted into healthy PLBs 12 32 67 88 85 (±2.5) Nodular swelling followed by PLBs formation 14 48 76 90 55 (±2.5) Nodular swelling followed by poorer PLBs formation 16 50 - - - Only swelling but no PLBs formation * On MS medium containing 2% sucrose, 5% CW, 3µM NAA + 3µM BA in combination. # Standard error. Data represents the mean of five replicates. Table 2 Effect of different basal media on in vitro germination of C. iridioides embryos Basal media Germination % germination Type of response * Time (days) (±SE)** Gamborg (B 5 ) 63 50 (±2.0) Few PLBs formed but failed to differentiate into plantlets Knudson C 65 45 (±1.5) As above Mitra et al. 70 65 (±2.5) Moderate germination and healthy and green PLBs formed but regeneration delayed MS 58 95 (±2.0) Most of the germinated embryos converted into healthy PLBs with white hairy structures SH 75 50 (±2.5) Germinated embryos formed PLBs but failed to differentiate and degenerated * Media containing 2% sucrose, 5% CW and NAA + BA (3 +3 µm respectively in combination). ** Standard error. Data represents the mean of five replicates. 6
Apart from green pod age and nutrient media, incorporation of other factors, like quality and quantity of organic carbon sources, CW and PGRs exhibited pronounced effect on successful in vitro germination of immature embryos and subsequent differentiation of plants. Amongst the different organic carbon sources used in the present study, dextrose and glucose did not support healthy germination and delayed germination. Compared to dextrose and glucose, sucrose rich media supported better germination and optimum germination was registered on medium containing 2.0% sucrose while, at both lower and higher concentration of sucrose germination was delayed (Table 3). Table 3 Effect of different organic carbon sources on in vitro embryo culture on C. iridioides * Organic carbon Conc. (%) Days taken to Type of response sourses germinate - 0 98 Few PLBs formed Dextrose 1 71 Very few PLBs formed 2 70 As above 3 65 Very few healthy PLBs formed 4 60 As above Glucose 1 62 Few green PLBs formed 2 60 As above 3 55 Moderate germination but few green PLBs formed 4 60 As above Sucrose 1 62 Moderate germination but few green PLBs formed 2 58 Most of the germinated embryos converted into healthy PLBs with white hairy structures 3 65 As above but cultures were healthier in the above treatment 4 95 Germination delayed and few healthy PLBs formed * On MS medium containing 5% CW, NAA + BA (3 + 3 µm respectively in combination) and embryos scoped out from green pod of 10 MAP.Data represents the mean of five replicates. Amongst the different PGRs incorporated, the optimum germination was registered on MS medium containing sucrose (2%), and a combination of NAA and BA (Table 4). The treatment with NAA and BA 7
individually, could not exhibit optimum response though the singly treatment with NAA out-performed BA rich media. About 80% germination was registered on MS medium containing 3 µm NAA after 68 days of culture. The cultures on BA rich media delayed germination and about 50% germination was achieved after similar duration. About 95% of the cultured immature embryos/seeds responded positively on MS medium with a combined treatment of both NAA and BA (3 µm each) + sucrose (2%) + CW (5%) and exhibited optimum response. Incorporation of CW (5%) (v/v) exhibited faster initiation of germination and the responding seeds started swelling within three wk of culture. Regeneration and mass multiplication The germinating seeds on germination media converted into PLBs (Fig 1 c). The PLBs so developed were maintained on the same initiation medium with optimum growth adjuncts for further differentiation before transferring to regeneration medium. Within 7-8 wk on regeneration medium the advanced stage PLBs started differentiating into rooted plantlets. The PLBs formed on germination medium turned green and started differentiating upon transfer to full light condition. Amongst the different nutrient media and different levels of PGRs tested for plant regeneration and mass multiplication, optimum regeneration as well as multiple shoot bud formation was achieved on MS medium supplemented with NAA (3 µm) + BA (6 µm) and 3% sucrose (Table 5). Amongst the three media studies in the present study, Knudson C medium did not support regeneration, and cultures degenerated subsequently. On Mitra et al, medium regeneration was delayed, and 1 st leaf and root formed after 18 and 80 days of culture respectively. On the other hand about 20 shoot buds/plbs formed per explants per subculture and within 15 and 16 days of culture 1 st leaf and root respectively formed (data not presented). Under optimum regeneration condition as many as 20 shoot/buds developed per subculture (Fig 1d). None of the auxins supported healthy differentiation while, the cultures supplemented with BA singly started releasing the first leaflet within 4 wk of culture. Amongst all the five different PGRs studied in the present investigation, TDZ (3µM) supported best regeneration and multiple shoot buds formation but plantlets growth was stunted. For multiple shoot bud formation and healthy growth of plantlets, a combination of NAA (3 µm) and BA (6 µm) supported maximum shoot bud formation. 8
Table 4 Effect of PGRs on in vitro culture of immature embryos of C. iridioides PGRs Conc. (µm) # Germination % germination Type of response * NAA BA time (days) (±SE) ** 0 0 63 40 (±1.5) Small green PLBs formed 3-68 80 (±2.5) Healthy green PLBs formation 6-75 60 (±2.0) Green PLBs formed 9-85 60 (±1.5) Delayed germination and few PLBs formed 12-85 50 (±1.5) Small green PLBs formed and degenerated subsequently - 3 67 50 (±2.0) Few PLBs formed - 6 79 50 (±2.5) As above 3 3 58 95 (±2.0) The germinated embryos converted into green PLBs followed by release of first leaflets 3 6 64 70 (±2.0) Green PLBs formed 3 9 70 60 (±2.5) As above but delayed germination 3 12 75 50 (±1.5) Few PLBs formed but degenerated subsequently 6 3 103 50 (±2.0) Delayed germination 6 6 64 65 (±1.5) Fewer Green PLBs formed 6 9 105 55 (±2.5) As above 9 3 64 45 (±2.0) Few green PLBs formed 9 6 75 40 (±2.0) As above but degenerated 12 3 105 35 (±1.5) As above # Only significant treatments are computed here. * On MS medium containing 2% sucrose, 5% CW and embryos of 10 MAP. ** Standard error Data represents the mean of five replicates 9
Table 5 Effect of different levels of PGRs on plantlet regeneration and mass multiplication of C. iridioides * PGRs Conc. (µm) No. of shoot buds Days taken for Type of response NAA IAA BA Kn TDZ formed/explants 1 st leaf 1 st root 0 0 0 0 0 - - - No response and culture degenerated 3 - - - - 4 29 34 Plantlet healthy and stunted 6 - - - - 2 30 35 Plantlets stunted 9 - - - - 2 30 35 As above - 3 - - - 2 80 45 Few PLBs formed - 6 - - - 2 75 45 As above - 9 - - - 3 80 50 Stunted growth with smaller leaf - - 3 - - 2 30 50 As above - - 6 - - 7 30 45 Plantlets healthy with few secondary PLBs - - 9 - - 3 35 45 Few PLBs formed - - - 3-4 16 30 Plantlets with thin leaves - - - 6-3 60 60 Plantlets stunted - - - - 3 10 58 65 Plantlets healthy - - - - 6 8 58 65 As above 3-3 - - 3 16 56 Plantlets healthy with small leaves 3-6 - - 20 15 16 Well rooted healthy plantlets with multiple shoot buds and secondary PLBs 3-9 - - 6 16 20 Few shoot buds formed 6-3 - - 7 60 80 Plantlets etiolated with light green, thin and long leaves 9-3 - - 3 80 90 Plantlets stunted 9-6 - - 4 80 95 As above - 3-3 - 2 80 80 As above - 3-6 - 2 16 30 Plantlets with small leaves - 3-9 - 2 59 - Slightly elongated PLBs - 6-6 - 3 80 70 Plantlets stunted with smaller leaves and roots 3 - - 3-5 45 70 Stunted plant growth 10
3 - - 6-3 16 16 Slightly etiolated plantlets 3 - - 9-2 70 70 As above 6 - - 3-4 45 45 Plantlets with healthy leaves and roots * On MS medium containing 3% sucrose. Data represents the mean of five replicates. Hardening of plantlets and transferring to community potting mix The well rooted plantlets (about 6-7 cm long with 2-3 roots) were taken out from the regeneration medium and transferred on culture vials containing ½MS medium supplemented with 1% sucrose freed from any PGRs. In the culture vials charcoal pieces, brick pieces and chopped mosses (at 1:1 ratio) was used as substratum (Fig 1e). The cultures were maintained for 4-6 wk in normal laboratory condition before transferring to community potting mix (CPM) containing sand: brick pieces: coconut husk: charcoal pieces: decayed wood at 1:1 ratio with a moss topping (Fig 1f). The potted plants were exposed in normal day light for about 1 hour in a day for initial one wk and subsequently increased the exposure period by 2 hours from the second week and finally after one month the plantlets were left in the normal full day light condition. About 80% of the transplants survived after two months of potting. During this process plantlets turned deep green. About 80% of the transplants survived after two months of potting. DISCUSSION For mass propagation of orchids, immature embryos serve as the best material which allows producing thousands of plantlets in a relatively short period of time. In the present study the cultured immature embryos exhibited nodular swelling within three wk of culture as the first sign of germination from the dark cultured embryos but the diffused light 11 and full light grown seeds delayed initial response. Though there was a delayed response from the embryos cultured in light condition, but registered highest germination (95%) subsequently and outperformed other two conditions (Figure 2). The age of the green pod/immature embryos was found to be the key factor for successful asymbiotic germination of seeds. Seeds age up to 8 MAP delayed germination; it may be due the fact that embryos have not been reached proper stage of maturation as reported in Vanda coerulea 17. While, the embryos from the green pod age >14 MAP did not germinate at all. The optimum germination was recorded with green pod age of 10 MAP (Table 1). The importance of time interval between pollination and fertilization has also been stressed 2,9. The relative time taken by ovules after pollination for successful germination seems to vary with species 2. The present observation with C. iridioides is in agreement with some of the previous reports 2-3,18-19. Of the different nutrient basal media tested not all the media were equally effective for successful culture initiation. In the present study, optimum germination was occurred on MS medium (95%). Besides MS medium, other media supported moderate germination but in most of the cases either delayed differentiation or failed to differentiate into either PLBs or plantlets. The relative time taken for germination varies with species 2-3,8,20. The nutrient regime for orchid culture is species specific and no single culture medium is universally applicable for
all the orchid species. e.g. Dactylorhiza hatagirea on Knudson C medium 19, Aerides rosea on Knudson C, VW and MS media 21, Arachnis labrosa on Mitra et al, medium 8, Cleisostoma racemifefum on MS medium 22, Malaxis khasiana on MS medium 5, Coelogyne suaveolens on MS medium 3 were reportedly most suitable over other nutrient media. Apart from green pod age and nutrient media, incorporation of other factors, like quality and quantity of organic carbon sources, CW and PGRs exhibited pronounced effect on successful in vitro germination of immature embryos and subsequent differentiation of plants. Amongst the different organic carbon sources used in the present study, dextrose and glucose did not support healthy germination and delayed germination. Compared to dextrose and glucose, sucrose rich media supported better germination and optimum germination was registered on medium containing 2.0% sucrose while, at both lower and higher concentration of sucrose germination was delayed (Table 3). Earlier, while working with Cymbidium elegans and Coelogyne punctulata, Sharma and Tandon 11 found that at a range of (2-3%) sucrose, fructose and D-glucose supported optimum seed germination. While, Temjensangba and Deb 2 reported incorporation of 3% sucrose in the germination medium supported optimum germination in Cleisostoma racemiferum. On media devoid of any PGRs, the cultured seeds exhibited only nodular swelling but failed to germinate and formed healthy PLBs. Amongst the different PGRs incorporated; the optimum germination was registered on MS medium containing sucrose (2%), and a combination of NAA and BA. The treatment with NAA and BA individually, could not exhibit optimum response though the singly treatment with NAA out-performed BA rich media. Incorporation of CW (5%) (v/v) exhibited faster initiation of germination and the responding seeds started swelling within three wk of culture. However, addition of CW in the germination medium had little effect on germination and subsequent development except the early swelling of the seeds. The synergistic effect of NAA and BA in asymbiotic seed germination as in the present study has been reported in Dendrobium aphyllum 23, Aerides odorata 24, C. racemiferum 2, C. suaveolens 3 where NAA and BA in combination were found superior over other treatments. However, combined treatment of NAA and KN favored optimum germination in Vanda coerulea 7. The germinating seeds subsequently converted into PLBs on the same initiation medium and PLBs were allowed to differentiate on the medium with optimum adjuncts. The advanced PLBs differentiated into rooted plantlets within 7-8 wk on regeneration medium. Amongst the different nutrient media and different levels of PGRs tested for plant regeneration and mass multiplication, optimum regeneration as well as multiple shoot bud formation was achieved on MS medium supplemented with NAA (3 µm) + BA (6 µm) and 3% sucrose (Table 5). On the other hand cultures on Knudson C medium did not support regeneration, and cultures degenerated subsequently while, on Mitra et al, medium regeneration was delayed. Of the different PGRs used for regeneration,none of the auxins supported healthy differentiation while, the cultures supplemented with BA singly started releasing the first leaflet within 4 wk of culture. Amongst all the five different PGRs studied in the present investigation, TDZ (3µM) supported best regeneration and multiple shoot buds formation but plantlets growth was stunted. For multiple shoot bud formation and healthy growth of plantlets, a combination of NAA (3 µm) and BA (6 µm) 12
supported maximum shoot bud formation. The synergistic effect of auxin and cytokinin in plant regeneration and multiple shoot buds formation as in the present study has also been reported in Malaxix khasiana 5, M. acuminata 25, C. suaveolens 3. In the present study a successful attempt was made to culture immature embryos in vitro and mass multiplication of C. iridioides. The protocol described in this communication reveals the different factors controlling the immature embryo germination of this horticultural important orchid. The organizations/individual associated in horticulture can use this protocol for commercial production of this species. Further work on in vitro mass multiplication and short- to medium-term conservation of the species is in progress. REFERENCES 1. J. L. Harley. The Biology of mycorrhiza, Leonard Hill, London, 1959. 2. Temjensangba and C. R. Deb. Effect of different factors on non-symbiotic seed germination, formation of Protocorm-like bodies and plantlet morphology of Cleisostoma racemiferum (Lindl.) Garay, Indian J. Biotech., 5: 223-228, (2006). 3. Sungkumlong and C. R. Deb. Effects of different factors on immature embryo culture, PLBs differentiation and rapid mass multiplication of Coelogyne suaveolens (Lindl.) Hook. Indian J. Exp. Biol., 46: 243-248, 2008. 4. L. Knudson. Non-symbiotic germination of orchid seeds. Bot. Gaz.., 73: 1-25, 1922. 5. C. R. Deb and Temjensangba. In vitro propagation of threatened terrestrial orchid, Malaxis khasiana Soland ex. Swartz through immature seed culture. Indian J. Exp. Biol., 44: 762-766, 2006. 6. C. R. Deb and Sungkumlong. In vitro regeneration and mass multiplication of Taenia latifolia (Lindl.) using immature seeds : A threatened terrestrial orchid. J. Pl. Biol., 38: 1-6, 2008. 7. C. G. Devi, M. Damayanti and G. J. Sharma. Aseptic embryo culture of Vanda coerulea Grief. J. Orchid Soc. India, 12: 83-87, 1998. 8. Temjensangba and C. R. Deb. Regeneration and multiplication of Arachnis labrosa (Lindl.ex. Paxt) Reichb : A rare and threatened orchid. Curr. Sci., 88: 1966-1969, 2005. 9. H. L. Valmayor and Y. Sagawa. Ovule culture in some orchids. American Orchid Soc. Bull., 36: 766-769, 1967. 10. S. P. Vij and P. Pathak. Asymbiotic seed germination of the saprophytic orchid, Cymbidium macrorhizon : A study in vitro. J. Orchid Soc. India, 2: 25-32, 1988. 11. S. K. Sharma and P. Tandon. Asymbiotic germination, seedling growth of Cymbidium elegans Lindl. and Coelogyne punctulata Lindl. as influenced by different carbon sources. J. Orchid Soc. India, 4: 83-87, 1990. 12. T. Murashige and K. Skoog. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plantanum, 15: 473-497, 1962. 13. G. C. Mitra, R. N. Prasad and A. R. Roy Chowdhury. Inorganic salts and differentiation of protocorm in seed callus of an orchid and correlation changes in its free amino acid content. Indian J. Exp. Biol., 14: 350-351, 1976. 14. L. Knudson. A new nutrient solution for germination of orchid seeds, American Orchid Soc. Bull., 15: 214-217, 1946. 13
15. O. L. Gamborg, R. A. Miller and K. Ojima. Nutrient requirements of suspension cultures of soyabean root cells. Exp. Cell. Res., 50: 151-158, 1968. 16. R. U. Schenk and A. C. Hildebrandt. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot., 50: 199-204, 1972. 17. J. Sharma. Studies on Vanda: Effect of age capsules (pods) on in vitro seed germination. J. Orchid Soc. India, 12: 43-45, 1998. 18. P. Pathak, S. P. Vij and K. C. Mahant. Ovule culture in Goodyera biflora (Lindl.) HK. F.: A study in vitro. J. Orchid Soc. India, 6: 49-53, 1992. 19. S. P. Vij, P. Pathak and K. C. Mahant. Green pod culture of a therapeutically important species Dactylorhiza hatagirea (D. Don) Soo. J. Orchid Soc. India, 9: 7-12, 1995. 20. S. P. Vij. Genetic Resources of Orchids. In: K. L. Chadha and S. K. Bhattacharjee (eds.), Advances in horticulture, Vol. 12. (Ornamental plants I), Malhotra Publishing House, New Delhi, India., 1995, pp. 153-181. 21. S. K. Sinha, L. S. Singh and S. N. Hegde. In vitro multiplication of Aerides rosea Loddiges ex. Paxt. through asymbiotic seed germination. Arunachal For. News, 16: 38-44, 1998. 22. C. R. Deb and Temjensangba. Rapid mass multiplication of Cleisostoma racemiferum (Lindl.) Garay: An endangered orchid. J. Pl. Biol., 34: 99-105, 2007. 23. A. Talukdar. Multiple shoot induction in Dendrobium aphyllum Roxb. J. Orchid Soc. India, 15: 35-38, 2001. 24. B. Pant and R. Gurung. In vitro seed germination and seedling development in Aerides odorata Lour. J. Orchid Soc. India, 19: 51-55, 2005. 25. S. P. Vij and S. Kaur. Micropropagation of the therapeutically important orchids: Malaxix acuminata D. Don. J. Orchid Soc. India, 12: 8 14
ASYMBIOTIC CULTURE OF IMMATURE EMBRYOS, MASS MULTIPLICATION OF CYMBIDIUM IRIDIOIDES D. DON. AND THE ROLE OF DIFFERENT FACTORS 15