In vitro Rapid Propagation of Cymbidium aloifolium (L.) Sw.:A Medicinally Important Orchid via Seed Culture

MATERIALS AND METHODS

The present study was carried out at Department of Botany, Panjab University, Chandigarh from September, 2004 to June, 2006. Green unripe capsules of C. aloifolium were collected 24 weeks after pollination from forested areas of Manipur in North Eastern part of India. The capsules are then thoroughly washed under the running tape water with 20% teepol for 10-15 min. They were then surface sterilized for 7-8 min in 0.4% HgCl₂ solution with 1-2 drops of teepol as wetting agent. This was followed by repeated washing of treated capsules for 3-4 times in sterile distilled water to remove HgCl₂ completely from its surface. The capsules were finally dipped in 70% ethanol for 15-20 min followed by flaming for 3-4 sec. The surfaced sterilized capsules were subsequently split opened by using a sharp sterilized surgical blade and the immature seeds were scooped out to be used as explants for initiating culture. The whole procedure was performed in aseptic condition under laminar flow to prevent any form of contaminations.

The extracted seeds were inoculated on agar gelled Mitra et al. (1976) and Murashige and Skoog (1962) medium to determine the most appropriate medium for seed germination and development. The culture media contained 0.9% agar and 2% sucrose at pH of 5.8. The growth response was monitored with or without 0.2% of activated charcoal in the culture media. When AC was incorporated, complete dispersion was required by swirling the vessels before the medium got solidified. Plant growth regulators like Indole-3-butyric Acid (IBA), 6-Benzyl Amino Purine (BAP) and 1-Nathyl Acetic Acid (NAA) with different concentration at 0.5 and 1 mg L^-1 were supplemented either singly or in combination to study their effect on the growth of the culture in vitro. The growth regulators were added prior to autoclaving and definite quantities of the medium were dispensed to test tubes and conical flasks and closed tightly with cotton plugs. The medium was autoclaved at 1.1 kg cm² pressure and 121°C for 15-20 min. After autoclaving, the culture vessels with sterilized medium were then placed in an appropriate position (test tubes in slanting and flask in vertical) to allow the medium to gel. The cultures were then maintained at 25±2°C which was illuminated at 60 μmol/m²/sec for 12 h a day using white fluorescent tubes.

Subculturing was carried out after every 4 weeks and 15 replicates were used and each treatment was repeated twice. The rate of seed germination was recorded after 3-5 weeks of culture as the seeds started swelling due to absorption of nutrients. The seed germination percentage was calculated by dividing the number of germinated seeds by total number of seeds inoculated multiplied by 100. The formation of globular structure spherules as well as the subsequent protocorm development in weeks was recorded to analyse different stages of development leading to seedling formation. The number of shoots and roots formed in the seedlings were also recorded to determine suitable medium and growth hormone combinations for appropriate shooting and rooting.

The data recorded from the present study was subjected to Analysis of Variance (ANOVA) and significant differences were determined by Duncan’s multiple range test at p = 0.05 (Duncan, 1955). The statistical data analysis was conducted using the SPSS (SPSS Inc., Chicago, USA). The well grown seedlings complete with leaves and roots were subsequently hardened by culturing them for 2 weeks in a medium (full strength) without any growth regulators followed by culturing them in the similar condition for few weeks without sucrose and vitamins in the medium. The hardened well rooted seedlings were removed and rinsed thoroughly with luke warm sterile water to remove agar and nutrients sticking to them and treat with 0.01% fungicide solution for 15-20 min. The plants are then transplanted to plastic/clay pots containing brick pieces, pine bark, charcoal pieces and moss (1:1:1) as potting mixture. The transplanted plants were finally kept in the glass house for further acclimatization to nursery condition.

RESULTS AND DISCUSSION

The immature seeds of C. aloifolium showed varied response depending upon the type of medium used as well as different plant growth regulators employed at varied concentrations. The two culture media used in the present study have differential combination of minerals, micro and macro elements. MS has richer contents of macro and micro-elements but lower vitamins as compared to M medium. Seed germination in the two culture basal media without any growth regulators was found to be high at around 83.52% in Mitra medium and 74.53% in MS medium (Table 1, 2).The positive influence of basal MS medium on seed germination was also observed in Vanda coerulea (Roy et al., 2011) and Dendrobium transparens (Alam et al., 2002). There are reports of species-specific media for successful seed germination of orchids in vitro (Bhadra et al., 2002). The seeds started swelling in two weeks times indicating successful germination. The swelling was due to imbibition of water and nutrients and the embryos eventually underwent several divisions to produce irregularly shaped parenchymatous cell mass called spherules which emerged out from the cracked seed coat. These globular spherules with hairy growth at the basal portion developed into protocorms which are a chlorophylous initially but subsequently turned green in color after acquiring chlorophyll in 6-7 weeks (Fig. 1a). This phenomenon was also observed by Sheelavanthmath and Murthy (2001) and Gayatri et al. (2006) in some terrestrial orchids. The protocorms are round, oval, elongated, branched or spindle shaped bodies which are considered as an intermediate structure between the embryos and the plants. These protocorms then underwent further morphogenetic changes resulting in the development of leaf primordia and finally to the formation of complete seedling in 14-15 weeks in Mitra and 15-16 weeks in MS medium. Nayak et al. (1998) reported regeneration of C. aloifolium by using rhizomes developed from the immature seeds when grown on the MS medium supplemented with different growth regulators like IAA, IBA and NAA. They observed the development of rhizomes from protocorms after 2-3 months which subsequently developed into the whole plant. However, in present study the chlorophyllous protocorms directly differentiated into seedlings without rhizome formation in relatively shorter time of only 14-16 weeks (Fig. 1b). Das and Bhadra (1998) had similarly reported direct seedling formation but the time required for seedling development was comparatively longer as compare to this report. When AC was incorporated, the seed germination was enhanced followed by rapid protocorm development in 6-7 weeks. This significantly reduced time required for seedling formation by another 2-3 weeks. Complete seedlings with well developed shoots and roots were regenerated in 12-13 weeks in both the Mitra and MS media. Hossain et al. (2009) also reported the improvement of seed germination in C. aloifolium when AC was incorporated in Mitra and MS media in the absence of any additives and plant growth regulators. The presence of AC might help in adsorbing the toxic substances released in the medium due to autoclaving and also by germinating seed explants. The importance of AC in asymbiotic seed germination was earlier reported by Ernst (1975) as it helped in adsorbing phenolic exudates, aerating the medium and providing near natural conditions for seed germination by absorbing light. Supplementation of 0.5 mg L^-1 of BAP singly or in combination with 0.5 mg L^-1 of IBA in both the culture media drastically reduced in vitro germination of seeds. Presence of BAP showed reduced germination percentage of 53.40±0.35 in Mitra medium and 57.67±0.78 in MS medium. However, Hossain et al. (2009) observed significantly high rate of seed germination in C. aloifolium when 1.0 mg L^-1 of BAP was supplemented in both the culture media. This change in germination frequency may result due to difference in the concentration of plant growth regulator employed for the present study. The few germinated seeds developed into globular structure spherules which subsequently developed into small insignificant protocorms in 15-16 weeks in Mitra (Table 1) and 11-12 weeks (Table 2) in MS medium.

Fig. 1:	In vitro growth response of C. aloifolium under different culture conditions (a) rapid protocorm proliferation in basal Mitra medium, (b) direct seedling formation from protocorms in basal MS medium, (c) robust root multiplication in Mitra medium supplemented with 0.5 mg L-1 of NAA and (d) enhanced leaf development in Mitra medium incorporated with 1.0 mg L-1 of NAA along with AC

Table 1:	In vitro germination response of Cymbidium aloifolium on Mitra medium with different growth regulators
Results based on the average of 15 replicates. Means followed by same letter are not significantly different at p = 0.05. * AC was incorporated into the medium

Table 2:	In vitro germination response of Cymbidium aloifolium on MS medium with different growth regulators
Results based on the average of 15 replicates. Means followed by same letter are not significantly different at p = 0.05. * AC was incorporated into the medium

This is in contrast to earlier reports in other orchids which indicated the promotive effect of BAP in seed germination, protocorm multiplication as well as shoot multiplication (De Pauw et al., 1995; Chowdhury et al., 2003; Nhut et al., 2005). The presence of both BAP and IBA also produced poor percentage of seed germination (50.20±0.45 in Mitra and 49.51±0.34 in MS medium) leading to development of small underdeveloped protocorms. These stunted protocorms did not survive long which turned brown in colour and finally died even after several subcuturing. Similar finding was reported by Hossain et al. (2009). Incorporation of AC with increased concentration of plant growth regulators (1 mg L^-1) significantly improved germination rate and protocorm formation in both the culture media. Large protocorms with high chlorophyll content were evident in short span of time. This showed that either the inhibitory effect of BAP when used singly or its antagonistic response to IBA when present together was alleviated by combined action of AC and increased concentration of growth regulator in the medium. This is also indicative of the high adsorptive nature of AC in eliminating the potential growth inhibitors released in the culture thereby producing increased germination rate and large healthy protocorm formation. Roy et al. (2007) earlier demonstrated the stimulatory effect of AC on culture growth of Thunia marshalliana by reducing the inhibitory effect of other compounds present in either MS or Phytamax media. MS medium supplemented with 1 mg L^-1 of BAP and 2 g L^-1 of AC was found to be most suitable for shoot multiplication as the surface of protocorms with several small green protuberances subsequently developed into multiple shoots in 7-8 weeks time. Maximum number of shoots (5.71±0.96) per seedling was recorded in this combination (Table 4). The induction of multiple shoot proliferation directly from the protocorms in presence of cytokinins was similarly reported in other orchids (Nayak et al., 1997; Chang and Chang, 1998). Rooting (No. of roots per seedling) was also improved with AC (3.82±0.42 in Mitra and 2.81±0.81 in MS medium) in addition to enhanced shoot multiplication (Table 3). This finding substantiated the earlier report by Eymar et al. (2000) which stated that the addition of AC helped in maintaining the pH level during culture, increased nitrogen uptake and improved culture development thereby reducing the inhibitory effect of exogenous cytokinin on root growth. Seed germination was found to be significantly improved when NAA was incorporated into the culture media. The enhanced effect of NAA in seed germination was also reported in Calanthe tricarinata by Godo et al. (2010). The germinated seeds subsequently developed into healthy chlorophyllous protocorms in 5-6 weeks which was followed by rapid root initiation in 2 weeks resulting into the formation of stout root system (Fig. 1c).

Table 3:	Effect of different growth regulators on shoot and root formation of C. aloifolium after 16 weeks of culture on Mitra medium
Values are Mean±SD, N = 15. Means followed by same letter are not significantly different at p = 0.05. *Activated Charcoal (AC) was incorporated into the medium

Table 4:	Effect of different growth regulators on shoot and root formation of C. aloifolium after 16 weeks of culture on MS medium
Values are Mean±SD, N = 15. Means followed by same letter are not significantly different at p = 0.05. *Activated Charcoal (AC) was incorporated into the medium

The promotive effect of auxin in producing enhanced rooting was also demonstrated in Cymbidium devonium (Das et al., 2007), Vanda coerulea (Malabadi et al., 2004), Oncidium taka (Rahman et al., 2005b) and Encylia maraie (Santos Diaz and Alvarez, 2009). The presence of 0.5 mg L^-1 IBA similarly provided a conducive environment for rigorous root formation with 5.41±0.69 and 4.61±1.01 (roots per seedling) recorded in Mitra and MS medium respectively. The effectiveness of IBA in inducing rooting in orchids had earlier been reported by many workers (Giridhar et al., 2001; Nongdam and Nirmala, 2009). However, NAA was found to be more effective in producing rooting than IBA in Cymbidium aloifolium. Medium supplemented with 0.5 mg L^-1 of NAA was found to be most suited for root development with maximum number of root formation recorded in Mitra medium (5.80±1.76). Surprisingly when AC was added, there was reduction in root formation even after the concentration of either NAA or IBA was increased to 1 mg L^-1. This might suggest the detrimental affect of AC in rooting initiation in C. aloifolium in the presence of auxin while promoting shoot formation. Leaf formation was enhanced and more prominent with AC in the culture medium (Fig. 1d). Such observation was also earlier reported in Dendrobium orchid by Nagaraju et al. (2004).

CONCLUSION

The present study showed that the immature seeds of C. aloifolium responded in accordance to the various stimuli present in the culture environment. Maximum germination response was recorded on basal Medium supplemented with AC. BAP singly or in association with IBA in absence of AC did not provide appropriate culture condition for seed germination, protocorm formation and seedling development. There was delay in the development of healthy protocorms. Instead stunted and insignificant protocorms were produced which ultimately died due to loss of chlorophyll. However, 1 mg L^-1 BAP with AC in MS medium produced maximum shooting while rooting was significantly high when either 0.5 mg L^-1 of NAA or IBA was used in both the culture media. The in vitro plants with 2-3 well developed leaves and roots were gradually hardened and acclimatized to the nursery conditions. The in vitro regeneration protocol established from the present investigation can be employed for the propagation of this useful orchid in larger scale which may not only help in addressing the urgent local needs for effective conservation but also meet the high pharmaceutical and ornamental requirements of this region.

ACKNOWLEDGMENT

Authors are thankful to CSIR, New Delhi for providing financial assistance for carrying out the present study.