re observed on 0.5 Kn C medium whereas maximum length of shoot (5.61 ± 0.82), leaf (3.23 ± 0.63) and roots (2.95 ± 0.78) were found on 0.25 Kn C medium (Table 1). One way ANOVA showed that the value of all the growth parameters except number of root and length of root varied significantly among different conditions of Kn C medium (p < 0.05; Table 1).
3.2. Effect of Storage Conditions: Temperature and Duration for Viability
In this investigation, the artificial seed of protocorm contained only calcium alginate and were devoid of any other nutrients or plant growth regulators. In both MS and Kn C media, artificial seeds stored at 4˚C were green, fresh and remained intact inside the calcium alginate beads until 90 days; whereas those stored at RT became pale and reduced its size. In RT, artificial seeds ruptured slowly after 10 weeks of inoculation and most of them become contaminated; while at 4˚C the germination was started within 7 - 8 weeks of inoculation on MS medium. In case of Kn C medium, stored artificial seeds germinated within 8 weeks of culture at 4˚C and 10 - 11 weeks of culture at RT. On MS medium, 83.3% of artificial seeds stored for 30 days at 4˚C were viable but it declined substantially with time. A similar trend was evident in Kn C medium as well. On the other hand, the artificial seed of protocorm stored at RT showed only 30% viability on day 30 with no viability or regeneration after 60 days (Table 2).
Artificial seeds offer many advantages and opportunities especially in cross pollinating species where seed production and germination is difficult and expensive  . Therefore, the present investigation was carried out for the purpose of germplasm storage, delivery and conservation of the threatened medicinal orchid Cymbidium aloifolium through artificial seeds. Both MS and Kn C media have been found suitable for artificial seed germination and regeneration of plantlets. The germination time of artificial seeds was found to be different with media and growth conditions.
Although all tested media were successfully supported the conversion of encapsulated protocorms into whole plantlet with shoot, leaf and root growth showed varied responses. It might be due to the compositional variation of two different media including plant hormones. Many shoot buds were regenerated directly from a single encapsulated protocorm which later developed into a whole plant (Figure 1(a) and Figure 1(b)). Though earlier germination was observed on hormone-free Kn C medium, healthy and chlorophyllous shoots and roots were developed earlier on hormone free full strength of MS medium (Figure 1(c)). It could be due to the low amount of macro
Table 2. Effect of media, storage temperature and duration of storage on germination and plantlet development from artificial seeds of Cymbidium aloifolium.
Culture conditions: MS & Kn C liquid media, 90 days of storage, RT-room temperature (21˚C ± 2˚C).
(a) (b)(c) (d) (e)
Figure 1.Production, germination and plantlet regeneration of artificial seed containing protocorms of Cymbidium aloifolium. (a) Production of artificial seed using 4% sodium alginate and 0.2M calcium chloride solution; (b) Germinated artificial seeds gave rise to shoot bud on 0.25 MS medium; (c) Development of healthy roots and shoot buds on hormone free 1.0 MS medium; (d) Multiplication of shoots with well developed roots on hormone free 1.0 MS medium; (e) Hardening of in vitro plantlets on potting mixture of cocopeat, clay and sphagnum moss of 2:1:1 ratio.
and micro nutrients present in Kn C medium. Whereas MS medium was enriched with high concentration of nutritional compounds which made the suitability for efficient regeneration of healthy plantlet from artificial seed of Cymbidium aloifolium. Hence, MS medium was significantly more effective for germination and seedling development rather than Kn C medium. In this medium, shoot buds were developed by cracking the alginate beads of artificial seeds of protocorms after 8 weeks of inoculation; complete plant with shoot and root were developed after 14 - 15 weeks of culture (Figure 1(d)). The most appropriate condition for the germination of the artificial seed was dependent on the time taken for germination, efficiency of germination, growth and development of seedlings. It can be concluded from this investigation that plants get physiologically stressed in hormone attributed both MS and Kn C media as a result they could not undergo proliferation sufficiently as much as like hormone free MS and Kn C media. In case of lower strength of MS media i.e. 0.25 MS & 0.5 MS, plant didn’t get sufficient nutrition as they required for their optimum development  .
The present result was similar to the findings of other researchers: Gupta (2016) in Renanthera imschootiana  , Teixeira da Silva (2012) in hybrid Cymbidium  , Nagananda et al. (2011) in Flickingeria nodosa  , Sarmah et al. (2010) in Vanda coerulea  , Mohanraj et al. (2009) in Coelogyne breviscapa  in the aspect of production of artificial seed or Synseed from protocorm or protocorm like bodies for regeneration. However, the present findings are in contrast with the result obtained by other researchers in the aspect of culture of artificial seed in high concentration of hormone attributed medium. Zhang et al. (2011) reported that Maltose 4%, 6-BA/NAA 12:1, active carbon 0.3%, sodium alginate 4% and time of ion exchange for 5 minutes were optimum condition for artificial seed production with PLB’s in Dendrobium candidum  . As well, Qin et al. (2008) found that modified MS with 4% maltose and BA (1 mg/l) and NAA (1 mg/l) favoured germination and seedling of orchid Dendrobium huoshanesises by using 4% sodium alginate  . Moreover, Datta et al. (1999) found that modified Knudson medium supplemented with BAP (1 mg/l) and NAA (1 mg/l) was the most effective condition for germination of artificial seed of Geodorum densiflorum (Lan) Schltr.  . Further, Saiprasad and Polisetty (2003) reported 100% conversion of encapsulated PLBs when cultured on MS medium supplemented with BA (4.44 μM) and NAA (0.54 μΜ) on Dendrobium and NAA alone on Oncidium and Cattleya  . Finally, Mohanty and Das (2013) found that MS medium supplemented with 2 mg/l BAP was efficient for 100% conversion of encapsulated PLB’s into plantlets on Dendrobium densiflorum Lindl. ex Wall.  .
Besides germination and mass propagation of artificial seeds containing protocorms of Cymbidium aloifolium, the viability of stored artificial seeds were observed until 90 days in two different temperatures i.e. room temperature (RT, 21˚C ± 2˚C) and 4˚C. In both media, it was found that the viability percentage of artificial seeds of protocorms decreased gradually with increase in storage time. The decline in germination and plant development as a result of prolonged storage of tissue could be due to the inhibited respiration of tissues   . The maximum percentage of viability was observed in 30 days stored artificial seed (83.33%) at 4˚C inoculated on MS medium. This result revealed that the germination and regeneration capacity of the stored artificial seeds at 4˚C was quite higher rather than stored at RT. It might be due to the chilling effect of low temperature, which not only store the calcium alginate beads for prolonged period by reducing their growth but also enhance its regeneration capacity when transferred to re-growth medium containing essential nutrients. Here, the storage temperature and duration of storage were played an important role on conversion of artificial seeds into plantlets (Table 2). The present result was supported by the findings obtained by various researchers: Pradhan et al. (2014) successfully stored the artificial seed of Cymbidium aloifolium up to 28 days on MS medium with 97.5% conversion rate  . Nagananda et al. (2011) encapsulated the PLBs of Flickingeria nodosa and achieved 95% conversion after 3 months storage at 4˚C  . Zhang et al. (2011) produced artificial seeds of Dendrobium candidum with protocormlike bodies as propagator and achieved the germination rate 87.70 ± 0.46 after 5 days and 14.87 ± 0.31 after 20 days of storage in refrigerator at 4˚C which was lower compared to the present investigation  . Similarly, Datta et al. (1999) found 86% viability of artificial seeds of Geodorum densi-
florum when stored at 4˚C and 44% viability when stored at room temperature for 120 days. Nutrient medium also plays a crucial role on germination and development of artificial seed  . In present investigation, MS medium was found to be the most favourable condition for regeneration of plantlet from fresh as well as stored artificial seed containing protocoms of C. aloifolium. The use of MS medium only for regeneration of plantlet from stored artificial seed also provides a way to reduce the costs by eliminating the need for plant hormones.
The ultimate objective of producing artificial seed was to recover the whole plantlet from artificial seed under in vitro condition. In present investigation, in vitro plant with well-developed roots obtained from artificial seeds, cultured on both MS and Kn C media after attaining the height of 4 - 5 cm was selected for acclimatization. They were transferred to earthen pot containing different acclimatization substrates. The combination of cocopeat, clay and sphagnum moss in ratio 2:1:1 was found to be a suitable potting mixture for hardening (Figure 1(e)). Eighty five percentage of seedlings were successfully survived under this condition. Hence, this result suggests that the mixture of cocopeat, clay and moss will be favourable for the acclimatization of epiphytic orchids as in Cymbidium aloifolium.
Many researchers have successfully produced and propagated artificial seed of many crops but very limited in orchids. The present investigation revealed that full strength of liquid MS medium without plant growth regulators was found to be the most effective condition for germination as well as for development of efficient plant regeneration from artificial seeds of Cymbidium aloifolium than other tested conditions of MS and Kn C medium. 30 days stored artificial seeds have showed highest percentage of viability i.e. 83.33% at 4˚C on MS medium as compared to 60 and 90 days of storage. The development of protocol for direct recovery of plant from artificial seeds may have greater impact on tissue culture methods. Therefore, the present investigation concluded that artificial seeds have the excellent potential to be used as alternative planting materials for in vitro clonal mass multiplication, short to medium term storage, easy handling and transportation and ex-situ conservation of economically important species like Cymbidium aloifolium. Hence, the protocol established from present investigation can be employed for large scale propagation of C. aloifolium which may be not only useful for fulfilling the urgent local needs for effective conservation but also reliable for plant breeders and local orchid growers for storage of plant genetic resources, easy delivery and their commercial production by using minimum space with lower labour and maintenance cost.
Authors are grateful to Central Department of Botany, Tribhuvan University, Kirtipur, Nepal for providing all laboratory facilities for this investigation. We are also thankful to Nepal Academy of Science and Technology (NAST), Khumaltar, Lalitpur, Nepal for providing the financial assistance to carry out the present research work.
Cite this paper
Pradhan, S., Tiruwa, B.L., Subedee, B.R. and Pant, B. (2016) Efficient Plant Regeneration of Cymbidium aloifolium (L.) Sw., a Threatened Orchid of Nepal through Artificial Seed Technology. American Journal of Plant Sciences, 7, 1964- 1974. http://dx.doi.org/10.4236/ajps.2016.714179