In Vitro Propagation of Endangered Orchid, Vanda pumila Hook.f. through Protocorms Culture

The Vanda pumila is a monopodial orchid with beautiful flowers that are native to Thailand but now found across South Asia. The immature seeds of Vanda pumila were used for in vitro culture and then the protocorms developed were used as explants for seedling development and mass propagation. Protocorms were cultured on 1/2 MS (Murashige and Skoog, 1962) medium fortified separately with Kinetin (Kn), 6-Benzyl amino purine (BAP) and Gibberellic Acid (GA3) each in different concentrations as (0.5 mg/L, 1.0 mg/L and 2.0 mg/L) well as each on each concentrations of each medium supplemented with 5% and 10% coconut water (CW) respectively. The greatest number of shoots (9.50 ± 0.29 shoots per culture) was developed on 1/2 MS medium fortified with 1.0 mg/L Kn plus 10% CW and the longest shoots (0.78 ± 0.07 cm per culture) developed on 1/2 MS medium fortified with 2.0 mg/L BAP plus 10% CW. The shoots derived from protocorms were then developed on 1/2 MS medium fortified with three different rooting hormones viz. Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) and α-Naphthalene acetic acid (NAA), each in four concentrations (0.5 mg/L, 1.0 mg/L, 1.5 mg/L and 2.0 mg/L) as well as 1.0 mg/L of each hormone supplemented with 10% CW. The 1/2 MS medium fortified with 0.5 mg/L IAA was found to be the most effective condition for the development of maximum number of root (5 ± 0.0 roots per culture) and root length (0.93 ± 0.07 cm). Hence, the present study could be useful for standardizing the protocol for mass propagation of the endangered orchid V. pumila.


Introduction
Orchids are an immensely beautiful group of plants with extraordinary variety.
The colour of the flowers of these members of Orchidaceae family is undoubtly their greatest charm. As few orchid flowers are pollinated in nature, the few seed pods are formed and those seeds that are produced are unlikely to germinate [1].
In nature, orchid seeds require specific mycorrhizal fungal associations to germinate, so less than 1% do that [2]. In vitro culture in contrast, fungal associations are not required for germination, as first demonstrated by Knudson in 1922 [3] [4].
Six species of Vanda, including Vanda pumila have been recorded in Nepal [5] and its synonym is Trudelia pumila. V. pumila, which is closely related to V. cristata, is commonly known as "The Dwarf Vanda". This orchid species is also found in North East India, Nepal, Myanmar, China, Laos, Thailand and Vietnam at the elevation of 1200 -2300 m [5]. V. pumila is important for breeding cultivars [6] and most of the species have great medicinal value. The root of Vanda tessellata, for example is traditonally used for an antidote for scorpion stings and a remedy for bronchitis and rheumatism. A paste of its leaves is used to treat fever, cough, piles and other aliments [7] [8] [9]. In addition, the leaves of Vanda teres and Vanda cristata are used to treat fever [10]. Vanda cristata also has anti-cancer property [11].
Because of the restricted population size and expected medicinal properties of V. pumila, it is required to develop an effective conservation strategy to save this plant for future. Plant tissue culture technique provides an opportunity to preserve and commercialize the number of rare and threatened orchid species [12]. Hence, the present study was carried out to standardize the protocol for mass propagation and ex-situ conservation of this species.

Plant Material and Protocorm Culture
Immature capsules of Vanda pumila were collected from the garden of the Central Department of Botany, Kirtipur, Kathmandu. The capsules were surface sterilized with 1 -2 drops of TWEEN 20 and washed with running tap water for at least half an hour. The capsules were then dipped in 1% sodium hypochlorite solution for 15 minutes before they held with forceps and submerged in 70% ethanol where they were rapidly set a flame for a few seconds. The capsules were then rinsed three times in sterile water. These surface sterilized capsules were then cut longitudinally with a sterile scalpel and the exposed seeds were removed with forceps and transferred to an agar gel (0.8% per liter) nutrient medium for seed germination. Different strength of MS [13] medium (full strength, 1/2 and 1/4 th strength), were used to germinate the seeds and then the protocorms developed from the seeds were used for shoot proliferation. The sequential growth and proliferation of Vanda pumila was then evaluated.
The pH of the medium was maintained at a slightly acidic 5.

Statistical Analysis
Average value of week was taken for in vitro seed germination. Data of shooting and rooting were presented as mean and standard error. Data were analyzed by one way ANOVA with F-statistics at 95% confidence interval using SPSS version 20.

Shoot Development through Protocorm Culture
To develop the shoots of Vanda pumila, the eight month old protocorms measuring 3 mm -4 mm in diameter derived from the in vitro seed germination of V. pumila were sub-cultured on 1/2 MS medium supplemented with different finding is encouraging as, according to Teixeira da Silva et al. [14], young PLBs are highly meristematic in nature and can be used to improve plant regeneration when they are used as primary explant.
Of the three concentrations of BAP supplemented medium, 1/2 MS medium fortified with 2.0 mg/L BAP yielded the greatest number of shoots (6 shoots per American Journal of Plant Sciences culture) and the longest shoots (0.58 ± 0.25 cm) as well as the greatest number of leaves (9 ± 0.57 leaves per culture) and the longest leaves (0.43 ± 0.48 cm per culture). This result was similar to the findings of David et al. [15] who reported that in case of Vanda helvola. They reported that MS medium fortified with either 1.0 mg/L BAP or 2.0 mg/L BAP were the best for protocorms proliferation.
Similarly, Luo et al. [16] reported that BAP alone was more effective for the proliferation of PLBs of Dendrobium densiflorum when combined with NAA and Kn. Regmi et al. [17] also reported that MS medium fortified with both 2.0 mg/L BAP and 0.5 mg/L NAA was effective for the protocorms proliferation of Cymbidium aloifolium. In the present study, the development of shoot was found to be the highest (0.78 ± 0.07 cm per culture) on 2 mg/L BAP fortified with 10% CW while the greatest number of shoots (7.5 ± 0.65 shoots per culture), the greatest number of leaves (11.25 ± 0.48 leaves per culture) and the longest leaves Hrahsal and Thangjam [19] reported that with respect to Vanda coerulea, BAP    ( Figure 4 and Figure 5). Ng and Saleh [20] reported that 1/2 MS medium fortified with 2.0 μM Kn resulted in the highest proliferation of PLBs of Paphiopedilum. They also observed that 1/2 MS medium fortified with 20% CW promoted tertiary PLBs proliferation in Paphiopedilum. Similarly, Dutta et al. [21] reported that, in case of in Dendrobium aphyllum, IAA supplemented with Kn was more effective for protocorm proliferation.
In the present study, 1/2 MS medium fortified with different concentrations of GA3 (0.5 mg/L, 1.0 mg/L and 2.0 mg/L) alone resulted in very little multiplication of protocorms as most of the cultures started to become necrotic within five weeks after seedling ( Figure 6). Dohling et al. [22] reported that seed germination and seedling development in Dendrobium species were inhibited when GA3 in medium. Of the 1/2 MS medium supplemented with different concentrations    As Jualang [23] was found the similar report with Vanda dearie, all the hormone concentrations supplemented with CW study of Vanda pumila, white hair papillae was appeared. Clearly, coconut water plays a vital role in the protocorm proliferation. According to Gnasekaran et al. [24], coconut water is added to the tissue culture medium because it contains diphenyl urea, a growth factor which exhibits cytokinin-like responses. The result of this study is partially supported by Saiprasad et al. [25], who reported that 0.5 mg/L Kn supplemented with 10% CW was ideal for the protocorm multiplication of Dendrobium Oncidium and Catteleya, but shoot length was the greatest on 1/2 MS medium supplemented with BAP. The present results were also in accordance with the findings regarding Dendrobium, Oncidium and Catteleya orchid as well as Paphiopedilum and Cymbidium pendulum [20] [25] [26]. The present study of V. pumila revealed that kinetin helped to convert protocorms into shoots and this result was in

Root Development of Vanda pumila
Once they were about 0.5 -0.8 cm. long, the shoots derived from protocorms grown in vitro were cultured on 1/2 MS medium fortified with different concen- Bhattacharjee and Islam [29] reported that, with respect to Acampe premorsa, Agrostophylum khasianum and Phalaenopsis cornorerri roots were longer on a full strength MS medium fortified with 0.5 mg/L IAA than on full strength MS medium supplemented with 1.0 mg/L IAA. Hossain et al. [30] reported that for Cymbidium aloifolium, a medium fortified with 0.5 mg/L IAA promoted the development of stout roots. Similarly, Pant and Thapa [31] found that in case of Dendrobium primulinum, MS medium supplemented with 0.5 mg/L IAA resulted in the greatest proliferation of roots. This finding, however, contrasted with that of Rahman et al. [32] on Vanda tessellata. Bhattacharjee and Islam [33] recorded that Vanda tessellate produced the most roots on 1/2 MS medium fortified with 1.0 mg/L IAA and Hrashel and Thangjam [19] reported that 1/2 MS medium fortified with 28.50 μM IAA yielded many roots in Vanda coerulea.  shoots (0.70 ± 0.23 cm) were found on 1/2 MS medium fortified with 1.0 mg/L IBA. In addition, the roots on this medium were stouter and healthier than they were on mediums made with other concentrations. Rafique et al. [34] and Aktar et al. [35] found that 1.0 mg/L IBA was maximized both the number and the lengths of roots in the case of Dendrobium orchids and Rahman et al. [32] found the Vanda tessellate also developed good roots on IBA supplemented mediums. Among the tested conditions of Auxin, 1/2 MS medium fortified with 0.5 mg/L IAA was found to maximize both the number and length of the roots of Vanda pumila. It was better than other concentrations of IAA, than other rooting hormones (IBA and NAA) and mediums with coconut water. However, in the present study, all the IBA supplemented mediums produced satisfactory number of roots from 0.5 to 3 roots per culture. Similar findings were reported by Pradhan et al. [37] who found that IBA was better rooting hormone for Dendrobium densiflorum as compare to IAA and NAA. American Journal of Plant Sciences

Conclusion
The present study concluded that among the different tested conditions for shoot development, the 1/2 MS medium fortified with 1.0 mg/L Kn plus 10% CW and 2.0 mg/L BAP plus 10% CW was found to be the more effective for the proliferation of large number of shoots (9.50 ± 0.29 shoots per culture) and shoot length (0.78 ± 0.07 cm per culture) respectively from the in vitro culture protocorms of Vanda Pumila. For rooting, a great number of strong and healthy roots of this species were developed on 1/2 MS medium fortified with 0.5 mg/L IAA (5 ± 0.0 roots per culture and 0.93 ± 0.07 cm root length per culture).
Hence the present result could be useful for the establishment of the standard protocol for the mass propagation and ex-situ conservation of this endangered orchid species.