In-vitro Conservation of Some Endangered Orchids of Himalayan Region

Abstract

Orchids (28000 species belonging to 763 genera), family Orchidaceae, are well known for their colorful flowering, fragrance, ornamental and some medicinal uses (Pillon and Chase 2007). By the end of 2020, the IUCN Global Red List included assessments for 1641 orchid species, of which 0.30% are extinct, 45.52% are threatened, 12.00% are critically endangered, 21.63% are endangered, and 5.79% are vulnerable. IUCN Global Red List (IUCN 2020). Due to destruction of natural habitat and the damages caused by locals or tourists, the many orchid species are at verge of extinction. It is difficult to multiply orchids in their natural habitats as their propagation rate is very slow. So there is a felt need to develop faster the propagation techniques to prevent the orchids from their extinction. This study is focused to develop the methods of propagation and conservation of the orchids through artificial seed technology under in-vitro conditions. In present study, the shoots of Dendrobium nobile Lindl., Coelogyne flaccida Lindl. and Rhyncostylis retusa (L.) were inoculated on MS medium supplemented with 87 mM sucrose and different concentrations of 6-benzylaminopurine (BAP-0, 1, 2.5 and 5 μM) and Indole acetic acid (IAA-0, 1, 2.5 and 5 μM). The highest number of shoots were recorded on medium containing 2.5 μM BAP and 5 μM IAA in all three orchids species under study. For the formation and multiplication of PLBs, the leaf explants were inoculated on MS medium supplemented with different concentrations of BAP (0, 1, 2.5 and 5 μM). The optimum result for both formation and multiplication of PLBs in all three orchid species were recorded on medium containing 2.5 μM BAP. After PLBs formation, the PLBs were inoculated on MS medium supplemented with different concentrations of sorbitol (0, 82.5, 165 and 247 mM) for maturation. Most PLBs of D. nobile (31%), C. flaccida (40%) and R. retusa (29%) matured on 247 mM sorbitol concentration followed by 165 mM sorbitol concentration. After maturation, the effect of plant growth regulators and sucrose on conversion of PLBs into plantlets was also studied. Mature PLBs were inoculated on MS medium supplemented with different concentrations and combinations of BA (0, 0.1 μM), GA3 (0, 1 μM) and sucrose (87, 174, 261 and 348 mM). The conversion rate was higher on MS medium supplemented with 0.1 μM BA, 1 μM GA3 and 261 mM sucrose concentration. The matured PLBs were also encapsulated in sodium alginate-calcium chloride beads to produce artificial seeds. The effect of different concentrations of sodium alginate (1%, 2%, 3%, 4%) and calcium chloride (50 mM, 100 mM, 200 mM) was studied. The best bead formation was observed Page | 11 in 3% sodium alginate and 100 mM calcium chloride. The storage effect on artificial seeds at two different temperatures 4 ℃ and 25 ℃ was also recorded at 10, 20, 30, 40, 50 and 60 days. It was observed that the artificial seeds of Dendrobium nobile were viable for longer period at 25 οC, whereas artificial seeds of Coelogyne flaccida and Rhyncostylis retusa were viable for longer period at 4 οC rather than 25 οC. The clonal fidelity of in-vitro germinated seeds of D. nobile, C. flaccida and R. retusa was also compared with that of mother plants using RAPD marker. All the marker scored using 26 RAPD primers were monomorphic showing clonal fidelity of the regenerated plants.