Delignification of Seeds and Culture as a Tool for Early Asymbiotic Seed Germination in Habenaria panchganiensis, A Critically Endangered Terrestrial Orchid of Western Ghats (India)

Terrestrial orchids are very difficult to germinate and cultivate in vitro. Orchid seeds are hydrophobic in nature which limits the interaction of seed with culture medium, as a result very low or no germination occurs in vitro. In present study, a protocol was established for early asymbiotic seed germination of Habenaria panchganiensis. Delignification of seeds was achieved by using variable concentrations of sodium hypochlorite (NaOCl) for a variable time duration. The nutritional requirement of seeds was quantified by subjecting seeds to six different media for asymbiotic seed germination experiments in solid and suspension cultures along with variable concentrations of cytokinins (6-benzylaminopurine & kinetin). BM1 medium proved superior for asymbiotic seed germination in suspension culture (along with 4mgL^-1 Kin). Different concentrations of sucrose were used to increase the tuber size because tuber reintroduction is easy and effective for the survival of in vitro developed plants. BM1 supplemented with 4 mg L^-1 Kin and 40 g sucrose showed higher tuber length (5.15 ± 0.37 cm), breadth (0.96 ± 0.17 cm) and fresh weight (2.42 ± 0.23 g). This study may help in mass propagation of the species which, in turn, may prove helpful in its conservation.