Cost-Effective in vitro Multiplication and Phenolic Profile of an Important Medicinal Orchid, Satyrium nepalense D. Don

Abstract In this study we report a cost-effective and reproducible protocol for large-scale multiplication of an important medicinal orchid, Satyrium nepalense through a four-step protocol, involving asymbiotic seed germination, multiplication of protocorm-like bodies (PLBs), development and multiplication of shoots, and finally in vitro rooting of the developed shoots. Out of various media tested for seed germination, Mitra’s medium supplemented with 0.1 % peptone (BM) supported the highest (79.19 %) seed germination. Eight-weekold protocorms were sub-cultured on BM alone and supplemented with different concentrations (1-8 μM) of BAP, KN, or TDZ. The highest number of shoots was developed on the medium containing 4 μM KN. Here we also observed that the highest number of shoots were produced from protocorms cultured on a 2 % isubgol gelled medium out of the seven different low-cost gelling agents used. Further, optimal elongation of shoots was observed on BM alone and 2 % guar gum, among the tested gelling agents. Elongated shoots were transferred for rooting on BM containing 0-2 μM of IAA, IBA, or NAA. The best rhizogenic response was observed on BM fortified with 0.5 μM IBA and isubgol (3 %). The rooted plantlets showed a 76 % survival rate on acclimatization after transfer to the potting mixture of sand and vermiculite (1:1). HPLC analysis of therapeutically important phenolic acids of leaves and tubers of in vitro regenerated and in vivo plants revealed the presence of higher levels of selected phenolic acids in in vitro tissues than those from their native habitat. This protocol could facilitate the conservation and propagation of this important medicinal orchid.