Axenic rhizome culture and genetic fidelity assessment of Eulophia dabia (D. Don) Hochr: an endangered terrestrial orchid species

Eulophia dabia (D. Don) Hochr is an endemic, critically endangered, terrestrial orchid species having profound importance in traditional herbal drug preparations and pharmacopoeia worldwide. In the present investigation, we have developed an efficient and genetically stable micropropagation protocol of E. dabia using axenic rhizome as explant. Seeds inoculated on half-strength Murashige and Skoog (MS) medium showed 5% germination efficiency. Scanning electron microscopy analysis revealed differential effects of various sterilizing agents (NaOCl 2 and ethanol) on the seed coat integrity leading to a significant improvement in seed germination rate. The rhizome-like bodies (RLBs) developed after 6 mo of seed germination showed best growth of rhizome (size: 3.3 × 1.5 cm and 5.0 nodes per rhizome) in MS medium containing casein hydrolysate and activated charcoal. One-year-old mature axenic rhizomes were further sub-cultured for multiple shoot induction, and maximum 96.1% response was observed in MS medium fortified with 4.4 μM BAP and activated charcoal with 4.3 mean shoot number and 13.4 cm shoot length. After 3 to 4 sub-cultures using the same medium, bipolar structures (plantlets with shoot and root) were developed. The properly developed plantlets were successfully acclimatized to open environment with 98% survival. Genetic stability of the discussed protocol was evaluated by random amplified polymorphic DNA (RAPD) markers, which proved true to typesets of the in vitro raised plants. In the present study we optimized in vitro propagation system for the E. dabia which can be successfully applied further for the genetic improvement studies and commercial scale propagation.