<em>In vitro</em> propagation and genetic stability assessment of an endangered terrestrial jewel orchid <em>Anoectochilus elatus </em> Lindl.

Abstract

<em>Anoectochilus elatus</em> Lindl. is an endangered terrestrial jewel orchid habituated in the Eastern and the Western Ghats of Tamil Nadu, India. It is endangered due to lack of suitable pollinators for ovary fertilization and low fruit development. Moreover, issues such as its germinationwhich depends up on thesymbiotic association with mycorrhizal fungus with their tiny embryosand indiscriminate collection cause scarcity of this species in nature. Hence, in the present study we have developed an efficient and reproducible micropropagation protocol for eco-restoration programmes for this species. The explants axillary bud and shoot tip responded positively in Murashige and Skoog medium (MS) supplemented with different concentrations of thidiazuron (TDZ) alone and combination with cytokinins, auxins, complex extracts and antioxidants at various concentrations. Among the different experimental trails, MS medium supplemented with 1.5 mg/L TDZ and 50 mg/L peptone proved beneficial for producing multiple shoots. A maximum of 17.2 shoots/explant were derived from axillary bud followed by shoot tip (14 shoots/explant) explants. <em>In vitro</em> rooting of micro shoots was observed on MS medium supplemented with 3 g/L activated charcoal (AC). Genetic stability of <em>in vitro </em> raised plantlets was analysed through Inter Simple Sequence Repeats (ISSRs) molecular markers. Band patterns revealed 2.38% polymorphism and 97.61% monomorphism with genomic uniformity that of the mother plant. The regenerated plantlets were successfully acclimatized in a greenhouse environment with 68.42% survival rate. Finally, acclimatized plants were shifted to the Vattakanal Conservation Trust, Pambarpuram, Kodaikanal, Tamil Nadu for further maintenance and establishment under natural conditions.