In vitro regeneration by callus culture of Anoectochilus elatus Lindley, an endangered terrestrial jewel orchid

A process of organogenesis via callus with successful plantlet formation was developed for Anoectochilus elatus. Indirect organogenesis was achieved from in vitro-derived node, internode, and leaf explants. The explants were cultured on Mitra medium fortified with different concentrations and combinations of plant growth regulators such as cytokinins (N-6-benzyl adenine [BA], thidiazuron [TDZ], kinetin [KN], N-6-(2-isopentyl) adenine [2ip] and zeatin [ZEA]), auxins (2,4-dichlorophenoxyacetic acid [2,4-D], alpha-naphthalene acetic acid [NAA], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and 4-amino-3,4,6-trichloro picolinic acid [Pic]), and additives (citric acid, trisodium citrate, peptone, coconut water, potato extract, and banana pulp). Organogenic callus proliferation was highest from internode (77.8 %), followed by node (69.7 %) and leaf explants (64.2 %), on Mitra medium supplemented with TDZ (1.0 mg L-1) and NAA (0.5 mg L-1). Organogenic callus derived from internodal explants produced an average of 41.8 shoots per explant, with average length of 2.5 cm, on Mitra medium supplemented with BA (1.0 mg L-1), NAA (0.5 mg L-1) and coconut water (10 %). In rooting experiments, a maximum of 3.2 roots per shoot was observed with an average length of 2.1 cm with 97.8% response on Mitra medium amended with AgNO3 (1.0 mg L-1). The rooted plantlets were acclimatized in a mixture of garden soil, sand, vermicompost, and used tea waste (8:4:2:1 [w/w/w/w]) in a greenhouse environment, with a 72.3% survival rate. Finally, the well-developed plants were transferred to the National Orchidarium, Yercaud, Tamil Nadu, a unit of the Botanical Survey of India, Southern Regional Centre, for further maintenance and establishment under natural conditions for conservation.