An efficient in vitro propagation protocol for Habenaria edgeworthii Hook. f. ex. Collett using seedderived callus was established. The maximum seed germination was observed in Murashige and Skoog (MS) medium supplemented with 1.0 µM α-naphthalene acetic acid (NAA). Induction of callus was achieved on full and ½-strength MS medium supplemented with 1.0 µM NAA. The highest number of shoot (11.9 shoots/explant) was achieved in MS medium supplemented with 0.1 µM 6-benzyladenine (BA) and 0.01 µM NAA. Further, elongated shoots when transferred to ½-strength MS rooting medium with different auxin concentrations induced roots (41.6–83.3%) and tubers (0–20.8%); however, a maximum of 87.5% rooting was achieved in a plant growth regulator (PGR)-free MS medium. Rooted shoots (plantlets) when transferred to a mixture of soil:sand:perlite (1:1:1 ratio) resulted in 68% survival. Inter-simple sequence repeats (ISSR) markers confirmed the genetic stability among regenerated plants. The phytochemical analysis of tissue culture-raised tubers showed higher phenolic content than wild tuber. The regeneration protocol developed in this study provides a basis for germplasm conservation and harnessing the total phenol and phenolic compounds of H. edgeworthii. Further, the methods can open avenues for application in other Orchidaceous plants of the Indian Himalayan region.
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