The application of modern biotechnology for mass propagation of rare and endangered species needs to develop a proper in vitro protocol. Here, a protocol was developed for high frequency in vitro multiplication of an endangered orchid, Orchis catasetum. Protocorms, as explants were cultured on Murashige and Skoog (MS) medium fortified with different concentrations of N⁶-benzyladenine (BA), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) either individually or in combination. A combination of 0.5 mg·L BA and 0.5 mg·L NAA was found to be suitable for maximum protocorm-like bodies (PLBs) regeneration (20.40 × plantlet^-1). The largest number of root (7.16 × plantlet^-1) and leaf (10.10 × plantlet^-1), also the highest plant height (114.20 mm × plantlet^-1) and root length (193.40 mm × plantlet^-1) were obtained on MS medium supplemented with 0.5 mg·L BA a long with 0.5 mg · L NAA. Plantlets with well-developed leaves and roots were transplanted to pots filled with perlite, wood pieces, ionolite and mineral cartridge shell (1:1:1:1), also perlite individually and transferred to the greenhouse. Upon ex vitro transfer, 100% of plants survived.

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