In vitro propagation and asymbiotic seed germination for the conservation of the endangered orchid Eulophia andamanensis Rchb.f.

Eulophia andamanensis Rchb.f., an endangered terrestrial orchid endemic to the Andaman and Nicobar Islands, is severely threatened by habitat loss, over-collection and climate change. The present study developed an optimized in vitro propagation protocol encompassing seed germination, protocorm-like body (PLB) formation, shoot multiplication, root induction and acclimatization to support ex situ conservation and potential reintroduction. Immature seeds cultured on various media showed the best response in full-strength Orchimax medium, with early germination observed at 60.30 days after culturing (DAC), maximum seed germination (92.20 %) at 90 DAC and early PLB formation at 74.90 DAC. Later, PLBs were transferred to full-strength Orchimax medium supplemented with 1.0 mg L-1 6-benzylaminopurine (BAP) and 0.5 mg L-1 α-naphthalene acetic acid (NAA) initiated shoots within 10.20 DAC, producing 4.73 shoots per explant with an average shoot length of 4.67 cm and 4.05 leaves after 90 DAC in the shooting medium. Rooting was optimal in Orchimax half-strength medium containing 2.0 mg L-1 indole-3-butyric acid (IBA), which induced root initiation at 10.30 DAC, with 4.77 roots per shoot and an average root length of 4.40 cm at 90 DAC in rooting media. Hardened plantlets grown in a red soil: sand: vermicompost: cocopeat (2:1:1:1) mixture exhibited an 85 % survival rate after 60 days of secondary hardening in the greenhouse. This efficient and reproducible protocol provides a reliable platform for the largescale propagation, genetic conservation and restoration of E. andamanensis in its natural habitats.