Asymbiotic Germination and Leaf Explant-Based Regeneration of the Endangered Medicinal Orchid <em>Hemipilia cucullata</em> from Mature Seeds

Hemipilia cucullata (Orchidaceae) is a terrestrial orchid widely valued for its medicinal properties and ornamental appeal, yet its wild populations are endangered due to habitat degradation, low seed viability, and limited pollination success. To support its conservation and sustainable utilization, this study developed an optimized protocol for the asymbiotic germination of mature seeds, protocorm development, shoot elongation, and protocorm-like body (PLB) induction from sterile leaf explants. Seeds cultured in 1/2 MS liquid medium supplemented with 0.5 mg/L NAA showed a germination rate of 72 ± 6%, significantly higher than the control (46%) and solid medium (31%), with germination initiating approximately 21 days earlier in liquid culture. Protocorms were subcultured on B5 medium containing 0.5 mg/L BA, 0.2 mg/L NAA, and 100 mL/L coconut water, leading to efficient proliferation. After 5-6 weeks, plantlets were transferred to shoot elongation medium (B5 supplemented with 0.5 mg/L BA and 1 mg/L NAA), where they exhibited robust shoot elongation and healthy leaf formation. For PLB induction, the highest induction rate (44.3 ± 5.1%) was achieved on MS medium containing BA (3 mg/L) and NAA (0.2 mg/L). This comprehensive and reproducible protocol can be an effective platform for the large-scale propagation and ex situ conservation of H. cucullata, offering valuable support for recovery efforts of this endangered orchid.