Efficient plant regeneration via protocorm-like body induction from stem nodes in Anoectochilus roxburghii (Wall.) Lindl.

Anoectochilus roxburghii (Wall.) Lindl., a highly valued yet endangered medicinal orchid, faces significant challenges in propagation due to its limited natural regeneration capacity. This study established an efficient in vitro regeneration system to achieve rapid large-scale propagation, with clear culture cycles for each stage: stem segment explants were subjected to 110 d of initiation culture to obtain sterile materials, 160 d of protocorm-like body (PLB) induction culture to achieve a high proliferation coefficient, 90 d of rooting culture, and 60 d of acclimatization and transplantation. Stem segment explants with nodes were used as explants and cultured on an initiation medium. Through combined full-factorial and orthogonal experiments, the optimal hormone combinations for different regeneration pathways were identified. Notably, a high frequency of PLBs was directly induced from axillary bud meristems without an intervening callus phase on the medium supplemented with 1.5 mg/L 6-BA, 1.0 mg/L NAA, and 0.1 mg/L KT, a regeneration mode that is rare in A. roxburghii, achieving a remarkably high proliferation coefficient of over 30.0. Furthermore, the optimal rooting medium was full-strength Murashige and Skoog (MS) medium supplemented with 0.5 mg/L NAA, yielding a 100% rooting rate. The rooted plantlets were successfully acclimatized with a survival rate exceeding 95%. Flow cytometry analysis showed no ploidy variation in regenerated materials at different culture stages, confirming the genetic stability of the system. The findings highlight a novel and efficient PLB-mediated regeneration pathway, which provides a robust technical foundation for the conservation and commercial production of this threatened medicinal plant.