Abstract: INTRODUCTION: Changnienia amoena is listed as a national second-class protected plant. It is a rare orchid species unique to China, a potential wild potted flower germplasm resource with great development potential, and a valuable medicinal plant as well. Currently, wild resources of C. amoena are rapidly declining, making conservation efforts crucial. RATIONALE: Research on C. amoena regeneration technology has mainly focused on different combinations of plant growth regulators, and a complete regeneration system has not yet been established. Tissue culture technology plays a crucial role in the conservation of wild C. amoena resources, and research on its regeneration system can promote the sustainable development and utilization of these resources. RESULTS: We found that: (1) 75% ethanol had a significant effect on the sterilization of C. amoena pseudobulbs, and the best sterilization treatment for C. amoena pseudobulbs was to use 75% ethanol for 30 seconds, followed by 0.1% mercuric acid solution for 12 minutes; (2) The pseudobulbs were able to induce adventitious buds under the different blocking treatments, but the effect of the adventitious buds was the following order: the complete pseudobulbs>the halved pseudobulbs>the cruciform block pseudobulbs, and the induction rate of the complete pseudobulbs was much higher than that of the blocking treatment; (3) Considering the browning rate, contamination rate, survival rate and induction rate, the optimal collection time for C. amoena pseudobulbs to be used as explants for inducing adventitious shoots was in May; (4) The optimal medium for inducing adventitious shoots of C. amoena pseudobulbs was 1/2MS+1.0 mg·L^–1 6-BA+0.5 mg·L^–1 NAA, with an induction rate of 75.56%; the optimal rooting medium was 1/2MS+0.4 mg·L^–1 6-BA+1.0 mg·L^–1 NAA, with a rooting rate of up to 93.33%; (5) After treatment with a 50 mg·L^–1 6-BA solution, the histocultured plantlets were transplanted into humus soil and covered with plastic film for hardening. The survival rate was as high as 94.44%. CONCLUSION: In this study, we used the pseudobulbs of C. amoena as explants to explore the effects of sterilization conditions, the way of explant cutting, different collection times, and the concentration of plant growth regulators on the sterilization effect of explants, inducing adventitious shoots and rooting of histocultured plantlets. The regeneration system of C. amoena can help to protect the germplasm resources of C. amoena, and provide a theoretical basis and technical references for the future propagation of C. amoena.

Key words: Changnienia amoena, pseudobulb, in vitro regeneration, adventitious buds, rooting culture