Abstract: INTRODUCTION: Changnienia amoena is listed as a national second-class protected plant. It is a rare orchid species unique to China, as well as a valuable medicinal plant and a potential wild potted flower germplasm resource with great development potential. 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: The results showed 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 that the complete pseudobulbs> the halved pseudobulbs> the cruciform block pseudobulbs and the rate of germination was much higher than that of the blocking treatment; (3) Considering the browning rate, contamination rate, survival rate and induced germination rate, the optimal collection time for C. amoena pseudobulbs to be used as explants for adventitious shoots induced in May; (4) The optimal medium for the adventitious shoots of C. amoena pseudobulbs was 1/2 MS+1.0mg∙L–1 6-BA+ 0.5mg∙L–1 NAA, with an induction rate of 75.56%; the optimal rooting medium is1/2 MS+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, the pseudobulbs of C. amoena were used 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, and to conduct a preliminary study on the regeneration system of C. amoena, which 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, indeterminate buds, rooting culture