独花兰 ( Changnienia amoena) 是兰科单种属植物, 被列为国家二级珍稀濒危植物。独花兰既是珍贵的药用植物和潜在可开发的优良野生花卉资源, 也是研究兰科植物系统发育的优良材料。但由于独花兰野生资源匮乏, 目前缺少对其再生体系的研究。该研究以独花兰假鳞茎为外植体, 探索不同消毒方式、外植体切块方式、不同采集时间及植物生长调节剂浓度对外植体灭菌效果、不定芽诱导和组培苗生根的影响, 初步建立了独花兰再生体系。结果表明, 用 75% 乙醇处理 30 秒再用 0.1% 升汞溶液消毒 12 分钟为独花兰假鳞茎最佳消毒方法; 用不同切块方式处理假鳞茎皆能诱导不定芽, 但完整假鳞茎的诱芽率显著高于切块处理后的假鳞茎; 每年 5 月采集假鳞茎进行不定芽诱导效果最好。独花兰假鳞茎不定芽诱导最适培养基为 1/2MS+1.0 mg·L^–1 6-BA+0.5 mg·L^–1 NAA, 诱导率为 75.56%; 最适生根培养基为 1/2MS+0.4 mg·L^–1 6-BA+1.0 mg·L^–1 NAA, 生根率可达 93.33%; 组培苗经 50 mg·L^–1 6-BA 处理后移栽到腐殖土中覆膜炼苗 7 天, 可显著提高成活率, 最终成活率达 94.44%。

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.