目的 优化青叶胆Swertia mileensis组织培养快速繁殖技术,同时比较不同交配方式后代植株的再生能力,从而探讨青叶胆片断化居群繁殖保障机制。方法 以自交、近交、远交和自然结实种子为材料,相同方法消毒后接种于空白MS培养基上,60 d后统计各组萌发率;在此基础上,以各组无菌苗带节茎段为外植体,采用正交试验考察不同来源外植体在添加不同植物激素及质量浓度组合的MS培养基中诱导愈伤组织及丛芽分化能力。结果 自交、近交、远交和自然结实种子萌发率之间无显著性差异(P>0.05);适宜的愈伤组织诱导培养基为MS+ZT 0.5 mg/L+2,4-D 0.05 mg/L,在此培养条件下,自交、近交、远交和自然结实种子苗带节茎段愈伤组织诱导率分别达100%、96.67%、96.55%和96.29%,各组间无显著性差异;适宜的丛芽发生培养基为MS+BA 2.0 mg/L+KT 0.1 mg/L+NAA 1.0 mg/L,4种来源不同的愈伤组织丛芽分化率均为100%;生根则在1/2 MS+NAA 1.0 mg/L中进行,4种来源不同的试管苗生根率均为100%,生根苗在生长势上亦无明显差别。结论 优化的青叶胆组织培养快速繁殖技术,为保护其野生资源、种苗繁殖提供了有效途径,同时从植物生理学角度初步推测青叶胆近交的获益可以补偿与近交相伴的适合度损失,其后代植株在再生能力方面与异交无显著差异。

Objective To establish and optimize the technique of tissue culture and rapid propagation, and compare the progeny regeneration capacity among different mating patterns in Swertia mileensis, meanwhile, to explore the reproductive assurance of the fragment populations. Methods Sterilized seeds from different mating patterns were cultured on MS media for germination, which included self-breed, inbreeding, outbreeding, and nature pollinating, and counted the germination rate 60 d later. The study used the different source explants maintained on MS medium supplemented with different types and concentration of plant growth regulator combinations to explore its capacity of callus induction and adventitious shoots differentiation with orthogonal design. Results There is no significant difference in the germination rates among all kinds of seed as touched above. The optimal medium for callus induction is MS + ZT 0.5 mg/L + 2,4-D 0.05 mg/L, the callus induction rates were 100%, 96.67%, 96.55%, and 96.29%; The optimal medium for adventitious shoots differentiation was MS + BA 2.0 mg/L + KT 0.1 mg/L + NAA 1.0 mg/L, and the differentiation rates all reached up to 100%; The optimal medium for rooting was 1/2 MS + NAA 1.0 mg/L, the rooting rate both were as high as 100% and there was no significant difference in the growth of plantlets. Conclusion This study optimizes the cultivation conditions and provides an effective solution for protecting the wild resources and sprouts multiplication of S. mileensis. Meanwhile, from the plant physiological basis, it could be preliminarily predicted that the advantage of inbreeding would make up the fitness cost associated with it and the progeny regeneration capacity shows no significant difference between inbreeding and outbreeding.

国家自然科学基金资助项目(31260077);云南省教育厅重点项目(2012Z005);湖南省高校创新平台开放项目(11K053)