以密花石斛(Dendrobium densiflorum Lindley ex Wallich)为研究试材,对植物SDS方法提取基因组DNA中的若干影响因子诸如药品、试剂配制方法、DNA取材部位、蛋白质去除次数、DNA析出时间、提取样品水浴时间等进行了比较分析。结果表明:不同的SDS缓冲液配制方法提取效果差异较大,Wang's配方产率及纯度都较高;石斛花苞的DNA提取产率较叶片高,但纯度较叶片低;提取过程中,适宜的水浴时间当为30～40min,过短DNA产率下降,过长会引起DNA的降解,或最后产物中增加其他杂质;氯仿/异戊醇抽提次当为2～3次,对去除DNA中蛋白质、多糖及其它杂质有一定的作用,过多则DNA损失也明显;异丙醇沉淀时间10min时核酸析出量在80%以上,纯度相对较高,继续沉淀的产物中DNA含量及纯度明显变低,同时采取挑取絮状沉淀或再用无水乙醇纯化10min的方法,可减少蛋白质、多糖等其它杂质的影响。
[Objective] This study was to germinate the hybrid seeds of [Den.Burana Green Star × Den.Rainbow-compactum] under aseptic condition and to explore the parameters for rapid propagation of F1 plantlets via tissue culture.[Method]Hybridization between Den.Burana Green Star（female parent）and Den.Rainbow-compactum（male parent）was performed and the in vitro culture and proliferation of F1 hybrids were studied using eight different basic media including MS,1/2MS,1/3MS,1/4MS,B5,N6,modified Knudson and H.[Result]Improved Knudson medium appended with 1.0 mg/L 6-BA,1.0 mg/L NAA and 10% mature banana puree performed best in F1 seed germination under aseptic condition,as well as the rapid propagation of protocorm-like body.Of all the eight media tested,1/2MS is the medium most suitable for the in vitro rapid propagation of the F1 seedlings.Efficiency of eight media in the in vitro rapid propagation was in order 1/2 MS MS1/3 MS1/4 MS ≈N6 improved Knudson ≈B5H.NAA presented better rooting and growth-promoting effect in the in vitro rapid propagation of the F1 seedlings than IBA.And the optimal NAA concentration to recommend from our experiment results was 2.0 mg/L.[Conclusion]Our experimental results provided mature method and important technological information for hybrid breeding dendrobium.
