巴西橡胶树游离小孢子培养

Isolated Microspore Culture in Hevea brasiliensis Müll.Arg

以橡胶树品种海垦2号为材料,对橡胶树游离小孢子培养体系进行初步研究,为进一步建立其成熟培养体系提供基础。小孢子的提取采用直接研磨法和间接研磨法进行,结果发现以间接研磨法为佳,虽然工作量大,但污染率低、杂质少、培养效果好。对小孢子分别进行高温热击、Starvation Medium B溶液、对照预处理试验,发现用Starvation Medium B溶液预处理小孢子2 d有利于小孢子分化。不同的碳源比较发现麦芽糖培养效果较蔗糖好。对诱导培养基的组分、pH研究发现,以改良N6为基本培养基添加外源激素2,4-D 0.5 gm/L、KT 0.5 mg/L,pH 6.6时诱导效果好,小孢子分化率达8.33%。小孢子的分化以B途径为主,初步获得小孢子分化的细胞团和微愈伤。同时以橡胶树小孢子为受体进行电激转化试验,瞬时表达结果证明外源基因已导入部分小孢子基因组中。

A protocol of isolated microspore culture in Hevea brasiliensis Mtill. Arg（Haiken 2）was described. Two different isolation methods of indirectly grinding anthers and directly grinding anthers were tested and the former was optimal with lower contaminative rate, less impurity, and better culturing effect compared to the latter although it had a low efficiency. Furthermore, different pretreatment techniques were also studied and the pretreatment of microspores with Starvation Medium B for two days was the optimized one. In addition, the effects of different carbon sources and pH and components in induction media were investigated. The results showed maltose as carbon source and pH6.6 and modified N6 medium supplemented with 2,4-D 0.5 mg/L and KT 0.5 mg/L in induction media had an optimal induction effect with 8.33% differentiation rate of mierospores which differentiated mainly through B way. Cell masses and microcalli derived from microspores were obtained. An electroporation test using microspores as receptors were carried out and the results of transient express confirmed the exogenous gene were expressed in microspores of Hevea by GUS staining.