Isolated Microspore Culture in Hevea brasiliensis Müll.Arg
A protocol of isolated microspore culture in Hevea brasiliensis Mull.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 microspores 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.