以甘露糖作为筛选剂的棉花遗传转化

Cotton Transformation with Mannose as Selective Agent

以pmi基因作为筛选标记基因,以甘露糖作为筛选剂,通过农杆菌介导法将GFP基因导入棉花细胞并得到再生植株,经过PCR检测、Southern杂交证实外源基因已经整合到棉花基因组中,Westbloting与荧光显微镜检测证明GFP基因得到表达。本文研究讨论了甘露糖作为棉花转化细胞的筛选剂在农杆菌介导的转化中的应用浓度及方法,即:甘露糖的筛选浓度在30～50g·L 1之间,在愈伤组织诱导初期适当低一点,随着愈伤组织的生长而加大筛选浓度。由于甘露糖不利于再生胚的分化,当愈伤组织转入分化培养基时,要以葡萄糖代替甘露糖。

Employing pmi as a selection marker gene and mannose as a selection agent, the GFP gene was transformed into cotton via agrobacterium and regeneration plants were acquired. Transgenic plants were confirmed by southern hybridization and PCR analysis, expression of the GFP gene was showed with fluorescence microscope and West blotting. The concentration and method for employing mannose as selection agent had been studied. The reasonable selection concentration of mannose should be 30~50 g·L^(-1). The start concentration of mannose in callus-induced medium is less and the concentration increased as callus grow up. Because mannose is not beneficial for embryo differentiation and development, mannose will be replaced with glucose when the callus was transferred to differazation medium.