转抗除草剂基因小麦植株的筛选方法研究

Screening of Transgenic Wheat Plants Resistant to Herbicide

为了降低小麦转bar基因时产生较高比例的假阳性植株,采用测定T0代转基因植株叶片释放NH4+浓度和对T1代植株喷施Basta除草剂等辅助筛选方法,建立了简便有效的转bar基因小麦筛选体系。研究结果表明,2叶期和5叶期叶片释放的NH4+浓度与bar基因PCR检测结果呈极显著相关,相关系数分别为0.86和0.75。对391株T0代再生植株进行叶片释放NH4+浓度测定,筛选到34株转基因植株,其中27株为bar基因的PCR检测所证实。用100 mg/L Basta除草剂对381株T0代植株的后代(3 800余株)进行喷施筛选,根据后代抗性分离筛选到13株T0代阳性植株,与PCR检测结果吻合度达到100%。这两种方法可用于以bar基因为选择标记基因的转基因植株的筛选。

Screening and identification of transgenic plants are the key factors for successful transformation. To decrease the ＂escape＂ plants in wheat transformation experiments, the detection of ammonium ion released from the leaves of T0 transgenic plants and spraying Basta herbicide on T1 transgenic plants were studied to establish a simple and efficient way to screen transgenic wheat plants with bar gene. The results showed the concentrations of ammonium ion from transgenic plants had significant correlations of r= 0.86 at two-leaf stage, and r=0.75 at five-leaf stage with the result from PCR analysis. Thirty-four transgenic plants were obtained by ammonium assay from 391 To plants regenerated on selection medium, among which 27 transgenic plants were confirmed by PCR analysis for bar gene. Thirteen transgenic T0 lines obtained by spraying 100 mg/L Basra herbicide on the progenies derived from 381 To plants were all validated by PCR analysis. The result indicated that the two methods can be used to screen transgenic wheat plants when the bar gene is used as the selectable marker.