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以萌发大豆幼苗顶芽为外植体,经纵切及农杆菌侵染后种植到大田中,转化植株用除草剂进行表型鉴定,存活植株进行PCR验证,并分析目的基因EPSPS在T2代转基因植株中的遗传情况;总计获得草铵膦涂抹表型鉴定阳性T0植株75株,草甘膦喷雾鉴定阳性T1植株65个,PCR测序阳性T1植株6个,PCR测序检测转化效率为0.14%;获得T2代PCR阳性植株52个,初步证明目的基因EPSPS能在子代中遗传;该方法能有效解决基因型依赖及再生植株困难等问题,缩短转化周期,为根癌农杆菌阶段的大豆遗传转化体系的优化与改良提供了参考。
The top buds of the germinating soybean seedlings were used as explants and the plants were inoculated into the field after being cut and infected with Agrobacterium tumefaciens. The transformed plants were phenotyped by the herbicide, and the surviving plants were verified by PCR. The target gene EPSPS was analyzed in the T2 generation The results showed that there were 75 positive T0 plants identified by glufosinate-resistant smear phenotype, 65 positive T1 plants identified by glyphosate spray and 6 positive T1 plants by PCR sequencing. The transformation efficiency was 0.14% T2-positive PCR-positive plants 52, initially proved that the target gene EPSPS can be inherited in the offspring; the method can effectively solve the problem of genotype-dependent and difficult plant regeneration and shorten the conversion cycle for Agrobacterium stage of soybean genetic transformation system The optimization and improvement provide a reference.