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利用C58c1农杆菌菌株(其携带的pPTN290质粒载体上含有nptⅡ基因和GUS基因)为供体材料和小麦幼胚为受体材料,对我国35个春小麦基因型进行了农杆菌敏感性研究和转化研究。组织化学染色结果表明,PM97034、P187、巴140105、扬麦158、新春9号和克丰6号等基因型农杆菌感染后GUS,基因瞬时表达率达到了15.0%～30.0%,显著高于模式基因型Bobwhite。其余幼胚分别在含10～25mg/LG418培养基上诱导愈伤组织和分化植株,抗性再生植株经ELISA检测、PCR检测、叶片离体退绿检测、XGluc染色检测和Southernblot检测,最终从新春9号、PM97034和Bobwhite获得了转基因植株,转化效率分别为3.3%、2.0%和1.1%,新春9号和PM97034的农杆菌转化效率高于Bobwhite。Southernblot检测结果同时表明,外源基因在多数转基因植株中表现为单拷贝整合。T1代植株ELISA检测结果和PCR检测结果表明,外源基因在个别株系中的分离符合孟德尔遗传规律,而在多数株系中的分离偏离了孟德尔遗传规律。阳性植株与阴性植株分离比经χ2c适合性检测,表明外源基因在多数株系中的遗传符合3∶1分离规律。本文首次对不同小麦基因型进行了农杆菌敏感性和转化效率研究,筛选到新春9号和PM97034两个对农杆菌比较敏感且转化效率比较高的基因型,可望广泛用于小麦转基因研究。
Agrobacterium-mediated transformation and transformation of 35 spring wheat genotypes in China were studied by using C58c1 Agrobacterium tumefaciens strain carrying pptN290 plasmid vector containing nptⅡ gene and GUS gene as donor material and wheat immature embryo as acceptor material . The results of histochemical staining showed that the transient expression rate of GUS gene after infection with genotypes PM97034, P187, Ba 140105, Yangmai 158, Xinchun 9 and Kefeng 6 reached 15.0% ~ 30.0%, which was significantly higher than that of model Genotype Bobwhite. The other immature embryos induced the callus and differentiated plants in medium containing 10 ~ 25mg / L LG418 respectively. The resistant regenerated plants were detected by ELISA, PCR, in vitro green leaves detection, XGluc staining and Southern blot detection, Transgenic plants were obtained on the 9th, PM97034 and Bobwhite, with transformation efficiencies of 3.3%, 2.0% and 1.1%, respectively. The Agrobacterium transformation efficiency of New Year 9 and PM97034 was higher than that of Bobwhite. Southern blot results also showed that exogenous genes showed single copy integration in most transgenic plants. T1 generation of the ELISA test results and PCR results showed that exogenous gene isolation in individual lines in line with Mendelian genetic law, but in most strains of the deviation from Mendelian inheritance. The ratio of positive plants to negative plants was tested by χ2c fitness test. The results showed that the inheritance of foreign genes in most strains was in accordance with the 3: 1 separation rule. In this paper, the Agrobacterium sensitivity and transformation efficiency of different wheat genotypes were studied for the first time. Two genotypes, Xinchun 9 and PM 97034, which were more sensitive to Agrobacterium and more efficient in transformation, were selected and could be widely used in transgenic studies of wheat.