摘要
采用携带GUS基因双元表达载体p1301-Pubi-Ecppc的根癌农杆菌菌株C58c1、LBA4404和EHA105对4个春小麦品种的幼胚愈伤组织进行了遗传转化。结果表明,3M1.5为最佳诱导培养基;扬麦158和扬麦10是用于小麦遗传转化的良好基因型。对农杆菌转化条件进行进一步优化,得出用农杆菌C58c1侵染预培养15d的扬麦158幼胚愈伤组织,当侵染液浓度为OD6000.8、侵染40min、共培养3d时,可以提高农杆菌介导小麦遗传转化的筛选频率和PCR阳性率。对所得到的具有hyg抗性的愈伤和抗性植株进行GUS基因化学组织检测和PCR分子鉴定,初步证明Ecppc基因已整合到小麦再生植株基因组中。本研究初步建立了农杆菌介导小麦的遗传转化体系。
Calli were induced from immature embryos of 4 spring wheat genotypes. The immature embryogenic calli were infected by A. tumefaciens （C58cl, LBA4404 and EHA105） harboring binary plasmid vector p 1301-Pubi-Ecppc. The results showed that 3M1.5 was the best medium, and Yangmai 158 and Yangmai 10 were better genotypes for wheat genetic transformation;and studies were conducted on the response of various genotypes and mediums to separate culture and factors that influence Agrobacterium-mediated transformation of wheat. After optimizing these factors, we found that it can improve the Agrobacterium-mediated transformation selective frequency of wheat and PCR positive frequency to make use of strains of Agrobacterium tumefaciens C58c1, pre-cuhure 15d wheat immature embryos-derived calli from Yangmai 158, concentration of bacterium suspension OD6000.8, time of infection 40min, co-culture 3 days. Some hyg-resistant calli and hyg-resistant plants were obtained. By means of PCR amplification and GUS histochemical analysis of regenerated plants indicated that Ecppc has been inserted into wheat genome, and efficient transformation system of Type Ⅱ calli of wheat mediated via Agrobacterium tumefaciens has been developed. These proved the established genetic transformation system of the wheat through Agrobacterium- mediated.
出处
《核农学报》
CAS
CSCD
北大核心
2007年第2期124-127,131,共5页
Journal of Nuclear Agricultural Sciences
基金
国家自然科学基金(30370853)
粮食丰产科技工程重大科技专项(2004BA520A12)
关键词
小麦
愈伤组织
农杆菌
遗传转化
wheat
calli
Agrobacterium tumefaciens
genetic transformation
