农杆菌介导遗传转化获得转CP4基因籼稻的研究

  目的  建立籼稻‘中恢161’Oryza sativa subsp. indica ‘Zhonghui 161’农杆菌Agrobacterium tumefaciens介导的转化体系。  方法  以籼稻‘中恢161’的成熟胚为材料，设置了5个草甘膦质量浓度(100、200、300、400和500 mg·L?1)进行胚性愈伤组织的草甘膦敏感性试验。利用农杆菌介导法，将草甘膦抗性基因CP4-EPSPS导入‘中恢161’的胚性愈伤组织中，转化后的胚性愈伤组织分别在含有300、350和400 mg·L?1草甘膦的选择培养基上进行抗性筛选。抗性愈伤组织进一步分化、成苗。  结果  草甘膦质量浓度为300~400 mg·L?1时，愈伤组织褐化率约50%，具有很好的选择效果。经统计，300、350和400 mg·L?1草甘膦抗性筛选后，愈伤组织阳性率分别为40.16%、61.72%和84.04%，抗性愈伤组织的分化率为46.43%，成苗率为32.84%。共获得67株再生小苗，经PCR检测，43株成功转入CP4基因，再生植株阳性率为64.18%。  结论  建立了‘中恢161’农杆菌介导的转化体系。图5参20

Agrobacterium-mediated transformation of CP4 gene into indica rice

Objective]The objective was to establish the Agrobacterium-mediated transformation system of Oryza sativa subsp.indica’Zhonghui 161’.Method]5 groups of glyphosate concentrations(100,200,300,400 and 500 mg·L^-1)were used to test the sensitivity of embryogenic callus to glyphosate.The glyphosateresistant gene(CP4-EPSPS)was introduced into the embryogenic callus of’Zhonghui 161’by Agrobacteriummediated method.The transformed embryogenic callus was screened for glyphosate resistance on the selective medium containing 300,350 and 400 mg·L^-1glyphosate.The resistant callus was further differentiated and seeded.Result]When the concentration of glyphosate was 300-400 mg·L^-1,the browning rate of callus was about 50%,showing a good selection effect.The positive rates of callus on 300,350 and 400 mg·L^-1glyphosate were 40.16%,61.72%and 84.04%,respectively.The further differentiation rate was 46.43%,and the seedling rate was 32.84%.A total of 67 regenerated plantlets were obtained,and 43 of them were successfully transformed into CP4 gene by PCR detection.The positive rate of regenerated plantlets was 64.18%.Conclusion]Agrobacterium-mediated transformation system of’Zhonghui 161’was established.