Agrobacterium-mediated transformation of CP4 gene into indica rice
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摘要:
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 Abstract: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 glyphosate-resistant gene (CP4-EPSPS) was introduced into the embryogenic callus of ‘Zhonghui 161’ by Agrobacterium-mediated method. The transformed embryogenic callus was screened for glyphosate resistance on the selective medium containing 300, 350 and 400 mg·L−1 glyphosate. 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−1 glyphosate 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. [Ch, 5 fig. 20 ref.] -
图 1 籼稻‘中恢161’胚性愈伤组织在不同质量浓度草甘膦培养基中培养20 d后褐化率
Figure 1 Browning rate of O.sativa subsp.indica ‘Zhonghui 161’ callus cultured in different concentrations of glyphosate for 20 days
图 2 籼稻‘中恢161’胚性愈伤组织在不同质量浓度草甘膦培养基中培养20 d后的褐化情况
Figure 2 Browning rate of O. sativa subsp. indica ‘Zhonghui 161’ callus cultured in different concentrations of glyphosate for 20 days
图 3 籼稻‘中恢161’再生植株CP4基因的PCR检测
Figure 3 PCR result of CP4 gene from glyphosate-resistance plants
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