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以吉林小粒7号大豆品种为材料,切取无菌幼苗下胚轴诱导愈伤组织,初步建立了大豆悬浮细胞系,测定不同硼酸浓度下悬浮细胞生长变化;采用农杆菌介导法对大豆悬浮细胞进行外源GUS基因的转化,先通过不同程度的超声处理确定最佳超声时长,然后在最佳超声处理时长的基础上,探讨了硼酸浓度对转化效率的影响;最后采用PCR和Southern blot杂交对抗性愈伤做分子鉴定。结果表明:大豆悬浮细胞正常生长硼酸浓度在2.0~10 mg·L-1,过高或过低均会抑制大豆细胞正常的生长,高于100 mg·L-1时抑制作用最显著。但在转化过程中,适当增加硼酸浓度(10~30 mg·L-1)则能够提高悬浮细胞的转化效率,并且有助于转化组织的筛选,借以3 min超声的辅助作用,30 mg·L-1硼酸浓度下转化效率达到1 mg转化细胞团中含28个转化体。
Using the Jilin 7 small soybean varieties as material, the hypocotyl-induced callus of the sterile seedlings was cut off and the suspension cell lines of soybean were initially established to measure the growth of suspension cells under different concentrations of boric acid. Agrobacterium-mediated transformation of soybean suspension cells The transformation of exogenous GUS gene was carried out. The optimal ultrasonic duration was determined by ultrasonic treatment at different degrees. Then the effect of boric acid concentration on the conversion efficiency was discussed based on the optimal ultrasonic treatment time. Finally, PCR and Southern blot hybridization Sexual callus make molecular identification. The results showed that the normal growth of soybean suspension cells with boric acid concentration of 2.0 ~ 10 mg · L-1, too high or too low will inhibit the normal growth of soybean cells, higher than 100 mg · L-1 inhibition most significantly. However, the appropriate concentration of boric acid (10-30 mg · L-1) could improve the transformation efficiency of the suspension cells and help the screening of the transformed tissues during the transformation process. Assisted by 3 min ultrasound, 30 mg · L -1 conversion efficiency of 1 mg at a boric acid concentration of 28 transformants in the transformed cell mass.