用PEG法把外源基因导入甘蓝型油菜原生质体再生转基因植株

PEG-MEDIATED GENE TRANSFER INTO BRASSICA NA- PUS PROTOPLASTS AND REGENERATION OF TRANSGENIC PLANTS

通过PEG处理把外源基因导入甘蓝型油菜原生质体。转化介质中二价阳离子的种类和浓度、携带DNA及PEG溶液的pH值都会影响基因导入效率。以潮霉素抗性和卡那霉素抗性作标记,均成功地筛选到了抗性愈伤组织。相对转化频率分别为1.3%和0.2%。前者明显高于后者。把抗性愈伤组织转到分化培养基上,分化出芽。诱导生根后移栽到土壤中,生长状况良好。酶活性测定和Southern blotting分析表明外源基因已插入到植物细胞基因组中。该遗传转化系统存在的主要问题是抗性愈伤组织分化频率较低。本文对其原因作了初步分析。

Foreign genes were introduced into protoplasts of Brassica napus L. by means of PEG treatment. Gene transfer efficiency was found to be influenced by two-valence cations and their concentrations in transformation mixture, by addition of carrier DNA, and by pH of PEG solution. With hygromycin or kanamycin resistance as selection markers, resistant calli were successfully recovered. The relative transformation frequencies for hygromycin resistance was 1.35-6, being distinctly higher than 0.2% for kanamycin resistance. Thereafter, resistant calli were transferred onto differentiation medium for shoot induction. After regenerated shoots were rooted on rooting medium, they were transplanted into pots. Most of them grew well. Enzyme assay and Southern blotting analysis showed that foreign genes had been integrated into plant genomes. Lower frequency of shoot differentiation from resistant calli was the main problem in the gene transfer system reported here, some reasons for which were suggested.