电击法诱导的欧白英原生质体转化和转化植株再生

Transformation of Solatium dulcamara Protoplasts and Regeneration of Transgenic Plants

本研究使用电击法成功地将带有标记基因NPTⅡ的质粒pCaMVNEO转入欧白英的原生质体,并获得了再生转化植株。通过用pDW2质粒进行的CAT基因短暂表达研究,确定了欧白英原生质体转化的电击条件为:电容30nF、电场强度1500V/cm、时间衰变常数59.4微秒;质粒DNA浓度为20μg/2×10~6原生质体。在以上条件下,欧白英原生质体的相对转化率为12.4％,绝对转化率为2.4×10^(-4)。在大多数抗性愈伤组织和从抗性愈伤组织再生的植株中检测到新霉素磷酸转移酶活性。分子杂交结果也证明了在转化植株中存在NPTⅡ基因序列,而未转化的对照植株则没有。

We have achieved successful transformation of Solanum dulcamara protoplasts by direct DNA uptake and regeneration of transgenic plants. The plasmids pDW2 carrying CAT gene and pCaMVNEO carrying NPT I gene were used. The electropora-tion voltage was 1 500V,which gave a field strength of 1 500V/cm with a time decay constant of 59. 4u sec. The concentration of plasmid was 20ug/2× 106 protoplasts. Under these conditions,a very high transformation efficiency was obtained,with relative transformation frequency being up to 12.4% and absolute transformation frequency 2. 4×10-4. The activity of NPT I was detected in 75% of the kanamycin resistant calli and all of the plants regenerated from resistant calli. The southern blotting analysis showed that the DNA sequence of NPT I gene derived from pCaMVNEO plasmid existed in the transformed plants of Solanum dulcamara.