Wang G, He P, Davey M R
Beijing Agricultural University, China.
Chin J Biotechnol. 1994;10(1):33-41.
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 NPTII gene were used. The electroporation voltage was 1500 V, which gave a field strength of 1500 V/cm with a time decay constant of 59.4 sec. The concentration of plasmid was 20 micrograms/2 x 10(6) 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 x 10(-4). The activity of NPTII was detected in 75% of the kanamycin resistant calli and all of the plants regenerated from resistant calli. Southern blot analysis showed that the DNA sequence of NPTII gene derived from pCaMVNEO plasmid existed in the transformed plants of S. dulcamara.
我们通过直接摄取DNA成功实现了欧白英原生质体的转化以及转基因植株的再生。使用了携带CAT基因的质粒pDW2和携带NPTII基因的质粒pCaMVNEO。电穿孔电压为1500V，场强为1500V/cm，时间衰减常数为59.4秒。质粒浓度为20微克/2×10⁶个原生质体。在这些条件下，获得了非常高的转化效率，相对转化频率高达12.4%，绝对转化频率为2.4×10⁻⁴。在75%的卡那霉素抗性愈伤组织以及所有从抗性愈伤组织再生的植株中检测到了NPTII的活性。Southern杂交分析表明，来自pCaMVNEO质粒的NPTII基因的DNA序列存在于欧白英的转化植株中。
