利用激光微束穿刺法将pBI121质粒DNA导入百脉根。该方法是用高渗缓冲液对百脉根子叶进行预处理,通过激光微束对子叶细胞进行穿刺,然后在卡那霉素培养基上筛选出具抗性的绿色小苗。转化植物在含卡那霉素浓度为100 μg/ml条件下筛选培养三代后,获四株转基因植物,取其中两株进行总DNA PCR扩增分析,皆为阳性。证实GUS基因已导入百脉根中。通过研究建立了激光微束穿刺法转化百脉根的技术体系,从而证明该转化方法操作简便,重复性好,转化频率较高,为高等植物遗传转化提供了一个新途径。

In several ways of introduction of foreign gene into plants, laser microbeam puncture is a potential and available method for transformation of foreign genes directly into plant tissue. The purpose of this research is uptaking the laser microbeam puncture technique to introduce pBI121 plasmid DNA containing GUS gene into Lotus. The laser microbeam irradiation source was a third harmonic Nd∶YAG laser. Before irradiation, the cells of cotyledon must be pretreated in high osmotic buffer. Then cultured the punctured tissues on screening media containing kanamycin to select green transgenic plantlets. After the seedings were selected on screening medium (km 100 mg/l) for three generations, we got four transgenic plants. Two of them were tested by PCR with GUS special primer. The results were positive, and proved that the GUS gene had been introduced into Lotus. We have established a laser microbeam puncture transformation system of Lotus, and proved that this system was easy to operate and replicate with relative high transformation frequency, thus providing a new way for high plant genetic transformation.