子房注射法转化‘紫宝石’蝴蝶兰的研究

Genetic Transformation of Phalaenopsis ‘Purple Gem'(Orchidaceae)via Ovary-injection

采用子房注射法对‘紫宝石’蝴蝶兰进行外源DNA导入研究,结果表明:不同时期注射和不同注射处理方式对‘紫宝石’蝴蝶兰的落果率影响不同,对胚座组织和种子的GUS染色率的影响也不同,在授粉后65 d时,以2%DMSO为缓冲液,注射400μg/mL浓度质粒,种子的GUS染色率最高达6.14%。选取各注射时期无菌播种后获得的1 000个原球茎,经系列浓度的潮霉素筛选,结果发现授粉后65 d注射时获得的抗性植株最多,为31株,转化率为3.1%。切取转化植株幼叶及幼根进行GUS检测,均为阳性;随机挑选7株幼苗进行PCR检测,均能扩增出GUS基因和HPT基因条带,说明外源基因已经整合到植株的基因组内。此研究为兰科植物育种提供了一种简单高效的途径。

The method of ovary injection was reported for Phalaenopsis ‘Purple Gem＇ transformation in this study. Results showed that different injection treatments in different development stage of P. Purple Gem had different effect on the percentage of fertile fruit and GUS staining rate of seeds and placentas. The highest GUS staining rate of seeds from the mature fruit by ovary-injection was 6.14%. The highest 3.1% hygromycin-resistance plants were gained after hygromycin（Hyg） screening by a series of concentration in 65 DAP fruit. Seven plants selected randomLy were detected by GUS assay and PCR analysis of GUS and HPT, and all of them were positive. The results showed the integration and expression of foreign gene were integrated into hygromycin-resistance plants. The successful application of ovary injection for P. Purple Gem transformation could provide a simple and efficient method for the Orchidaceae breeding.