利用子房注射法进行西瓜遗传转化

Transgenic Watermelon Plants Transformed with Ovary Injection

摘要: 利用子房注射法，在西瓜自交系W1-4和W1-12自花授粉后的不同时间，分别导入几丁质酶基因和葡聚糖酶基因。果实成熟后对坐果率进行调查，结果显示，在授粉后20h进行子房注射坐果率最高。对T0代植株进行田间卡那霉素筛选和PCR检测，结果表明，不同西瓜自交系子叶期对卡那霉素敏感性存在显著基因型差异，西瓜自交系W1-4和W1-12卡那霉素临界筛选浓度分别为75mg∙L⁻¹和50mg∙L⁻¹；西瓜自交系在自交授粉后20h进行外源DNA导入有最高的阳性率，几丁质酶基因和葡聚糖酶基因对W1-4的转化率分别为2.15%和3.19%，几丁质酶基因和葡聚糖酶基因对W1-12的转化率分别为5.56%和6.74%。本试验为进一步研究子房注射法转基因技术在西瓜育种中的应用提供了参考依据。

Abstract: Several fungal pathogens have been identified in the past and have caused serious economic losses in watermelon. The expression of both chitinase and glucanase genes was attempted to achieve higher levels of fungal resistance and to broaden the spectrum of resistance against many fungal pathogens. Chitinase and glucanase genes were successfully transferred into two inbred line of watermelon W1-4 and W1-12 in different time by ovary injection in the greenhouse. The harvested rate is in the highest at 20h after self-pollination, but the harvested rate is zero at 4h after self-pollination. Transgenic plants were obtained by kanamycin screening and PCR amplification. The result of kanamycin screening showed that the sensitivity of seedling to kanamycin was significantly different between W1-4 and W1-12. In cotyledon stage, the optimal screening concentration of W1-4 and W1-12 were 75 mg∙L⁻¹ and 50 mg∙L⁻¹, respectively. Chitinase and β-1,3-gluco- sanase genes were confirmed to integrate into watermelon by PCR. The highest transformation efficiency for Chitinase and glucanase genes of W1-4 was 2.15% and 3.19% at 20 h after self-pollina- tion, respectively. And the highest transformation efficiency for Chitinase and glucanase genes of W1-12 was 5.56% and 6.74% at 20h after self-pollination, respectively. The research provided some references for the application of ovary injection transformation technology in watermelon.