Studies On Agrobacterium-mediated Transformation Of Welsh Onion With BT Gene

In recent years, the emergence and spread of pests on Welsh Onion causes enormous losses in production. Chemical control not only costs much and the result is not satisfactory, but also causes serious pollution to environment. In order to improve the resistance to pests of Welsh Onion, we have studied on its regeneration system and Agrobacterium-mediated transformation system. The success of pect-resistant Welsh Onion is expected to play a role in breeding and provide technical accumulation for insect-resistant breeding.In our study we selected seeds, kerf seeds and middle cut seeds as explants. Then we transformed Welsh Onion with BT gene and Gus gene mediated by Agrobacterium, and the main results were summarized as follows:1. The optimization of regeneration system of Zhangqiu Welsh Onion. Used seeds, kerf seeds and middle cut seeds as explants, the result showed that middle cut seeds was the best explant, and the calli induction rate could reach to 72.97%.2. The determination of the concentration of Cef. Cef was used to restrain the growth of Agrobacterium after directly infected the calli. We made sure that the best concentration was 300 mg/L determined by calli induction rate and calli browning rate.3. The optimization of transformation system. The calli were soaked in Agrobacterium tumefaciens suspension, of which the OD600 value was 0.6. Besides, the best infecting time was 30 minutes, after directly infecting the calli were transferred onto the solid co-cultivation medium with pH 5.6, and co-cultured 3 days under 25℃, dark conditions. Then the biggest rate of resistant calli was obtained which could reach to 43.99%. 4. The gain of transformed plants. It was proved by PCR and PCR-Southern blotting that there were only 4 transformed plants in 256 regenerate plants.5. Diversity of exogenous gene integrity. It was proved by PCR that there were 5 plants contained gus gene in the 256 regenerate plants, but only 4 plants contained Bt gene. It was showed that part of or all the T-DNA was integrated into plant DNA in the process of transformation. Therefore, the choice aimed on gus gene may lose the transformation of part of the target gene.