Transgenic Indica Rice Harboring Two Insect-resistance Genes Obtained By Agrobacterium Tumefaciens-mediated Transformation And The Preliminary Research Of Their Descendants

In recent years, transgenic insect-resistant plants harboring Bt gene have been planted in large scale. However, plantation of these transgenic plants are facing several problems, the first is it's narrow insect resistant spectrum, the second is that the insects has become more tolerance to Bt transgenic plants. More recently, the new-generation transgenic insect-resistant plant which contains two or more insect-resistance gene is available.Rice is one of the most important crops in the world. With the increasing population and decreasing cultivated land, to further promote rice output is a very critical question for rice industry. As we know, the insect damage is one of the main factors that constraints the rice yield, and the transgene technique could be an effective way to solve the mentioned problems. In order to realize our goal, to constructed multi-resistant transgenic rice is carried out and summarized as following:1. For the purpose of selecting long duration and more wide insect-resistant spectrum transgenic rice, two insect-resistance gene including Bt, sck and marker( hpt) gene were constructed in one expression vector-pCUBAC-Hyg, then the vector was transferred into Agrobacterium tumefaciens line LBA4404 by electroporation. The callus that derivated from the immature embryos of indica rice Minghui 86, which is one of the best hybrid rice restoring line, was transfected by transformed Agrobacterium tumefaciens. After 6 times of transformation, 70 resistant clones were obtained and the frequency of transformation is 3.22%, at last 53 clones were regenerated, 45 clones(include 162 plantlets) were transplanted living. The PCR and PCR-Southern blot suggested that 137 lines of tansgenic plants have the two insect-resistance genes, 16 havesck gene only, and 9 were false positive.2. In the ELISA analysis of Bt protein content in the leaves of transgenic plant's descendants, the result revealed that Bt protein content was about 0.05-0.12% in total dissoluble protein. The result also revealed that some lines were transgenic silence in the analysis of two generation transgene lines-TI and T2, they were found have Bt gene only but no Bt protein.3. In the investigation of insect resistance from two generation in field, we found that most lines of transgenic plants have obvious resistance to insect especially to lepidopteron.4. 13 homozygous lines were found in the Ta generation, they were good breeding materials.At last, we also discussed the confusion phenomenon of segregation ratio preliminarily.