Marker-free Transgenic Indica Rice Harboring Two Insect-resistant Genes Obtained By Agrobacterium Tumifaciens Mediated Transforming

Rice is one of the most important crops in the world, as we know ,the insect pests' damage is one of the main factors that restrict rice yield. Plant transgenic technology makes up for tradition breeding methods to breeding resistant rice. In recent years transgenic plants of insect-resistant have been widely popularized and applied. Bt and CpT I are most effectively transferred. However resistant genes transferred into plants are restricted by its resistant spectrum, and researches show that tolerance could be induced. So it's effective to breeding multiple -mechanism resistant rice to control insect pest damages. Transgenic rice harboring two insect-resistant genes is important germplasm for breeding effect rice varies.Marker genes are used to select transgenic cells and obtain transgenic plants. However it's unnecessary any more once transgenic plants are produced, even results other problems. Thus the demand for marker-free transgenic plants is one of the popular tendency. Several techniques to eliminate marker gene have been developed. The co-transformation system seemed to be the simplest one. Two independent T-DNA segments could integrate into different sites of plant genome when co-transformation, and marker-free plants could be obtained.Co-transformation strategy and was used to make the transgenic system of marker-free in the research:pCDMARUBAC-HYg was designed in vitro ,Interest genes Bt+sck were constructed in different T-DNA fragment from marker gene hyg m double T-DNA vector .Vector was transferred to embryonic calli derived from immature embryos of Indica rice by Agrobacterium tumefaciens strain LBA4404 in order to select marker-free plants. 1. Transferred calli by the double T-DNA vectors was about 9.1 percent, and 148 cloneswere obtained. About 454 seedlings were regenerated with the frequency of 25.7 percent.2. PCR screening results indicated that Co-transformation was found in 53.6 percent TO transgenic plants indicted. And different transformations were tested in the same calli, so several genotypes were found. Marker-free plants were selected also in TO generation.3. PCR screening was done to analyze Tl generations derived from TO inbred seeds. Separation between interest and marker gene happened to most transgenic plants ,and marker-free plants were obtained; co-integration were inspected. Separation was detected in Marker- free plants derived from marker-free TO generation.4. 42 homozygous lines of marker-free were obtained in T2 generations by PCR screening.5. ELISA analysis indicated that content of Bt protein was about 0.01-0.22 percent of dissolvent protein in transgenic plant leaves .In some lines, Bt was found super expression in individual rice plant after gene recombination and seperation in passing-down of descendents. Together with molecular analysis, gene silence were found . The super expression of Bt gene in some Tl generation was detected.6. Part of co-transformed plants were found obvious resistant to insect especially lepidopteron from insect investigations in field.