| Rice is one of three important crops worldwide, particularly, is leading crop in China. Rice disease has been resulted in severe damage over several decades. Breeders have pursued to develop new cultivars holding disease resistance and enhance productivity of rice in breeding programs. With the recent progress of transformation technology for rice and some available gene were cloned, it is to be possible to create any cultivar. to which people wanted.Indica rice is leading cultivar form. The ratio of producing area of Indice rice is 70%. However, the majority of Indica rice cultivars are recalcitrant in both tissue culture and attempt of transformation, which has restricted severely application of transferring breeding in indica rice. So, it is necessary that establishing highly efficient reproducible system and the technology for the simultaneous introduction of multiple genes into one plant in this kind of rice was necessary for developing a new cultivar holding traits, such as resistance to insects and diseases, high produce and good quality.Four important parents of hybrid rice were screened for calli indication and culture. R6547 was the best among four cultivars. So it was used to do co-transformation research. Therefore, T4 lysozyme gene and pyrophosphatase gene (ppase) were transferred simultaneously into R6547 by using two plasmid / two-strain. The results were following:1. Studied on highly efficient tissue culture and transformation: CC medium with 1ml/g ABA is suit to many Indica rice cultivars. The choice of plant tissues as starting material was one of the most important factors. Callous of some cultivars was difficult to induce from scutella, but much easy from immature embryos, which significantly increased indication frequency of calli. Adding 1 ml of a liquid medium (AAM medium) on co-culture medium might enhance transformation efficiency. Slightly osmotic treatment of calli (partial desiccation treatment) dramatically enhanced the regeneration ability of Indica rice. In our experiment, the transfomation efficiency by using EHA105 or LBA4404 was alike for R6547.2. Establishment of an efficient tissue culture and transformation system for R6547: Optimizing conditions of tissue culture, induction calli frequency of immature embryos reached highly at 99% and the ratio of differentiation of calli was 80%. Approximately four hundred transformants were regenerated under antibiotic selection. The onehundred forty transgenic plants of one hundred sixty-six putative transformed plants were confirmed by PCR analysis. The ratio of positive plants was 84.3%.3. The co-transformation frequency by a two plasmid/ two-strain approach: Many reports indicated that only one copy of a foreign gene was inserted into a plant cell in most situations when using Agrobacterium-mediated transformation. It is one of the reasons why genetic transformation by Agrobacteriume is superior to by Particle bombardment. But, it is also the reason why co-transformation frequency was lower by Agrobacteriume than by Particle bombardment. In our experiment, sixteen plants of all one hundred sixty-six putative transformed plants were successfully co-transformated with both plasmids containing in separated Agrobacterium, which gave the 9.6% of co-transfomation efficiency. The transformants contained either lysozyme gene or ppase genes were sixty-six and fifty-eight, respectively, which account for 49.4% or 44.6% of mono-transfomation efficiency.4. Southern blotting analysis of the total DNA extracted from transferring lysozyme gene plants indicated that the T-DNAs were integrated into the rice genome, and most transgenic plants had one copy of T-DNA insert. The test results of inoculation with Rhizoctonia solani showed that some transformants had exerted higher inhibitor efficiency on the development of sheath blight disease than non-transformed control plant. However, The disease on a few transformants was more serious than non-transformed control plant.5. Western blotting analysis of the total protein extract...
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