Establishment Of Effective Agrobacterium-mediated Mature Embryottransformation System For Indica Rice

Rice（Oryza sativa L.）is one of the most important food crops. Increasing of rice yield is anessential factor to secure sufficient food supply for the growing population. Japonica and indica are twosubspecies of rice growing in different regions of the world. About80%of world rice production isbased on indica varieties. It has been suggested that biotechnology, combined with traditional breedingmethods and other technologies, can contribute to the agronomic improvement of indica and make itpossible to achieve the production requirements. The role of genetic transformation of indica playingwill be more and more important in the improvements of exist elite varieties, characterization of genefunction and construction of new rice mutants.In recent years, Agrobacterium-mediated transformation of rice has been widely used in japonicatransformation. However, the transformation of indica rice is still difficult, mainly because the inductionof embryogenic callus is not routine and the rate of plant regeneration from transformed calli is low,which is determined by the genetic differences between the two subspecies. In this study, a highlyefficient gene transfer method mediated by Agrobacterium tumefaciens was developed for N22andQ4041using mature seeds. Some relevant parameters affecting transformation efficency were alsooptimized in different varieties such as Q25, IR64, MH86and PA64S. The main results are as follows:1. By analyzing3factors (the basic medium, ABA and hormones) and4levels of them on theinduction effect, we found that MS medium was more suitable for callus induction of Q25, IR64, MH86,PA64S and N6medium was suitable for N22and Q4041. We also found that the exogenous hormones2,4-D and ABA were essential substances for rice embryogenic callus induction.2. By comparing the selection effects of3common marker genes to resistant calli induction, theresult showed that the BAR gene is the most effective selectable marker in tested indica genotypes. Inaddition, comparison of two agents, G418and Paromomycin, we found that G418was more efficientthan Paromomycin (P).3. In the resistant calli regeneration, by choosing Agrobacterium-infection methods, soakingthrough Agrobacterium infection, shaking meantime, and adjusting drying time to25min afterco-culture of the calli, we improved the regeneration frequency of the rice varieties N22and Q4041.4. We optimized Agrobacterium-mediated transformation system of N22and Q4041, andimproved the transformation frequency from29%to44%.5. Based on the results of this study and well-established japonica rice transformation system inour lab, we propose some strategies to improve efficiency of indica transformation and also put forwardthe future development of transgenic rice.