Using The Right Border (drb) Binary Vector System Without Marker Gene In Transgenic Rice

Rice is one of the most important cereal crops. Thirty-fivepercent of the world population, especially the people in developing countries, are living on this crop. Bacterial blight (BB) caused by Xanthomonas oryzae pv.orzae(Xoo) is one of the most destructive diseases of rice throughout the world. The loss in rice production caused by BB in some areas of Asia can be as high as 50%. The incorporation of resistance genes into cultivars is the most economic and effective method to control this disease. Gene Xa2l has a wide spectrum of resistance to Xoo. However traditional backcross breeding is generally a tedious and time-consuming task and often produces linkage drags in spite of utilization of marker-assisted selection(MAS). So it is the best way to improve BB resistance of rice by genetic transformation.In order to obtain the optimization of Agrobacterium transformation system for indica rice. We studied the condition for initation and regeneration of embryonic callus varied, the 6 different indica rice varieties were used to this thesis for investigation on these aspects in detail. The results showed that for most indica rice MS medium was better for callus initiation. While CC medium was better for callus subculture, and the browning of calli could be mitigated significantly.The quality of calli can be improved and the rate of embryogenic calli were increased in the CC medium. We also found that the concentration of mannitol influence significantly the rate of callus induction. The rate of induction was decreased with theconcentration of mannitol increasing . We investigated the optimal transformatin and selection scheme. The results showed that MS medium was better for calli selection and regeneration than CC medium. The best concentration of Agrobacterium was OD600=0.2.Currently employed transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interesting (Xa21) to select transformed cells from a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as rice (Oryza sativa L) is of increasing public concern. Thus the demand for selectable marker- free(SMF) transgenic plants is one of the most popular tendency. We have constructed a double right-border(DRB) binary vector carrying two sets of T-DNA right-border(RB) sequence flanking a selectable marker gene, followed by a Xa21 and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first KB containing the selectable gene(hpt) and the Xa21,then the other initiated from the second RB containing the Xa21,were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny plants with only the Xa21.Using the DRB binary vector system, We abtained 41 transgenic plants , including japonica restorer line C418 and indica restorer line shuhui 527, chuanghui 11 .PCR and Southern blot analysis revealed that forein gene were intergrated into the genome of these plants. At the same time , the field bioassay revealed that all of transgenic plant wereobviously resistant to BB than control. In additional, We recovered SMF transgenic lines containing single copy Xa21 in the rice cultivars C418. Approximately 18% of the primary transformants of C418 produced SMF progeny. Thus, the DRB binary vector is an efficient vector for producing SMF plant.