Studies On Increasing Resistance Of Rice To Bacterial Blight (BB) By Transformation With Double Rightborder (DRB) Binary Vector And MAS

Rice is one of the most important cereal crops. Bacterial blight (BB) caused by Xanthomonas oryzae pv. Orzae (Xoo.) is one of the most destructive diseases of rice throughout the world. The incorporation of resistance genes into cultivars is the most economic and effective method to control the disease. Gene Xa21 which is the first resisted gene separated from rice has a wide spectrum of resistance to Xoo. Gene Xa23 which separeated from wild rice by Zhang Q. has a wide spectrum of resistance to Xoo. come from China and other countries. Furthermore, it resists BB during all the Growth duration.The condition for initation and regeneration of young embryonic and mature embryonic callus were studied in order to obtain the optimization of Agrobacterium transformation system for indica rice. The 6 different indica rice varieties, Shuhui527, Mianhui725, Chuanghuill, Peiai64S, D62B, and 9311 were used to as specimens . The results showed that MS medium was better for mature embryonic callus initiation for most indica rice, while CC medium was better for mature embryonic callus prociferation, and the browning of calli could be mitigated significantly. The quality of callus can be improved and the rate of embryogenic callus were increased on the CC medium. That the concentration of mannitol influences significantly the rate of callus induction was found. The rate of induction was decreased as the concentration of mannitol increased. The colour, the quality, the growth speed and the rate of regeneration of young embryonic callus are better than mature embryonic callus. But few callus were obtained from young embryonic for restrictance of choosing materias period .The sensitivity of rice to Hyg was also studied. The results showed that different varieties have different sensitivities to Hyg, and, the same variety has different sensitivitiesto Hyg when on different media. Sensitivity to Hyg of varieties on MS medium is less than that on CC medium. The most sensitivity date to Hyg is regenetation. It is enough to distinguish the transformated plants and the non-transformated ones when the Hyg concentration is at 20mg/L, while It is fitable during the rootage and stronging seedling when at 15mg/L Hyg in medium.The influence factors to the rate of transform during the co-culture stage is the activage and concentration of bacterium. High concentration makes bacterium congregate each other, while low concentration is disadvantage to transform. The results showed that the best concentration of Agrobacterium was OD6oo=0.2.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. The selectable gene(hpt) situated between two right-border. Using the DRB binary vector system, we obtained 27 transformed plants , including japonica restorer line C418 and 3 indica restorer line Minghui 86 and 11 maintainer line D62B.Fastness validate in field revealed that 17 C418 To transformed plants , 11 D62B and 3 Minghui 86 ones resist BB. PCR analysie proved that 7 D62B and 3 Minghui 86 transformed plants were masculine.PCR analysie plants. At the same time , the field bioassay proved that all of the transformed plants were obviously resistant to BB than control. In additional, Southern blot analysie proved that 5 C418 To transformed plants and their offspring only incluse Xa21 gene but not hpt gene . So we obtained SMF transgenic lines containing single copy Xa21 in the rice cultivars C418. Approximately 18.5% of the primary transformants of C418 produced SMF progeny. Thus, the DRB binary vector is an efficient vector for producing SMF plant.Molecular marker-assisted selection(MAS) was wildly used to meliorate the fastness of crops in produce. So it is a good way to improve BB resistance of rice by MAS. The Xa23 gene resistant to bacterial blight (BB) was introduced to restorer line C418 , Minghui86 and sterile line by MAS and traditional backcross breeding . PCR analysie using primer RM206 and the field bioassay indicated that C4\S-Xa23 , Minghui86- Xa23,PA64- Xa23 are all resist BB. New indica hybrids was obtained by combinated them with Jiazao312A , Peiai64, Xiushuill and 666. By field bioassay indicated that they are all highly resisted BB.