Effect Of Ipt-bar Binary Genes On Growth And Development In Rice(Oryza Sativa)

To regulate the leaf senescence, low temperature tolerance and obtain the herbicide resistant characters, binary vector containing ipt gene and bar gene was transformed into rice through Agrobacterium tumefaciences and micro bombardment, the physiological characters and main agronomic traits of ipt-bar transgenic rice were studied. The results are as the follows:1. Establishment of rice transformation system mediated by AgrobacteriumAt the basis of rice callus regeneration system of our lab, we optimized the influencing factors of rice transformation mediated by Agrobacterium. The callus from mature or immature embryo subcultured 1-3 times were immersed in Agrobacterium EHA105 haboring expression vector whose OD600 is about 0.5-0.7, and sip up with sterile filter paper after 15min, then transferred the callus to solid medium NBD2-As.Three days later, removing excess Agrobacterium, inoculating the callus on the NBS medium (with 4 mg·L~-PPT) for screening, and keeping selecting pressure subculture 3 times. When the seedlings are about 10cm high, refining and transplanting to garden pot in green house. The transformation frequency of indica rice is 4.4%, of japonica rice is 9.7%,and of the Guizhou Huishui black sticky rice reached 13.6%.2. Optimization of micro bombardment parameters transformed riceExpression vector pBinAipt-bar was delivered into rice immature embryo cells by using particle bombardment method. The effects of high-osmotic ways and pressure of rupture disks on transformation efficiency of rice immature embryo cells were studied Via computing the frequency of resistant callus. Cultivating the callus on high-osmotic medium 6 hours before bombarding and maintaining high-osmotic 18h after bombardment, and 1100 psi or 1350psi rupture disks is proper for rice transformation. However, for Guizhou Huishui black sticky rice, rupture disks 1100 psi is better, and 1350psi for Zhonghan21. 3. Detection select of transgenic rice plants and physiology charactersThe binary expression elements with ipt gene and bar gene is transformed to rice via Agrobacterium mediated method or micro bombardment.PCR,TR-PCR and herbicide spraying experiment demonstrated that target gene has integrated into rice genome. Target gene inheritated to T4 progeny in EY105 and T3 progeny in R527 .Transgenic plants live normally longer than control under low temperature (4'C). Analysis of physiological characters at normal temperature and low temperature stress revealed that there isn't markedly difference between the main physiological characters transgenic rice and that of control. However, under low temperature stress the electrolyte leakage of transgenic plants decreased compare with control, the chlorophyll content of leaf, soluble sugar content, soluble protein content and proline content of transgenic plants leaves increased, POD activity, SOD activity and MDA content behave good exhibition.At the later bearing days, the photosynthesis rate, POD activity and chlorophyll content of leaf are notably higher than that of control. The bearing time prolonged about 15days compared with control. Through analysis of agronomic traits, cold tolerance and characteristic of herbicide resistant,2 stable inheritated transgenic lines were obtained. Compared with respectively control, the output of transgenic R527ibl increased 25.4%,and of transgenic EY105ib6-19 increasedl25.3%.In this study, binary expression vector containing ipt gene control by Act promoter and bar gene promoted by Pnos were delivered to rice firstly, and analysis the physiological characters and agronomic traits of transgenic rice. The transgenic lines behave delaying leaf senescence, cold tolerance increased and herbicide resistant were primarily obtained, which can use as the new germ plasm in rice breeding.