| Agrobacterium-mediated rice genetic transformation has become an indispensable research method in rice molecular biology.Agrobacterium-mediated transformation is the main method of rice genetic transformation.However,Agrobacterium-mediated rice genetic transformation was strongly dependent on the genotype.In this study,japonica cultivar Nipponbare,indica cultivars Minghui86 and 9311 were used as genetic transformation materials to explore different selectable markers,different culture conditions,two embryonic morphogenic genes and ternary vector system on rice genetic transformation efficiency.It helps us to optimize the rice genetic transformation system,and establish a ternary vector system for improving the efficiency of Agrobacterium-mediated transformation of rice.The main results are as follows:The mature embryos derived from Nipponbare and Minghui86 were used as genetic transformation materials to research the effects of different selectable markers Hpt and Bar on genetic transformation efficiency.The results showed that the selectable efficiency of Hpt was superior to Bar in both cultivars,and the transformation cycle of Hpt was significantly shortened.The mature embryos derived from indica cultivar Minghui86 were used as genetic transformation material to explore the effects of morphological regulatory genes BBM and WUS2 on embryogenesis efficiency and genetic transformation efficiency of indica.However,the results showed that there was no significant difference in the genetic transformation efficiency between the constructed vector containing morphological regulatory genes and the common vector.The mature embryos of Nipponbare were used as the genetic transformation material to compare the double T-DNA vector(single plasmid double T-DNA)and ternary vector(double plasmid double T-DNA)containing the same target gene RFP.The result showed that the regeneration rate of double T-DNA vector was higher than that of ternary vector system.In the ternary vector system,p VS1 is more suitable as the replicon of helper plasmid than RK2.The mature embryos derived from Minghui86 and 9311 were used as genetic transformation materials to explore genetic transformation efficiency of indica under different culture conditions.It was determined that continuous 28 days of dark culture at phases before pre-regeneration,including cocultivation,resting,and selection,was more suitable for the transformation of indica.Four ternary vector systems were introduced into mature embryos derived from Minghui86 and 9311.The results showed that the ternary vector system with additional vir genes improved the genetic transformation efficiency of Minghui86.But in 9311,the transformation rate of ternary vector was still not high,the highest transformation rate is only 2.6%.The ternary vector system still needs further improvement. |
