T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices

Vita.A routine and rapid transformation system for rice using Agrobacterium-mediated gene transfer and the meristematic shoot apex has been established. Shoot apices of Oryza sativa L. cv. Maybelle were explants for cocultivation and gene transfer using Agrobacterium containing plasmids for bar gene expression driven by the CaMV 35S promoter (pGSFR781A) or by the rice actin 1 promoter (pBARNPT). Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinateammonium (PPT), have established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices had been cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Several key factors were identified as being essential for obtaining transgenic rice plants. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells were most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, Fl and F2 generation plants were confirmed by molecular analyses and herbicide application tests. PCR analysis of 7 primary rice plants and Southern blot analysis of DNA (Bam Hi digested) from 1 0 primary rice plants indicated that the bar gene was present in at least 5 plants. The correlation between PCR analysis and Southern blot analysis was high. Southern blot analysis (Xba I digested) and leaf application of IgniteR to progeny from one of the transgenic primary plants established that the bar gene and nptll gene were present, and the plant showed resistance to IgniteR. A high correlation between a positive hybridization signal and resistance to the application of IgniteR was confirmed. A germination test of F2 progeny from one of the transgenic plants (Fl) showed a non-Mendelian inheritance pattern. The inactivation of the transferred foreign gene appeared to be due to loss of the transgene rather than methylation