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

This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.