In this project we utilized Arabidopsis as an in vivo experimental system to analyze gene function using rice as gene source. Using the heterologous method, we identified rice cDNAs that exhibited the desired Arabidopsis phenotypes. These cDNAs were capable of producing the same phenotype in plants other than Arabidopsis. To analyze whether these rice cDNAs could function beyond the plant species from which they were isolated, we constructed transgenic tomato plants expressing these cDNAs via Agrobacterium-mediated transformation.
Rice full-length cDNAs amplified from the genomic DNA of selected FOX lines were cloned into the expression vector pBIG2113SF. The plasmids constructed were introduced into Agrobacterium EHA105 by electroporation. Segments of cotyledons and hypocotyls of tomato (Solanum lycopersicon cv. Micro-Tom) were infected with Agrobacterium containing the plasmids. Transformed cells were selected with hygromycin resistance as a selectable marker. Regenerated plants were grown under fluorescent light (16 h light/8 h dark) at 28 °C. Insertion of the hygromycin resistant gene was confirmed by a polymerase chain reaction (PCR) using the genomic DNA of the regenerated plants as template. To remove tetraploid plants that were frequently produced via the transformation procedure, ploidy of regenerated plants was analyzed using a flow cytometer.