Genetic transformation as a tool for the introduction of agronomically important genes into citrus plants.

Abstract

A procedure for increased efficiency of production of transgenic citrus plants was developed by using an Agrobacterium-mediated transformation and shoot tip grafting in vitro regeneration system. The disarmed Agrobacterium tumefaciens strain EHA 105 harboring the binary vector p35SGUSINT was cocultivated with stem and epicotyl segments from young seedlings. The beta-glucuronidase (GUS) gene in the T-DNA served as reported in the histochemical assay and the neomycin phosphotransferase II (NPT ii) gene provided resistance to kanamycin and was used as selectable marker. Shoots regenerated under kanamycin selection were harvested from the stem se,aments within 5 months. Shoot basal portions were GUS-assayed for screening transformants and the remaining portions were shoot tip grafted in vitro for production of whole plants. Integration of the GUS and NPT II genes and expression of their mRNAs in the regenerated plants was confirmed by polymerase chain reaction (PCR), Southern and Northern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for citrus. We are using this system to introduce the gibberellin-C20-oxidase gene, in sense and antisense, in citrus plants trying to modify tree size.