High efficiency Agrobacterium-mediated transformation and regeneration of citrus

A procedure for increased efficiency of production of transgenic citrus plants was developed by extending the exposure of the explants to the selection agent and by grafting in vitro the regenerated shoot apices onto seedling rootstocks. Carrizo citrange (Citrus sinensis L. Osbeck x Poncirus trifoliara L. Raf.) stem segments from in vitro grown seedlings were cocultivated with Agrobacterium tumefaciens EHA 105 carrying the binary vector p35SGUSINT. The introncontaining @-glucuronidase (GUS) gene in the T-DNA served as reporter in the histochemical assay and the neomycin phosphotransferase II (NPT II) gene provided resistance to kanamycin and was used as selectable marker. Regenerated shoots were harvested from the stem segments within 5 to 6 months. Extended time periods of selection greatly improved recovering of transformed shoots and reduced the occurrence of escapes. However, prolonged exposure to kanamycin favoured the regeneration of chimeric shoots. Shoot basal portions were GUS-assayed for screening transformants and the remaining portions were shoot tip grafted in vitro for production of whole plants. Citrus genetic transformation was confirmed by polymerase chain reaction (PCR). Southern and Northern analysis. The transformation efficiencies obtained are the highest reported so far for citrus.