Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus

An improved protocol for genetic transformation of juvenile explants of Carrizo (Citrus sinensis Osb. x Poncirus trifoliata L. Raf.), Duncan (Citrus paradisi Macf.), Hamlin (Citrus sinensis (L.) Osbeck) and Mexican Lime (Citrus aurantifolia Swingle) cultivars using a vector containing a bifunctional egfp-nptII fusion gene is described. Several parameters were investigated to optimize genetic transformation of these four cultivars. It was determined that a short preincubation in hormone rich liquid medium and subculture of Agrobacterium for 3 h in YEP medium containing 100 mu M acetosyringone were required for improvement of transformation efficiency. Co-cultivation duration as well as addition of acetosyringone to co-cultivation medium also played an important role in transformation efficiency as did OD600 value of the Agrobacterium suspension used for transformation. We regenerated numerous EGFP expressing transgenic lines from all four cultivars. Based on these results, we conclude that genetic transformation of citrus is cultivar specific and optimization of conditions for maximum transgenic production are required for each individual cultivar.