Bifunctional selection-reporter systems for genetic transformation of citrus: mannose- and kanamycin-based systems

Agrobacterium-mediated transformation of Carrizo citrange [Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.] with a binary vector containing a novel bifunctional reporter-selection fusion gene comprising an in-frame fusion between the manA gene and egfp gene is detailed. This system combined the phosphomannose isomerase positive selection system with the ability to monitor gene expression in a non-destructive manner using EGFP. Transgenic plants stably expressing the EGFP protein were regenerated following Agrobacterium-mediated transformation using a vector containing this fusion gene. We also obtained comparable transformation efficiencies when Carrizo explants were transformed using another Agrobacterium strain containing a binary vector with a bifunctional egfp-nptII fusion gene. Regenerating shoots were selected on medium containing 15 g L-1 mannose supplemented with 5 g L-1 sucrose for the manA-based selection or on medium containing 100 mg L-1 kanamycin for the nptII-based selection. Our results indicated that the mannose-based antibiotic-free selection combined with visual identification of transgenic shoots using EGFP allows for early elimination of escape non-transgenic shoots and can provide a viable alternative to antibiotic-based selection systems in the genetic transformation of citrus and other crops.