Publications - Faculty - Recently Submitted Faculty Publications 2009

Grosser - In Vitro Cellular & Developmental Biology

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

M. DUTT, D. H. LEE and J. W. GROSSER

Abstract. Agrobacterium-mediated transformation of carrizo citrange [Citrus sinensis (L.) Osbc. × Poncirus trifoliata (L.) Raf.] with a binary vector containing a novel bifunctional reporter-selection fusion gene comprising of an in-frame fusion between the manA gene with the 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.