Genetic Transformation

In Vitro Cellular & Developmental Biology - Plant

, Volume 46, Issue 6, pp 467-476

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

  • Manjul DuttAffiliated withCitrus Research and Education Center, University of Florida/IFAS
  • , Dong H. LeeAffiliated withCitrus Research and Education Center, University of Florida/IFASNational Institute of Horticultural and Herbal Science, Rural Development Administration
  • , Jude W. GrosserAffiliated withCitrus Research and Education Center, University of Florida/IFAS Email author 

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Abstract

Agrobacterium-mediated transformation of Carrizo citrange [Citrus sinensis (L.) Osbeck × 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 egfpnptII 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.

Keywords

Agrobacterium tumefaciens Bifunctional gene Citrus EGFP Genetic transformation Mannose selection Kanamycin selection