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

  • Genetic Transformation
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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 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.

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Acknowledgments

We thank Dr. Dennis J. Gray and Dr. Zhijian Li (MREC, University of Florida/IFAS, Apopka, FL, USA) for providing us with a construct containing the bifunctional egfp–nptII fusion gene. Gary Barthe and Monica Vasconcellos are thanked for their technical assistance. We also thank FCPRAC (Florida Citrus Production Research and Advisory Council) for grants supporting this research.

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Authors and Affiliations

  1. Citrus Research and Education Center, University of Florida/IFAS, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

    Manjul Dutt, Dong H. Lee & Jude W. Grosser

  2. National Institute of Horticultural and Herbal Science, Rural Development Administration, 1318 Harye, Namwon, Seogwipo, Jeju, 697-943, South Korea

    Dong H. Lee

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  1. Manjul Dutt
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  2. Dong H. Lee
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Correspondence to Jude W. Grosser.

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Editor: J. Ranch

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Dutt, M., Lee, D.H. & Grosser, J.W. Bifunctional selection–reporter systems for genetic transformation of citrus: mannose- and kanamycin-based systems. In Vitro Cell.Dev.Biol.-Plant 46, 467–476 (2010). https://doi.org/10.1007/s11627-010-9300-0

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