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

Genetic Transformation

DOI: 10.1007/s11627-010-9300-0

Cite this article as:
Dutt, M., Lee, D.H. & Grosser, J.W. In Vitro Cell.Dev.Biol.-Plant (2010) 46: 467. doi:10.1007/s11627-010-9300-0

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 

Copyright information

© The Society for In Vitro Biology 2010

Authors and Affiliations

  1. 1.Citrus Research and Education CenterUniversity of Florida/IFASLake AlfredUSA
  2. 2.National Institute of Horticultural and Herbal ScienceRural Development AdministrationJejuSouth Korea

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