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Elimination of the nptII marker gene in transgenic apple and pear with a chemically inducible R/Rs recombinase

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Abstract

The efficient production of marker-free transgenic plants is still a challenge in most fruit species even though such plants are a necessary component of many “new breeding technologies”, particularly cis- and intragenesis. Marker-free plant production is also necessary for the successive stacking of genes in an elite fruit transgenic line. Here, we used a R/Rs site-specific recombinase that is post-translationally regulated by dexamethasone through fusion with a ligand-binding domain for this hormone, and a bi-functional selectable marker gene coding for a cytosine deaminase/neomycin transferase (codA–nptII) protein; this enabled a first step of positive kanamycin selection, followed by a second step of negative 5-fluorocytosine selection. The aim of our study was to optimize this system on the apple cv. Galaxy and on the pear cv. Conference by conducting a detailed study of the effects of dexamethasone and 5-fluorocytosine treatments, and by comparing an early versus a delayed selection strategy. We were able to produce marker-free transgenic pear plants for the first time, and confirm the feasibility of producing marker-free transgenic apple plants using a chemically inducible recombinase system. We recommend the use of an early selection strategy for the pear cv. Conference and a delayed selection strategy for the apple cv. Galaxy.

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Acknowledgments

This project was funded by the ISAFRUIT EU Project (6th PCRD) and post-doctoral grants from Region Pays de la Loire and Angers Agglomération. In addition, this work was supported by the Grant ANR-11-BTBR-0006-GENIUS. The plasmid pRCNG was kindly provided by Dr. Schaart (PRI, Wageningen). The authors gratefully acknowledge the INEM team and particularly J. M. Boré, N. Dousset and L. Leclout for excellent technical assistance in the greenhouses, and B. Billy (SNES-GEVES) for technical assistance in flow cytometry. Technical contributions from J. G. Bertault, C. Cesbron, T. Clochard, F. Dupuis, F. Simoneau and S. Sourice are also gratefully acknowledged.

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Author notes

  1. S. Djennane

    Present address: UMR 1131 Santé de la Vigne et Qualité du Vin, INRA, Université de Strasbourg, 68000, Colmar, France

  2. K. Loridon

    Present address: UMR 1334 Amélioration, Génétique et Adaptation des Plantes, INRA, 34060, Montpellier, France

Authors and Affiliations

  1. UMR1345 Institut de Recherche en Horticulture et Semences, INRA, 49071, Beaucouzé, France

    L. Righetti, S. Djennane, P. Berthelot, R. Cournol, N. Wilmot, K. Loridon, E. Vergne & E. Chevreau

  2. UMR1345 Institut de Recherche en Horticulture et Semences, SFR 4207 QUASAV, PRES L’UNAM, Université d’Angers, 49045, Angers, France

    L. Righetti, S. Djennane, P. Berthelot, R. Cournol, N. Wilmot, K. Loridon, E. Vergne & E. Chevreau

  3. UMR1345 Institut de Recherche en Horticulture et Semences, AgroCampus-Ouest, 49045, Angers, France

    L. Righetti, S. Djennane, P. Berthelot, R. Cournol, N. Wilmot, K. Loridon, E. Vergne & E. Chevreau

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  1. L. Righetti
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  2. S. Djennane
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  8. E. Chevreau
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Correspondence to E. Chevreau.

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Righetti, L., Djennane, S., Berthelot, P. et al. Elimination of the nptII marker gene in transgenic apple and pear with a chemically inducible R/Rs recombinase. Plant Cell Tiss Organ Cult 117, 335–348 (2014). https://doi.org/10.1007/s11240-014-0443-2

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  • DOI: https://doi.org/10.1007/s11240-014-0443-2

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