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In planta agro-infiltration system for transient gene expression in grapevine (Vitis spp.)

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Abstract

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional analysis and gene silencing studies in plants. A reproducible protocol of Agrobacterium-mediated transient gene transfer was developed for gene expression analysis on greenhouse-grown grapevines, as a complementary approach towards functional genomics and alternative to transgenics. Non-detached leaves from green cuttings were used as the target organ and vacuum infiltrated for in planta inoculation with Agrobacterium tumefaciens harboring mgfp 5-ER gene construct as visual reporter gene. Step-by-step optimization was performed and showed that the quality of greenhouse material as well as agro-infiltration conditions were the major factors which influenced successful gene expression assays. Following the optimized protocol, up to half of the infiltrated leaf surface displayed green fluorescent foci found in the intercoastal areas. Monitoring of transient Green Fluorescent Protein expression daily achieved for 2 weeks post-infiltration with the highest expression level on day 6. Evidence of GFP silencing in transgenic GFP-expressing grapevine via agro-infiltration was found for the first time. The in planta infiltration system described here provides a powerful tool to explore easily gene function in grapevine avoiding tissue culture steps and the labor-intensive generation of transgenic plants.

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Acknowledgments

We thank Dr. Mark Thomas (CSIRO Plant Industry, Australia) for kindly providing the GFP-transgenic grapevines. We are also glad to David Pdietz (America Mideast Educational & Training Services, Inc.), Dr. Laura Hales and Dr. Andrea Devlin for English editing and scientific proofreading of this manuscript.

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Authors and Affiliations

  1. RLP-AgroScience.GmbH, AlPlanta—Institute for Plant Research, Breitenweg 71, 67435, Neustadt/Weintrasse, Germany

    Anis Ben-Amar, Pascal Cobanov, Guenther Buchholz & Goetz Reustle

  2. Laboratory of Plant Molecular Physiology, Center of Biotechnology, Borj Cedria BP 901, 2050, Hammam-Lif, Tunisia

    Anis Ben-Amar & Ahmed Mliki

Authors
  1. Anis Ben-Amar
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  2. Pascal Cobanov
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  3. Guenther Buchholz
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  4. Ahmed Mliki
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  5. Goetz Reustle
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Correspondence to Anis Ben-Amar.

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Communicated by Y. Wang.

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Ben-Amar, A., Cobanov, P., Buchholz, G. et al. In planta agro-infiltration system for transient gene expression in grapevine (Vitis spp.). Acta Physiol Plant 35, 3147–3156 (2013). https://doi.org/10.1007/s11738-013-1348-0

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  • DOI: https://doi.org/10.1007/s11738-013-1348-0

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