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Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine

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Abstract

Cisgenesis is one of the new plant breeding technologies emerging as a promising tool for the future, more publicly accepted than the traditional transgenic approach. One of the requirements for a cisgenic plant is the absence of selectable marker genes in the genome. In this study, a system for marker gene removal after selection of transgenic plants has been tested in grapevine. This is based on a binary vector containing a heat-shock-inducible promoter which, upon induction, activates a recombinase to produce the excision of a FRT-flanked box. After the removal of this cassette hosting both the marker gene, nptII, and the recombinase itself, the reporter gene gus may be expressed. Gene transfer experiments on grapevine embryogenic callus were carried out via Agrobacterium tumefaciens. Different heat-shock treatments with variable temperatures and heat incubations times were tested on a selected line and the optimal conditions for a complete removal of nptII with the subsequent gus transcription were found. Plants were analysed by means of qPCR on genomic DNA, to quantify nptII removal, and by a fluorimetric assay to measure gus activity. Our study is conceived as a proof-of-concept to investigate the feasibility of this method in grapevine in view of developing an efficient cisgenic approach in this valuable fruit crop.

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Acknowledgments

We thank Ivana Gribaudo for sharing with us ‘Brachetto’ embryogenic calli and Justin Lashbrooke for the excellent proof reading of the manuscript. This work was supported by the Autonomous Province of Trento

Author’s contribution

LDC propagated the transgenic lines, designed and performed heat-shock induction experiments, carried out PCR, qPCR, fluorimetric assays, statistical analysis, and wrote the manuscript. SP designed and performed heat-shock induction experiments, carried out the long range PCR, gus histochemical assay, and revised the manuscript. MC carried out grapevine gene transfer and revised the manuscript. HF developed the binary vector pB–Npt–Hsp–Flp–Gus and revised the manuscript. MVH conceived the project and revised the manuscript. MM conceived the project and revised the manuscript.

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  1. Manuela Campa

    Present address: Department of Genetics, Stellenbosch University, Van der Bijl Street, Stellenbosch, 7600, South Africa

Authors and Affiliations

  1. Research and Innovation Centre, Fondazione Edmund Mach, via E Mach 1, 38010, San Michele a/Adige, Italy

    Lorenza Dalla Costa, Stefano Piazza, Manuela Campa & Mickael Malnoy

  2. Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Fruit Crops, Julius Kühn-Institut, Pillnitzer Platz 3a, 01326, Dresden, Germany

    Henryk Flachowsky & Magda-Viola Hanke

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  1. Lorenza Dalla Costa
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Dalla Costa, L., Piazza, S., Campa, M. et al. Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine. Plant Cell Tiss Organ Cult 124, 471–481 (2016). https://doi.org/10.1007/s11240-015-0907-z

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