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Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes

Nature Biotechnology volume 18, pages 442–445 (2000)Cite this article

Abstract

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation. Once transgenic material has been selected, the marker gene is dispensable. We report a novel strategy to remove undesirable parts of a transgene after integration into the tobacco genome. This approach is based on the transfer of a vector containing a NPTII gene flanked by two 352 bp attachment P (attP) regions of bacteriophage λ, and the identification of somatic tissue with deletion events following intrachromosomal recombination between the attP regions. This system was used to delete a 5.9 kb region from a recombinant vector that had been inserted into two different genomic regions. As the attP system does not require the expression of helper proteins to induce deletion events, or a genetic segregation step to remove recombinase genes, it should provide a useful tool to remove undesirable transgene regions, especially in vegetatively propagated species.

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Figure 1: T-DNA region of pattP-ICR.
Figure 2: Selection of marker-free transgenic tobacco plants.
Figure 3: PCR analysis of the Kan-resistant plants derived from line 1 (lane 2) and line 2 (lane 4), and Kan-sensitive plants derived from white tissue of line 1 (lane 3) and line 2 (lane 5).
Figure 4: Southern blot analysis of DNA isolated from Kan-resistant (lane 2) and Kan-sensitive tissue of line 1 (lane 3).

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Acknowledgements

We would like to thank Dr Peter Urwin for providing the effector gene. This work was supported by a Scottish Office grant, “Transgenic control of nematodes.”

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Author notes

  1. Charles Scutt

    Present address: Réproduction et Développement des Plantes, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364, Lyon cedex, 07, France

Authors and Affiliations

  1. Leeds Institute for Plant Biotechnology and Agriculture (LIBA), Faculty of Biological Sciences, The University of Leeds, Leeds, LS2 9JT, United Kingdom

    Elena Zubko, Charles Scutt & Peter Meyer

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  1. Elena Zubko
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Correspondence to Peter Meyer.

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Zubko, E., Scutt, C. & Meyer, P. Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18, 442–445 (2000). https://doi.org/10.1038/74515

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