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A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop

Nature Biotechnology volume 21, pages 439442 (2003) | Download Citation

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Abstract

It is generally thought that transformation of plant cells using Agrobacterium tumefaciens occurs at a very low frequency. Therefore, selection marker genes are used to identify the rare plants that have taken up foreign DNA. Genes encoding antibiotic and herbicide resistance are widely used for this purpose in plant transformation1,2. Over the past several years, consumer and environmental groups have expressed concern about the use of antibiotic- and herbicide-resistance genes from an ecological and food safety perspective. Although no scientific basis has been determined for these concerns, generating marker-free plants would certainly contribute to the public acceptance of transgenic crops. Several methods have been reported to create marker gene–free transformed plants, for example co-transformation, transposable elements, site-specific recombination, or intrachromosomal recombination3,4,5,6,7,8,9. Not only are most of these systems time-consuming and inefficient, but they are also employed on the assumption that isolation of transformants without a selective marker gene is not feasible10. Here we present a method that permits the identification of transgenic plants without the use of selectable markers. This strategy relies on the transformation of tissue explants or cells with a virulent A. tumefaciens strain and selection of transformed cells or shoots after PCR analysis. Incubation of potato explants with A. tumefaciens strain AGL0 resulted in transformed shoots at an efficiency of 1–5% of the harvested shoots, depending on the potato genotype used. Because this system does not require genetic segregation or site-specific DNA-deletion systems to remove marker genes, it may provide a reliable and efficient tool for generating transgenic plants for commercial use, especially in vegetatively propagated species like potato and cassava.

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Acknowledgements

The authors wish to thank SENTER for financial support.

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Affiliations

  1. AVEBE, AVEBE-weg 1, 9607 PT Foxhol, The Netherlands.

    • Nick de Vetten
    •  & Renaldo ter Stege
  2. Laboratory of Plant Breeding, Wageningen University, P.O. Box 386, 6700 AJ Wageningen, The Netherlands.

    • Anne-Marie Wolters
    • , Krit Raemakers
    •  & Richard Visser
  3. Plant Research International, P.O. Box 16, 6700 AJ Wageningen, The Netherlands.

    • Ingrid van der Meer
  4. Breeding Institute KARNA, Valtherblokken Zuid 40, 7876 TC Valthermond, The Netherlands.

    • Els Heeres
    •  & Paul Heeres

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The authors declare no competing financial interests.

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Correspondence to Richard Visser.

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DOI

https://doi.org/10.1038/nbt801

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