Molecular Breeding

, Volume 7, Issue 3, pp 195–202 | Cite as

Marker gene elimination from transgenic barley, using co-transformation with adjacent `twin T-DNAs' on a standard Agrobacterium transformation vector

  • Peter R. Matthews
  • Ming-Bo Wang
  • Peter M. Waterhouse
  • Sarah Thornton
  • Sarah J. Fieg
  • Frank Gubler
  • John V. Jacobsen


We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Agrobacterium transformation Barley Co-transformation Marker-free Selectable marker Transgene segregation 


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Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Peter R. Matthews
    • 1
  • Ming-Bo Wang
    • 1
  • Peter M. Waterhouse
    • 1
  • Sarah Thornton
    • 1
  • Sarah J. Fieg
    • 1
  • Frank Gubler
    • 1
  • John V. Jacobsen
    • 1
  1. 1.CSIRO Plant IndustryCanberraAustralia

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