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
Article

Abstract

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 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Dale E.C. and Ow D.W. 1991. Gene transfer with subsequent removal of the selection gene from the plant genome. Proc Natl Acad Sci USA 88: 10558–10562.Google Scholar
  2. 2.
    Daley M., Knauf V.C., Summerfelt K.R. and Turner J.C. 1998. Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Rep 17: 489–496.Google Scholar
  3. 3.
    De Block M. and Debrouwer D. 1991. Two T-DNAs co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theor Appl Genet 82: 257–263.Google Scholar
  4. 4.
    De Framond A.J., Back E.W., Chilton W.S., Kayes L. and Chilton M. 1986. Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation. Mol Gen Genet 202: 125–131.Google Scholar
  5. 5.
    Deikman J. and Jones R.L. 1985. Control of α-amylase accumulation by gibberellic acid and calcium in barley aleurone layers. Plant Physiol 78: 192–198.Google Scholar
  6. 6.
    Depicker A., Herman L., Jacobs A., Schell J. and Van Montagu M. 1985. Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant interaction. Mol Gen Genet 201: 477–484 (1985).Google Scholar
  7. 7.
    Ebinuma H., Sugita K., Matsunaga E. and Yamakado M. 1997. Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci USA 94: 2117–2121.Google Scholar
  8. 8.
    Garvin D.F., Miller-Garvin J.E., Viccars E.A., Jacobsen, J.V. and Brown A.H.D. 1998. Identification of molecular markers linked to ant28-484, a mutation that eliminates proanthocyanidin production in barley seeds. Crop Sci. 38: 1250–1255.Google Scholar
  9. 9.
    Gleave A.P. 1992. A versatile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20: 1203–1207.Google Scholar
  10. 10.
    Goldsbrough A.P., Lastrella C.N. and Yoder J.I. 1993. Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Bio/technology 11: 1286–1292.Google Scholar
  11. 11.
    Horton R.M., Hunt H.D., Ho S.N., Pullen J.K. and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68.Google Scholar
  12. 12.
    Jacobsen J.V. and Close T.J. 1991. Control of transient expression of chimaeric genes by gibberellic acid and abscisic acid in protoplasts prepared from mature barley aleurone layers. Plant Mol Biol 16: 713–724.Google Scholar
  13. 13.
    Joersbo M., Donaldson I., Kreiberg J., Petersen S.G., Brunstedt J. and Okkels F.T. 1998. Analysis of mannose selection used for transformation of sugar beet. Mol Breed 4: 111–117.Google Scholar
  14. 14.
    Komari T., Hiei Y., Saito Y., Murai N. and Kumashiro T. 1996. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10: 165–174.Google Scholar
  15. 15.
    Kunkel T., Niu Q., Chan Y. and Chua N. 1999. Inducible isopentenyl transferase as a high-efficiency marker of plant transformation. Nature Biotech 17: 916–919.Google Scholar
  16. 16.
    Lazo G.R., Stein P.A. and Ludwig R.A. 1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Bio/technology 9: 963–967.Google Scholar
  17. 17.
    Matthews P.R., Gubler F. and Jacobsen J.V. 1997. A plant-based expression system for matching cDNA clones and isozymes. Plant Mol Biol Rep 15: 163–169 (1997).Google Scholar
  18. 18.
    McKnight T.D., Lillis M.T. and Simpson R.B. 1987. Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol 8: 439–445.Google Scholar
  19. 19.
    Olsen O. 1992. A rapid method for preparing multiple DNA fusions. Methods Mol Cell Biol 3: 159–160.Google Scholar
  20. 20.
    Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  21. 21.
    Tibbot B.K. and Skadsen R.W. 1996. Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley. Plant Mol Biol 30: 229–241.Google Scholar
  22. 22.
    Tingay S., McElroy E., Kalla R., Fieg S., Wang M., Thornton S. and Brettell R. 1997. Agrobacterium tumefaciens-mediated barley transformation. Plant J 11: 1369–1376.Google Scholar
  23. 23.
    Thomson D. and Henry R. 1995. Single-step protocol for preparation of plant tissue for analysis by PCR. BioTechniques 9: 394–400.Google Scholar
  24. 24.
    Wan Y. and Lemaux P.G. 1994. Generation of large numbers of independently transformed barley plants. Plant Physiol 104: 37–48.Google Scholar
  25. 25.
    Wang M.B., Li Z.-Y., Matthews P.R., Upadhyaya N.M. and Waterhouse P.M. 1998. Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants. Acta Hort 461: 401–407.Google Scholar
  26. 26.
    Wang M.B., Upadhyaya N.M., Brettell R.I.S. and Waterhouse P.M. 1997. Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J Genet Breed 51: 325–334.Google Scholar
  27. 27.
    Wang M.B. and Waterhouse P.M. 1997. A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes. Plant Mol Biol Rep 15: 209–215.Google Scholar
  28. 28.
    Yoder J.I. and Goldsbrough A.P. 1994. Transformation systems for generating marker-free transgenic plants. Bio/technology 12: 263–267.Google Scholar
  29. 29.
    Yon J. and Fried M. 1989. Precise gene fusion by PCR. Nucl Acids Res 17: 4895.Google Scholar
  30. 30.
    Zambryski P. 1988. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Ann Rev Genet 22: 1–30.Google Scholar

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

Personalised recommendations