Skip to main content
SpringerLink
Log in

Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. An G, Watson BD, Stachel S, Gordon MP, Nester EW: New cloning vehicles for transformation of higher plants. EMBO Journal 4: 277–284 (1985).

    Google Scholar 

  2. Bevan MW, Flavell RB, Chilton MD: A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304: 184–187 (1983).

    Google Scholar 

  3. Bevan MW: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12: 8711–8721 (1984).

    Google Scholar 

  4. Bradford MM: A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–254 (1976).

    Google Scholar 

  5. Caplan A, Herrera-Estrella L, Inze D, VanHaute E, VanMontagu M, Schell J, Zambryski P: Introduction of genetic material into plant cells. Science 222: 815–821 (1983).

    Google Scholar 

  6. Chilton MD, Drummond MH, Merlo DJ, Sciaky D, Montoya A, Gordon MP, Nester EW: Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crowngall tumorigenesis. Cell 11: 263–271 (1977).

    Google Scholar 

  7. Deak M, Kiss GB, Koncz C, Dudits D: Transformation of Medicago by Agrobacterium mediated gene transfer. Plant Cell Reports 5: 97–100 (1986).

    Google Scholar 

  8. Ditta G, Stanfiel S, Corbin D, Helinski DR: Broad host range DNA cloning system for Gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351 (1980).

    Google Scholar 

  9. Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Bittner ML, Brand LA, Fink CL, Fry JS, Gallup GR, Goldberg SB, Hoffmann NL, Woo SC: Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 80: 4803–4807 (1983).

    Google Scholar 

  10. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180 (1983).

    Google Scholar 

  11. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT. A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).

    Google Scholar 

  12. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).

    Google Scholar 

  13. Otten L: Lysopine dehydrogenase activity as an early marker in crown gall transformation. Plant Sci Lett 25: 15–27 (1982).

    Google Scholar 

  14. Otten LABM, Schilperoort RA: A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta 275: 497–500 (1978).

    Google Scholar 

  15. Reed KC, Mann DA: Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13: 7207–7221 (1985).

    Google Scholar 

  16. Reiss B, Sprengel R, Willi M, Schaller H: A new, sensitive method for qualitative and quantitative assay of the neomycin phosphotransferase in crude cell extracts. Gene 30: 211–218 (1984).

    Google Scholar 

  17. Sutton W: Some features of the DNA of Rhizobium bacteroids and bacteria. Biochim Biophys Acta 366: 1–10 (1974).

    Google Scholar 

  18. White DWR: Plant regeneration from long-term suspension cultures of white clover. Planta 162: 1–7 (1984).

    Google Scholar 

  19. Zambryski P, Joos H, Genetello C, VanMontagu M, Schell J: Ti-plasmid vector for the introduction of DNA into plant cells without altering their normal regeneration capacity. EMBO Journal 12: 2143–2150 (1983).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Grasslands Division, Department of Scientific and Industrial Research, Palmerston North, New Zealand

    Derek W. R. White & Denise Greenwood

Authors
  1. Derek W. R. White
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Denise Greenwood
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

White, D.W.R., Greenwood, D. Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors. Plant Mol Biol 8, 461–469 (1987). https://doi.org/10.1007/BF00017991

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00017991

Key words

Search

Navigation