Skip to main content
SpringerLink
Log in

Positive-negative selection and T-DNA stability in Arabidopsis transformation

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

We have analysed the application of positive-negative selection for the selection of homologous recombination interactions between the chromosome and a T-DNA molecule after transformation of plant cells. Two different genomic loci in a cell suspension of Arabidopsis thaliana were chosen to study gene targeting events. One was the chalcone synthase (CHS) gene present as a single copy and the second an hemizygous chromosomally inserted T-DNA containing the hpt gene, conferring resistance to hygromycin, flanked by CHS sequences. The target lines were transformed with replacement-type T-DNA vectors which contained a positive selectable marker flanked by the regions of the CHS gene and a negative selectable marker to counter-select random insertions. As negative marker we used the Escherichia coli codA gene encoding cytosine deaminase, conferring upon the cells sensitivity to 5-flourocytosine (5-FC). Doubly selected transformants represent 1–4% of the primary transformed cells. Targeting events were not found at the chalcone synthase locus nor at the artificial hpt locus in a total of 4379 doubly selected calli, corresponding to at least 109 475 individual primary transformants. We show by PCR and Southern analysis that the 5-FC resistance in the majority of these cells is associated with substantial deletions of the T-DNA molecule from the right-border end.

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. Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B: A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell culture. Plant Physiol Biochem 30: 1–5 (1992).

    Google Scholar 

  2. Berthold DA, Best BA, Malkin R: A rapid preparation for PCR from Chlamydomonas reinhardtii and Arabidopsis thaliana. Plant Mol Biol Rep 11: 338–344 (1993).

    Google Scholar 

  3. Church GM, Gilbert W: Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995 (1984).

    Google Scholar 

  4. Doutriaux M-P, Couteau F, Bergounioux C, White CI: Isolation and characterisation of the RAD51 and DMC1 homologues from Arabidopsis thaliana. Mol Gen Genet, in press (1998).

  5. Durrenberger F, Crameri A, Hohn B, Koukolikova-N, Z.: Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci USA 86: 9154–9158 (1989).

    Google Scholar 

  6. Feinbaum RL, Ausubel FM: Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol Cell Biol 8: 1985–1992 (1989).

    Google Scholar 

  7. Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).

    Google Scholar 

  8. Feinberg AP, Vogelstein B: Addendum to ‘A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity’. Anal Biochem 137: 266–267 (1984).

    Google Scholar 

  9. Hajdukiewicz P, Svab Z, Maliga P: The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25: 989–994 (1994).

    Google Scholar 

  10. Halfter U, Morris P-C, Willmitzer L: Gene targeting in Arabidopsis thaliana. Mol Gen Genet 231: 186–193 (1992).

    Google Scholar 

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

    Google Scholar 

  12. Keil RL, Roeder GS: Cis-acting, recombination-stimulating activity in a fragment of the ribosomal DNA of S. cerevisiae. Cell 39: 377–386 (1984).

    Google Scholar 

  13. Kempin SA, Liljegren SJ, Block LM, Rounsley SD, Yanofsky MF, Lam E: Targeted disruption in Arabidopsis. Nature 389: 802 (1997).

    Google Scholar 

  14. Koncz C, Nèmeth K, Rèdei GP, Schell J: Homology recognition during T-DNA integration into the plant genome. In: Paszkowski J (ed) Homologous Recombination and Gene Silencing in Plants, pp. 167–189. Kluwer Academic Publishers, Dordrecht, Netherlands (1994).

    Google Scholar 

  15. Kononov ME, Bassuner B, Gelvin SB: Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11: 945–957 (1997).

    Google Scholar 

  16. Lee KY, Lund P, Lowe K, Dunsmuir P: Homologous recombination in plant cells after Agrobacterium mediated transformation. Plant Cell 2: 415–426 (1990).

    Google Scholar 

  17. Mansour S, Thomas K, Capecchi M: Disruption of the protooncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336: 348–52 (1988).

    Google Scholar 

  18. Martineau B, Voelker TA, Sanders RA: On defining T-DNA. Plant Cell 6: 1032–1033 (1994).

    Google Scholar 

  19. Miao ZH, Lam E: Targeted disruption of the TGA3 locus in Arabidopsis thaliana. Plant J 7: 359–365 (1995).

    Google Scholar 

  20. Murray MG, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8: 4321–4325 (1980).

    Google Scholar 

  21. Nickoloff JA: Transcription enhances intrachromosomal homologous recombination in mammalian cells. Mol Cell Biol 12: 5311–5318 (1992).

    Google Scholar 

  22. Offringa R, Franke van dijk MEI, Degroot MJA, Vandenelzen PJM, Hooykaas PJJ: Nonreciprocal homologous recombination between Agrobacterium transferred DNA and a plant chromosomal locus. Proc Natl Acad Sci USA 90: 7346–7350 (1993).

    Google Scholar 

  23. Ohl S, Offringa R, Vandenelzen PJM, Hooykaas PJJ: Gene replacement in plants. In: Paszkowski J (ed) Homologous Recombination and Gene Silencing in Plants, pp. 191–217. Kluwer Academic Publishers, Dordrecht, Netherlands (1994).

    Google Scholar 

  24. Ooms G, Bakker A, Molendijk L, Wullems GJ, Gordon MR, Nester EW, Schilperoort RA: T-DNA organisation in homogenous and heterogenous octopine-type crown gall tissues of Nicotiana tabacum. Cell 30: 589–597 (1982).

    Google Scholar 

  25. Paszkowski J, Baur M, Bogucki A, Potrykus I: Gene targeting in plants. EMBO J 7: 4021–4026 (1988).

    Google Scholar 

  26. Perera RJ, Linard CG, Signer ER: Cytosine deaminase as a negative selection marker for Arabidopsis. Plant Mol Biol 23: 793–799 (1993).

    Google Scholar 

  27. Puchta H, Dujon B, Hohn B: Two different but related mechanisms are used in plants for the repair of genomic doublestrand breaks by homologous recombination. Proc Natl Acad Sci USA 93: 5055–5060 (1996).

    Google Scholar 

  28. Puchta H, Hohn B: From centiMorgans to base pairs: homologous recombination in plants. Trends Plant Sci 1: 340–348 (1996).

    Google Scholar 

  29. Ramanathan V, Veluthambi K: Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. Plant Mol Biol 28: 1149–1154 (1995).

    Google Scholar 

  30. Reiter RS, Young RM, Scolnik PA: Genetic linkage of the Arabidopsis genome: methods for mapping with recombinant inbreds and random amplified polymorphic DNAs (RAPDs). In: Koncz C, Chua N-H, Schell J (eds) Methods in Arabidopsis research, pp. 170–190. World Scientific, Singapore (z).

  31. Risseeuw E, Franke van Dijk MEI, Hooykaas PJJ: Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome. Plant J 11: 717–728 (1997).

    Google Scholar 

  32. Risseeuw E, Offringa R, Franke van dijk MEI, Hooykaas PJJ: Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus. Plant J 7: 109–119 (1995).

    Google Scholar 

  33. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual., 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  34. Stachel SE, Timmerman B, Zambryski P: Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 50 virD gene products. EMBO J 6: 857–863 (1987).

    Google Scholar 

  35. Stougaard J: Substrate-dependent negative selection in plants using a bacterial cytosine deaminase gene. Plant J 3: 755–761 (1993).

    Google Scholar 

  36. Taylor CB: Comprehending cosuppression. Plant Cell 9: 1245–1249 (1997).

    Google Scholar 

  37. Thomas BJ, Rothstein R: Elevated recombination rates in transcriptionally active DNA. Cell 56: 619–630 (1989).

    Google Scholar 

  38. Thyagarajan B, Johnson BL, Campbell C: The effect of target site transcription on gene targeting in human cells in vitro. Nucl Acids Res 23: 2784–2790 (1995).

    Google Scholar 

  39. Thykjaer T, Finnemann J, Schauser L, Christensen L, Poulsen C, Stougaard J: Gene targeting approaches using positivenegative selection and large flanking regions. Plant Mol Biol 35: 523–530 (1997).

    Google Scholar 

  40. Tinland B: The integration of T-DNA into plant genomes. Trends Plant Sci 1: 178–184 (1996).

    Google Scholar 

  41. Topfer R, Matzeit V, Gronenborn B, Schell J, Steinbiss HH: A set of plant expression vectors for transcriptional and translational fusions. Nucl Acids Res 15: 5890 (1987).

    Google Scholar 

  42. van der Graaf E, den Dulk-Ras A, Hooykaas PJJ. Deviating TDNAtransfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31: 677–681 (1996).

    Google Scholar 

  43. Voelkel-Meiman K, Keil RL, Roeder GS: Recombinationstimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I. Cell 48: 1071–1079 (1987).

    Google Scholar 

Download references

Author information

Author notes

  1. P. Sirand-Pugnet

    Present address: Laboratoire de Génétique et d'Amélioration des Champignons Cultivés, Université Victor Segalen Bordeaux 2-INRA, CRA de Bordeaux-, BP81, 33 883, Villenave d'Ornon Cedex, France

Authors and Affiliations

  1. Centre de Recherche sur les Plantes, CNRS ERS 569, Université de Paris XI, 91405, Orsay Cedex, France

    M.E. Gallego & C.I. White

Authors
  1. M.E. Gallego
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Sirand-Pugnet
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C.I. White
    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

Gallego, M., Sirand-Pugnet, P. & White, C. Positive-negative selection and T-DNA stability in Arabidopsis transformation. Plant Mol Biol 39, 83–93 (1999). https://doi.org/10.1023/A:1006192225464

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006192225464

Search

Navigation