Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

The SEV System: A New Disarmed Ti Plasmid Vector System for Plant Transformation

Bio/Technology volume 3, pages 629–635 (1985)Cite this article

Abstract

A derivative of the octopine-type Ti plasmid, pTiB6S3, was constructed in which the TL-DNA oncogenic functions, the TL-DNA right border sequence and all of TR-DNA were deleted and replaced with the kanamycin antibiotic resistance marker from Tn903 (601). The resulting avirulent plasmid, pTiB6S3–SE contains only the TL-DNA left border sequence and a region (1.6 kb) of homologous DNA (LIH) to allow recombination with intermediate vectors such as pMON120 or pMON200. Cointegrate formation between pTiB6S3–SE and pMON200, which contains a chimeric plant kanamycin resistance gene (Kmr), an intact nopaline synthase gene (NOS), a poly linker region to facilitate the insertion of foreign genes into the vector and a functional T-DNA right border sequence, results in the formation of a selectable, avirulent T-DNA. This vector system is referred to as the SEV system (Split End Vector) since the T-DNA border sequences are present on separate plasmids prior to recombination. Plant cells (tobacco, petunia and tomato) transformed with SEV T-DNA are easily identified by their resistance to kanamycin and their ability to synthesize nopaline. A major advantage of the new disarmed system is that all transformed colonies can potentially be regenerated into intact plants that normally express and segregate the inserted foreign DNA sequences.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Caplan, A., Herrera-Estrella, L., Inze, D., Van Haute, E., Van Montagu, M., Schell, J. and Zambryski, P. 1984. Introduction of genetic material into plant cells. Science 18: 815–821.

    Google Scholar 

  2. Bevan, M. and Chilton, M.-D. 1982. T-DNA of the Agrobacterium Ti and Ri plasmids. Ann. Rev. Genet. 16: 357–384.

    Article  CAS  PubMed  Google Scholar 

  3. Ream, L. and Gordon, M. 1982. Crown gall disease and prospects for genetic manipulation of plants. Science 218: 854–859.

    Article  CAS  PubMed  Google Scholar 

  4. Matzke, A. and Chilton, M.-D. 1981. Site-specific insertion of genes into the T-DNA of the Agrobacterium tumour-inducing plasmid: An approach to genetic engineering of higher plant cells. J. Mol. Appl. Genet. 1: 39–49.

    CAS  PubMed  Google Scholar 

  5. Leemans, J., Shaw, C., Deblaere, R., DeGreve, H., Hernalsteens, J.-P., Maes, M., Montagu, M. and Schell, J. 1981. Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J. Mol. Appl. Genet. 1: 149–164.

    CAS  PubMed  Google Scholar 

  6. Fraley, R., Rogers, S., Horsch, R., Sanders, P., Flick, J., Adams, S., Bittner, M., Brand, L., Fink, C., Fry, J., Galluppi, J., Goldberg, S., Hoffmann, N. and Woo, S. 1983. Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA 80: 4803–4807.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Comai, L., Schilling-Cordaro, C., Mergia, A. and Houck, C.M. 1983. A new technique for genetic engineering of Agrobacterium Ti plasmid. Plasmid 10: 21–30.

    Article  CAS  PubMed  Google Scholar 

  8. Van Haute, E., Joos, H., Maes, M., Warren, G., Van Montagu, M. and Schell, J. 1983. Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. The EMBO J. 2: 411–417.

    Article  CAS  PubMed  Google Scholar 

  9. Garfinkel, D., Simpson, R., Ream, R., White, F., Gordon, M. and Nester, E.W. 1981. Genetic analysis of crown gall; fine structure map of the T-DNA by site-directed mutagenesis. Cell 27: 143–155.

    Article  CAS  PubMed  Google Scholar 

  10. Leemans, J., Deblaere, R., Willmitzer, L., DeGreve, H., Hernalsteens, J., Van Montagu, M. and Schell, J. 1982. Genetic identification of functions of TL-DNA transcripts in octopine crown galls. The EMBO Journal 1: 147–152.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zambryski, P., Depicker, A., Kruger, K. and Goodman, H. 1982. Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J. Mol. Appl. Genet. 1: 361–370.

    CAS  PubMed  Google Scholar 

  12. Yadav, N., Vanderleyden, J., Bennet, D., Barnes, W. and Chilton, M.-D. 1982. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc. Natl. Acad. Sci. USA 79: 6322–6326.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang, K., Herrera-Estrella, L., Van Montagu, M. and Zambryski, P. 1984. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38: 455–462.

    Article  CAS  PubMed  Google Scholar 

  14. Klee, H.J., White, F.F., Iyer, V.N., Gordon, M.P. and Nester, E.W. 1983. Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J. Bacteriol. 87: 153–161.

    Google Scholar 

  15. Ooms, G., Klapwijk, P., Poulis, J. and Schilperoort, R. 1980. Characterization of Tn904 insertions in octopine Ti plasmid mutants of Agrobacterium tumefaciens. J. Bacteriol. 82: 144–150.

    Google Scholar 

  16. Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M. and Schell, J. 1983. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. The EMBO J. 2: 2143–2150.

    Article  CAS  PubMed  Google Scholar 

  17. Barton, K., Binns, A., Matzke, A. and Chilton, M.-D. 1983. Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell 32: 1033–1043.

    Article  CAS  PubMed  Google Scholar 

  18. Herrera-Estrella, L., DeBlock, M., Messens, E., Hernalsteens, J.-P., Van Montagu, M. and Schell, J. 1983. Chimeric genes as dominant selectable markers in plant cells. The EMBO J. 2: 987–995.

    Article  CAS  PubMed  Google Scholar 

  19. Bevan, M., Flavell, R. and Chilton, M.-D. 1983. A chimeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304: 184–187.

    Article  CAS  Google Scholar 

  20. Horsch, R., Fraley, R., Rogers, S., Sanders, P., Lloyd, A. and Hoffman, N. 1984. Inheritance of functional foreign genes in plants. Science 223: 496–498.

    Article  CAS  PubMed  Google Scholar 

  21. Davies, R.W., Botstein, D. and Roth, J.R. 1980. Advanced Bacterial Genetics, p. 116. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

  22. Ish-Horowicz, D. and Burke, J.F. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9: 2989–2998.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Colman, A., Beyers, M.J., Primrose, S.B. and Lyons, A. 1978. Rapid purification of plasmid DNAs by hydroxyapatite chromatography. Eur. J. Biochem. 91: 303–310.

    Article  CAS  PubMed  Google Scholar 

  24. Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular Cloning, p. 504. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

  25. Adams, S.P., Holder, S.B., Wykes, E.J., Kavka, K.S. and Galluppi, G.R. Hindered dialkylamino nucleoside phosphite reagents in the synthesis of two DNA 51-mers. J. Am. Chem. Soc. 105: 661–663.

    Article  CAS  Google Scholar 

  26. Murray, N.E., Bruce, S.A. and Murray, K.J. 1979. Molecular cloning of the DNA ligase gene from bacteriophase T4. J. Mol. Biol. 132: 493–505.

    Article  CAS  PubMed  Google Scholar 

  27. Rogers, S.G. and Weiss, B. 1980. Cloning of the exonuclease III gene of Escherichia coli. Gene 11: 187–195.

    Article  CAS  PubMed  Google Scholar 

  28. Bale, A., d'Alarcao, M. and Marinus, G.M. 1979. Characterization of a DNA adenine methylation mutant of Escherichia coli K12. Mutat. Res. 59: 157–165.

    Article  CAS  PubMed  Google Scholar 

  29. Messing, J., Crea, R. and Seeburg, P. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9: 309–321.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Oka, A., Sugisaki, H. and Takanami, M. 1981. Nucleotide-sequence of the kanamycin resistance transposon Tn 903. J. Mol. Biol. 147: 217–226.

    Article  CAS  PubMed  Google Scholar 

  31. Ditta, G., Stanfield, S., Corbin, D. and Helinski, D. 1980. Broad host range DNA cloning system for gram-negative bacteria: construction of a genebank of Rhizobium meliloti. Proc. Natl. Acad. Science USA 77: 7347–7351.

    Article  CAS  Google Scholar 

  32. Hirsch, P. and Beringer, J. 1984. A physical map of pPH1JI and pJB4JI. Plasmid 12: 139–141.

    Article  CAS  PubMed  Google Scholar 

  33. Otten, L.A.B.M. and Schilperoort, R.A. 1978. A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochem. Biophys. Acta 527: 497–500.

    CAS  PubMed  Google Scholar 

  34. Nagao, R., Shah, D., Eckenrode, V. and Meagher, R. 1981. Multigene family of actin-related sequences isolated from a soybean genomic library. DNA 1: 1–9.

    Article  CAS  PubMed  Google Scholar 

  35. Dhaese, P., DeGreve, H., Decraemer, H., Schell, J. and Van Montagu, M. 1979. Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucl. Acid Res. 7: 1837–1849.

    Article  CAS  Google Scholar 

  36. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503–517.

    Article  CAS  PubMed  Google Scholar 

  37. Rogers, S.G. et al. (Manuscript submitted for publication)

  38. Fraley, R., Horsch, R., Matzke, A., Chilton, M.-D., Chilton, W. and Sanders, P. 1981. In vitro transformation of petunia cells by an improved method of cocultivation with A. tumefacins strains. Plant Mol. Biol. 3: 371–378.

    Article  Google Scholar 

  39. Zambryski, P., Holsters, M., Kruger, K., Depicker, A., Schell, J., Van Montague, M. and Goodman, H. 1980. Tumor DNA structure in plant cells transformed by A. tumefaciens. Science 209: 1385–1391.

    Article  CAS  PubMed  Google Scholar 

  40. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. and Goodman, H. 1982. Nopaline synthase transcript mapping and DNA sequence. J. Mol. App. Genet. 1: 561–574.

    CAS  Google Scholar 

  41. Bevan, M., Barnes, W. and Chilton, M.-D. 1983. Structure and transcription of the nopaline synthase gene region of T-DNA, Nucleic Acids Res. 11: 370–385.

    Article  Google Scholar 

  42. Shaw, C., Watson, M., Carter, G. and Shaw, C. 1984. The right hand copy of the nopaline T-plasmid 25 bp repeat is required for tumor formation. Nucleic Acid Res. 12: 6031–6041.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. deFramond, A., Barton, K. and Chilton, M.-D. 1983. Mini-Ti: a new vector strategy for plant genetic engineering. Bio/Technology 1: 262–269.

    Google Scholar 

  44. Hoekema, A., Hirsch, P., Hooykaas, J. and Schilperoort, R. 1983. A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180.

    Article  CAS  Google Scholar 

  45. Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation, Nucleic Acids Res. 12: 8711–8721.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. An, G., Watson, B., Stachel, S., Gordon, M. and Nester, E. 1985. New cloning vehicles for transformation of higher plants. The EMBO J. 4: 277–286.

    Article  CAS  PubMed  Google Scholar 

  47. Chilton, M.-D., Tefler, D., Petit, A., David, C., Cusse-Delbart, F. and Tempe', J. 1982. Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295: 432–434.

    Article  CAS  Google Scholar 

  48. White, F., Ghidossi, G., Gordon, M. and Nester, E. 1982. Tumor induction by Agrobacterium rhizogenes involves the transfer of plasmid DNA to the plant genome. Proc. Natl. Acad. Sci. USA 79: 3193–3197.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Thomashow, M., Panagopoulos, C., Gordon, M. and Nester, E. 1980. Host range of Agrobacterium tumefaciens is determined by the Ti plasmid. Nature 283: 794–796.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO, 63198

    Robert T. Fraley, Stephen G. Rogers, Robert B. Horsch, David A. Eichholtz, Jeffery S. Flick, Cynthia L. Fink, Nancy L. Hoffmann & Patricia R. Sanders

Authors
  1. Robert T. Fraley
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen G. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert B. Horsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David A. Eichholtz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffery S. Flick
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Cynthia L. Fink
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nancy L. Hoffmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Patricia R. Sanders
    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

Fraley, R., Rogers, S., Horsch, R. et al. The SEV System: A New Disarmed Ti Plasmid Vector System for Plant Transformation. Nat Biotechnol 3, 629–635 (1985). https://doi.org/10.1038/nbt0785-629

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0785-629

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing