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. 1985 Feb;4(2):277–284. doi: 10.1002/j.1460-2075.1985.tb03626.x

New cloning vehicles for transformation of higher plants

G An 1, B D Watson 1,2, S Stachel 3,4, M P Gordon 2, E W Nester 3
PMCID: PMC554183  PMID: 16453603

Abstract

We have constructed a set of small vectors based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage λ; (iv) the border sequences from the ends of the T-DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild-type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T-DNA is necessary for DNA transformation.

Keywords: shuttle vector, plant transformation, selectable marker, T-DNA borders, Ti plasmid

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akiyoshi D. E., Morris R. O., Hinz R., Mischke B. S., Kosuge T., Garfinkel D. J., Gordon M. P., Nester E. W. Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci U S A. 1983 Jan;80(2):407–411. doi: 10.1073/pnas.80.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BRAUN A. C. The activation of two growth-substance systems accompanying the conversion of normal to tumor cells in crown gall. Cancer Res. 1956 Jan;16(1):53–56. [PubMed] [Google Scholar]
  3. Barton K. A., Binns A. N., Matzke A. J., Chilton M. D. Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell. 1983 Apr;32(4):1033–1043. doi: 10.1016/0092-8674(83)90288-x. [DOI] [PubMed] [Google Scholar]
  4. Barton K. A., Chilton M. D. Agrobacterium Ti plasmids as vectors for plant genetic engineering. Methods Enzymol. 1983;101:527–539. doi: 10.1016/0076-6879(83)01036-8. [DOI] [PubMed] [Google Scholar]
  5. Bevan M., Barnes W. M., Chilton M. D. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 1983 Jan 25;11(2):369–385. doi: 10.1093/nar/11.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Broglie R., Coruzzi G., Fraley R. T., Rogers S. G., Horsch R. B., Niedermeyer J. G., Fink C. L., Chua N. H. Light-regulated expression of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene in transformed plant cells. Science. 1984 May 25;224(4651):838–843. doi: 10.1126/science.6719112. [DOI] [PubMed] [Google Scholar]
  7. Caplan A., Herrera-Estrella L., Inzé D., Van Haute E., Van Montagu M., Schell J., Zambryski P. Introduction of genetic material into plant cells. Science. 1983 Nov 18;222(4625):815–821. doi: 10.1126/science.222.4625.815. [DOI] [PubMed] [Google Scholar]
  8. Chilton M. D., Drummond M. H., Merio D. J., Sciaky D., Montoya A. L., Gordon M. P., Nester E. W. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell. 1977 Jun;11(2):263–271. doi: 10.1016/0092-8674(77)90043-5. [DOI] [PubMed] [Google Scholar]
  9. Chilton M. D., Saiki R. K., Yadav N., Gordon M. P., Quetier F. T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4060–4064. doi: 10.1073/pnas.77.7.4060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  11. Depicker A., Stachel S., Dhaese P., Zambryski P., Goodman H. M. Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1982;1(6):561–573. [PubMed] [Google Scholar]
  12. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garfinkel D. J., Simpson R. B., Ream L. W., White F. F., Gordon M. P., Nester E. W. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell. 1981 Nov;27(1 Pt 2):143–153. doi: 10.1016/0092-8674(81)90368-8. [DOI] [PubMed] [Google Scholar]
  14. Gielen J., De Beuckeleer M., Seurinck J., Deboeck F., De Greve H., Lemmers M., Van Montagu M., Schell J. The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5. EMBO J. 1984 Apr;3(4):835–846. doi: 10.1002/j.1460-2075.1984.tb01894.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grosveld G. C., de Boer E., Shewmaker C. K., Flavell R. A. DNA sequences necessary for transcription of the rabbit beta-globin gene in vivo. Nature. 1982 Jan 14;295(5845):120–126. doi: 10.1038/295120a0. [DOI] [PubMed] [Google Scholar]
  16. Haas M. J., Dowding J. E. Aminoglycoside-modifying enzymes. Methods Enzymol. 1975;43:611–628. doi: 10.1016/0076-6879(75)43124-x. [DOI] [PubMed] [Google Scholar]
  17. Herrera-Estrella L., Block M. D., Messens E., Hernalsteens J. P., Montagu M. V., Schell J. Chimeric genes as dominant selectable markers in plant cells. EMBO J. 1983;2(6):987–995. doi: 10.1002/j.1460-2075.1983.tb01532.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Herrera-Estrella L., Van den Broeck G., Maenhaut R., Van Montagu M., Schell J., Timko M., Cashmore A. Light-inducible and chloroplast-associated expression of a chimaeric gene introduced into Nicotiana tabacum using a Ti plasmid vector. Nature. 1984 Jul 12;310(5973):115–120. doi: 10.1038/310115a0. [DOI] [PubMed] [Google Scholar]
  19. Hille J., van Kan J., Schilperoort R. trans-Acting virulence functions of the octopine Ti plasmid from Agrobacterium tumefaciens. J Bacteriol. 1984 May;158(2):754–756. doi: 10.1128/jb.158.2.754-756.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iyer V. N., Klee H. J., Nester E. W. Units of genetic expression in the virulence region of a plant tumor-inducing plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1982;188(3):418–424. doi: 10.1007/BF00330043. [DOI] [PubMed] [Google Scholar]
  21. Kleckner N. Transposable elements in prokaryotes. Annu Rev Genet. 1981;15:341–404. doi: 10.1146/annurev.ge.15.120181.002013. [DOI] [PubMed] [Google Scholar]
  22. Koncz C., Kreuzaler F., Kalman Z., Schell J. A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and alpha-actin in plant tumors. EMBO J. 1984 May;3(5):1029–1037. doi: 10.1002/j.1460-2075.1984.tb01923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kozak M. Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res. 1981 Oct 24;9(20):5233–5252. doi: 10.1093/nar/9.20.5233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lemmers M., De Beuckeleer M., Holsters M., Zambryski P., Depicker A., Hernalsteens J. P., Van Montagu M., Schell J. Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline grown gall tumours. J Mol Biol. 1980 Dec 15;144(3):353–376. doi: 10.1016/0022-2836(80)90095-9. [DOI] [PubMed] [Google Scholar]
  25. Meyerowitz E. M., Guild G. M., Prestidge L. S., Hogness D. S. A new high-capacity cosmid vector and its use. Gene. 1980 Nov;11(3-4):271–282. doi: 10.1016/0378-1119(80)90067-0. [DOI] [PubMed] [Google Scholar]
  26. Montoya A. L., Chilton M. D., Gordon M. P., Sciaky D., Nester E. W. Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumor cells: role of plasmid genes. J Bacteriol. 1977 Jan;129(1):101–107. doi: 10.1128/jb.129.1.101-107.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Murai N., Kemp J. D., Sutton D. W., Murray M. G., Slightom J. L., Merlo D. J., Reichert N. A., Sengupta-Gopalan C., Stock C. A., Barker R. F., Hall T. C. Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors. Science. 1983 Nov 4;222(4623):476–482. doi: 10.1126/science.222.4623.476. [DOI] [PubMed] [Google Scholar]
  28. Ream L. W., Gordon M. P. Crown gall disease and prospects for genetic manipulation of plants. Science. 1982 Nov 26;218(4575):854–859. doi: 10.1126/science.218.4575.854. [DOI] [PubMed] [Google Scholar]
  29. Rothstein S. J., Jorgensen R. A., Yin J. C., Yong-di Z., Johnson R. C., Reznikoff W. S. Genetic organization of Tn5. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):99–105. doi: 10.1101/sqb.1981.045.01.018. [DOI] [PubMed] [Google Scholar]
  30. Shaw C. H., Watson M. D., Carter G. H., Shaw C. H. The right hand copy of the nopaline Ti-plasmid 25 bp repeat is required for tumour formation. Nucleic Acids Res. 1984 Aug 10;12(15):6031–6041. doi: 10.1093/nar/12.15.6031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang K., Herrera-Estrella L., Van Montagu M., Zambryski P. 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. 1984 Sep;38(2):455–462. doi: 10.1016/0092-8674(84)90500-2. [DOI] [PubMed] [Google Scholar]
  32. Watson B., Currier T. C., Gordon M. P., Chilton M. D., Nester E. W. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1975 Jul;123(1):255–264. doi: 10.1128/jb.123.1.255-264.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wullems G. J., Molendijk L., Ooms G., Schilperoort R. A. Differential expression of crown gall tumor markers in transformants obtained after in vitro Agrobacterium tumefaciens-induced transformation of cell wall regenerating protoplasts derived from Nicotiana tabacum. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4344–4348. doi: 10.1073/pnas.78.7.4344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yang F., Simpson R. B. Revertant seedlings from crown gall tumors retain a portion of the bacterial Ti plasmid DNA sequences. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4151–4155. doi: 10.1073/pnas.78.7.4151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zambryski P., Joos H., Genetello C., Leemans J., Montagu M. V., Schell J. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 1983;2(12):2143–2150. doi: 10.1002/j.1460-2075.1983.tb01715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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