Molecular and General Genetics MGG

, Volume 163, Issue 2, pp 181–187 | Cite as

Transfection and transformation of Agrobacterium tumefaciens

  • M. Holsters
  • D. de Waele
  • A. Depicker
  • E. Messens
  • M. van Montagu
  • J. Schell


The freeze thaw transfection procedure of Dityatkin et al. (1972) was adapted for the transfection and transformation of A. tumefaciens. Transfection of the strains B6S3 and B6-6 with DNA of the temperate phage PS8cc186 yielded a maximum frequency of 2 10-7 transfectants per total recipient population. In transformation of the strain GV3100 with the P type plasmid RP4 a maximum frequency of 3.5 10-7 transformants per total recipient population was obtained. Agrobacterium Ti-plasmids were introduced in the strain GV3100 with a maximal efficiency of 4.5 10-8. These experiments provide further evidence that the Ti-plasmid is responsible for the oncogenic properties of A tumefaciens and for its capacity to induce “opine” synthesis in Crown-gall plant cells.


Plant Cell Agrobacterium Agrobacterium Tumefaciens Maximum Frequency Maximal Efficiency 
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  1. Bauer, W., Vinograd, J.: The interaction of closed circular DNA with intercalative Dyes. I. The superhelix density of SV40 DNA in the presence and absence of Dye. J. molec. Biol. 33, 141–172 (1968)Google Scholar
  2. Bomhoff, G., Klapwijk, P.M., Kester, H.C.M., Schilperoort, R.A.: Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Molec. gen. Genet. 145, 177–181 (1976)Google Scholar
  3. Clewell, D.B., Helinski, D.R.: Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc. nat. Acad. Sci. (Wash.) 62, 1159–1166 (1969)Google Scholar
  4. Chilton, M.D., Farrand, S.K., Levin, R., Nester, E.W.: RP4 Promotion of transfer of a large Agrobacterium plasmid which confers virulence. Genetics 83, 609–618 (1976)Google Scholar
  5. Chilton, M.D., Drummond, M.H., Merlo, 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 11, 263–271 (1977)Google Scholar
  6. Cosloy, S.D., Oishi, M.: Genetic transformation in Escherichia coli K12. Proc. nat. Acad. Sci. (Wash.) 70, 84–87 (1973)Google Scholar
  7. Datta, N., Hedges, R.W., Shaw, E.J., Sykes, R.B., Richmond, M.H.: Properties of an R factor from Pseudomonas aeruginosa. J. Bact. 108, 1244–1249 (1971)Google Scholar
  8. Dityatkin, S.Ya., Lisovskaya, K.V., Panzhava, N.N., Iliashenko, B.N.: Frozen-thawed bacteria as recipients of isolated coliphage DNA. Biochim. biophys. Acta (Amst.) 281, 319–323 (1972)Google Scholar
  9. Engler, G., Holsters, M., Montagu, M. van, Schell, J.: Agrocin 84 sensitivity: a plasmid determined property in Agrobacterium tumefaciens. Molec. gen. Genet. 138, 345–349 (1975)Google Scholar
  10. Genetello, C., Larebeke, N. van, Holsters, M., Depicker, A., Montagu, M. van, Schell, J.: Ti plasmids of Agrobacterium as conjugative plasmids. Nature (Lond.) 265, 561–563 (1977)Google Scholar
  11. Guerry, P., Leblanc, D. J., Falkow, S.: General method for the isolation of plasmid deoxyribonucleic acid. J. Bact. 116, 1064–1066 (1973)Google Scholar
  12. Hamilton, R. H., Fall, M.Z.: The loss of tumor-initiating ability in Agrobacterium tumefaciens by incubation at high temperature. Experientia (Basel) 27, 229–230 (1971)Google Scholar
  13. Holsters, M., Silva, B., Genetello, C., Engler, C., Vliet, F. van, Block, M. de, Villarroel, R., Montagu, M. van, Schell, J.: Spontaneous formation of cointegrates of the oncogenic Tiplasmid and the wide host range P-plasmid RP4. Plasmid, in press (1978a)Google Scholar
  14. Holsters, M., Silva, B., Vliet, F. van, Hernalsteens, J.P., Genetello, C., Montagu, M. van, Schell, J.: In vivo transfer of the Ti-plasmid of Agrobacterium tumefaciens to Escherichia coli. Submitted to Molec. gen. Genet. (1978b)Google Scholar
  15. Hotchkiss, R.D.: In. Methods in enzymology, Vol. 3, p. 708. New York: Academic Press Inc. 1957Google Scholar
  16. Hooykaas, P.J.J., Klapwijk, P.M., Nuti, M.P., Schilperoort, R.A., Rörsch, A.: Transfer of the Agrobacterium tumefaciens Tiplasmid to Avirulent Agrobacteria and to Rhizobium ex planta. J. gen. Microbiol. 98, 477–484 (1977)Google Scholar
  17. Humphreys, G.O., Willshaw, G.A., Anderson, E.S.: A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. biophys. Acta (Amst.) 383, 457–463 (1975)Google Scholar
  18. Kerr, A., Manigault, P., Tempé, J.: Transfer of virulence in vivo and in vitro in Agrobacterium. Nature (Lond.) 265, 560–561 (1977)Google Scholar
  19. Kleinschmidt, A.K.: Monolayer technique in electron microscopy of nucleic acid molecules. In: Methods in enzymology (Coldwich, S.P. and Kaplan, N.O., eds.), Vol. 12B, pp. 361–377. New York: Academic Press 1968Google Scholar
  20. Larebeke, N. van, Engler, G., Holsters, M., Elsacker, S. van den, Zaenen, I., Schilperoort, R.A., Schell, J.: Large plasmid in Agrobacterium tumefaciens essential for crown-gall inducing ability. Nature (Lond.) 252, 169–170 (1974)Google Scholar
  21. Larebeke, N. van, Genetello, C., Schell, J., Schilperoort, J.P., Hermans, A.K., Hernalsteens, J.P., Montagu, M. van: Acquisition of tumour-inducing ability by non-oncogenic agrobacteria as a result of plasmid transfer. Nature (Lond.) 255, 742–743 (1975)Google Scholar
  22. Larebeke, N. van, Genetello, C., Hernalsteens, J.P., Depicker, A., Zaenen, I., Messens, E., Montagu, M. van, Schell, J.: Transfer of Ti-plasmids between Agrobacterium strains by mobilisation with the conjugative plasmid RP4. Molec. gen. Genet. 152, 119–124 (1977)Google Scholar
  23. Levin, R.A., Farrand, S.K., Gordon, M.P., Nester, E.W.: Conjugation in Agrobacterium tumefaciens in the absence of plant tissue. J. Bact. 127, 1331–1336 (1976)Google Scholar
  24. Lippincott, J.A., Beiderbeck, R., Lippincott, B.B.: Utilization of octopine and nopaline by Agrobacterium. J. Bact. 116, 378–383 (1973)Google Scholar
  25. Mandel, M., Higa, A.: Calcium-dependent bacteriophage DNA infection. J. molec. Biol. 53, 159–162 (1970)Google Scholar
  26. Manigault, P., Kurkdjian, A.: Influence de la lumière sur le développement des tumeurs de la plantule de pois, Pisum sativum L., induites par Agrobacterium tumefaciens (Smith et Town.). Comm. C.R. Acad. Sci. (Paris), Sér. D 264, 2304–2306 (1967)Google Scholar
  27. Milani, V.J., Heberlein, G.T.: Transfection in Agrobacterium tumefaciens. J. Virol. 10, 17–22 (1972)Google Scholar
  28. Petit, A., Tempé, J.: Etude du métabolisme des guanidines des tissus de crown-gall par la souche T37 d'Agrobacterium tumefaciens. C. R. Acad. Sci (Paris) 281, 467–469 (1975)Google Scholar
  29. Schell, J.: The role of plasmids in crown-gall formation by A. tumefaciens. In: Genetic manipulations with plant material (L. Ledoux, ed.), pp. 163–181, New York: Plenum Publishing Co. 1975Google Scholar
  30. Stonier, T.: Agrobacterium tumefaciens Conn. II. Production of an antibiotic substance. J. Bact. 79, 889–898 (1960)Google Scholar
  31. Vervliet, G., Holsters, M., Teuchy, H., Montagu, M. van, Schell, J.: Characterization of different plaque forming and defective temperate phages in Agrobacterium strains. J. gen. Virol. 26, 33–48 (1975)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. Bact. 123, 255–264 (1975)Google Scholar
  33. Yamamoto, K.R., Alberts, B.M., Benzinger, R., Lawhorne, L., Treiber, G.: Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40, 734–744 (1970)Google Scholar
  34. Zaenen, L., Larebeke, N. van, Teuchy, H., Montagu, M. van, Schell, J.: Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J. molec. Biol. 86, 109–127 (1974)Google Scholar

Copyright information

© Springer-Verlag 1978

Authors and Affiliations

  • M. Holsters
    • 1
  • D. de Waele
    • 1
  • A. Depicker
    • 1
  • E. Messens
    • 1
  • M. van Montagu
    • 1
  • J. Schell
    • 1
  1. 1.Laboratorium voor Genetika en voor Histologie en GenetikaRijksuniversiteit GentGentBelgium

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