, Volume 188, Issue 3, pp 439–456 | Cite as

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

  • Rajbir S. Sangwan
  • Yvan Bourgeois
  • Spencer Brown
  • Gérard Vasseur
  • Brigitte Sangwan-Norreel


The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.

Key words

Agrobacterium Arabidopsis Cotyledon Root explant (in vitro culture) Transformation 





basal medium


2,4-dichlorophenoxyacetic acid






kanamycin resistant


naphthaleneacetic acid


periodic acid-Schiff's


GUS marker gene driven by the promoter of cauliflower mosaic virus


transferred DNA


GUS marker gene driven by TR-promoter


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  1. Alizadeh, S., Mantell, S.H. (1991) Early cellular events during direct somatic embryogenesis in cotyledon explants of Solanum aviculare. Forst. Ann. Bot. 67, 257–263Google Scholar
  2. An, G. (1985) High efficiency transformation of cultured tobacco cells. Plant Physiol. 79, 568–570Google Scholar
  3. Basiran, N., Armitage, P., Scott, R.J., Draper, J. (1987) Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: Regeneration of transformed shoots via a callus phase. Plant Cell Rep. 6, 396–399Google Scholar
  4. Binns, A.N. (1990) Agrobacterium-mediated gene delivery and the biology of host range limitations. Physiol. Plant. 79, 135–139Google Scholar
  5. Binns, A.N. (1991) Transformation of wall deficient cultured tobacco protoplasts by Agrobacterium tumefaciens. Plant Physiol. 96, 496–506Google Scholar
  6. Braun, A. (1954) The physiology of plant tumors. Annu. Rev. Plant Physiol. 5, 133–162Google Scholar
  7. Bronner, R. (1975) Simultaneous demonstration of lipids and starch in plant tissues. Stain Technol. 50, 1–4Google Scholar
  8. Brown, S.C., Devaux, P., Marie, D., Bergounioux, C., Petit, P.X. (1991a) Cytométrie de flux: application a l'analyse de la ploïdie chez les végétaux. Biofutur 105, 2–16Google Scholar
  9. Brown, S.C., Bergounioux, C., Tallet, S., Marie, D. (1991b) Flow cytometry of nuclei for ploidy and cell cycle analysis. In: A laboratory guide for cellular and molecular plant biology, pp. 326–345, Negrutiu, I., Gharti-Chhetri, G., eds. Birkhauser Verlag Basel, SwitzerlandGoogle Scholar
  10. Chriqui, D., David, C., Adam, S. (1988) Effect of the differentiated or dedifferentiated state of tobacco pith tissue on its behaviour after inoculation with A. rhizogenes. Plant Cell Rep. 7, 111–114Google Scholar
  11. Clark, G. (1984) Staining procedures. 4th ed., Williams & Wilkins, Baltimore LondonGoogle Scholar
  12. Deblaere, R., Bytebier, B., De Greve, H., Debock, F., Schell, J., Van Montagu, M., Leemans, J. (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium mediated gene transfer. Nucleic Acids Res. 13, 4777–4788Google Scholar
  13. De Cleene, M., De Ley, J. (1976) The host range of crown gall. Bot. Rev. 42, 389–466Google Scholar
  14. De Rocher, E.J., Harkins, K.R., Galbraith, D.W., Bohnert, H.J. (1990) Developmentally regulated systemic endopolyploidy in succulents with small genomes. Science 250, 99–101Google Scholar
  15. Dong, J.Z., Yang, M.Z., Jia, S.R., Chua, N.H. (1991) Transformation of melon (Cucumis melo L.) and expression from the cauliflower mosaic virus 35S promoter in transgenic melon plants. Bio-Technology 9, 858–863Google Scholar
  16. Douglas, C., Halperin, W., Gordon, M., Nester, E. (1985) Specific attachmment of Agrobacterium tumefaciens to bamboo cells in suspension cultures. J. Bacteriol. 161, 764–766Google Scholar
  17. Draper, J., Mackenzie, I.A., Davey, M.R., Freeman, J.P. (1983) Attachment of Agrobacterium tumefaciens to mechanically isolated Asparagus cells. Plant Sci. Lett. 29, 227–236Google Scholar
  18. Esau, K. (1965) Plant anatomy. 2nd edn., John Wiley & Sons, New YorkGoogle Scholar
  19. Fowke, L.C. (1986) Ultrastructural cytology of cultured plant tissues, cells, and protoplasts. In: Cell culture and somatic cell genetics of plants, vol. 3, pp. 323–339, Vasil, I.K. ed., Academic Press Inc., Orlando, FloridaGoogle Scholar
  20. Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., Firoozabady, E. (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051Google Scholar
  21. Gasser, C.S., Fraley, R.T. (1989) Genetically engineering plants for crop improvement. Science 244, 1293–1299Google Scholar
  22. Gheysen, G., Villarroel, R., Van Montagu, M. (1991) Illegitimate recombination in plants: a model for T-DNA integration. Gene Devel. 5, 287–297Google Scholar
  23. Halperin, W. (1986) Attainment and retention of morphogenetic capacity in vitro. In: Cell culture and somatic cell genetics of plants, vol. 3, pp. 3–37, Vasil, I.K. ed., Academic Press Inc., Orlando, FloridaGoogle Scholar
  24. Hinchee, M.A.W., Connor-Ward, D.V., Newell, C.A., McDonnell, R.E., Sato, S.J., Gasser, C.S., Fischhoff, D.A., Re, D.R., Fraley, R.T., Horsch, R.B. (1988) Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio-Technology 6, 915–922Google Scholar
  25. Hooykaas, P.J.J. (1989) Transformation of plant cells via Agrobacterium. Plant Mol. Biol. 13, 327–336Google Scholar
  26. Jefferson, R.A., Kavanagh, T.A., Bevan, M.W. (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907Google Scholar
  27. Jefferson, R.A. (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387–407Google Scholar
  28. Jensen, W.A. (1962) Botanical histochemistry. W.H. Freeman & Company, San FranciscoGoogle Scholar
  29. Koncz, C., Olsson, O., Langridge, W.H.R., Schell, J., Szalay, A.A. (1987) Expression and functional assembly of bacterial luciferase in plansts. Proc. Natl. Acad. Sci. USA 84, 131–135Google Scholar
  30. Kovats, K., Binder, A., Hohl, H.R. (1991) Cytology of induced systemic resistance of Cucumber to Colletotrichum lagenarium. Planta 183, 484–490Google Scholar
  31. Krens, F.A., Molendijk, L., Wullems, G.J., Schilperoort, R.A. (1985) The role of bacterial attachment in the transformation of cell wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens. Planta 166, 300–308Google Scholar
  32. Leemans, J., Deblaere, R., Willmitzer, L., De Greve, H., Hernalsteens, J.P., Van Montagu, M. (1982) Genetic identification of function of TL-transcripts in octopine crown gall. EMBO J. 1, 147–152Google Scholar
  33. Lida, A., Yamashita, T., Yamada, Y., Morikawa, H. (1991) Efficiency of particle-bombardement-mediated transformation is influenced by cell cycle stage in synchronized cultured cells of tobacco. Plant Physiol. 97, 1585–1587Google Scholar
  34. Lippincott, J.A., Lippincott, B. (1980) Microbial adherence in plants. In: Receptors and recognition. Ser. B, Vol. 6: Bacterial adherence, pp. 376–398, Beachey, E.W. ed., Chapman & Hall, LondonGoogle Scholar
  35. Matthysse, A.G. (1987) Initial interactions of Agrobacterium tumefaciens with plant host cell. Crit. Rev. Microbiol. 13, 281–307Google Scholar
  36. Mayerhofer, R., Koncz-Lalman, Z., Nawrath, C., Bakkeren, G., Crameri, A., Angelis, K., Redei, G.P., Schell, J., Hohn, B., Koncz, C. (1990) T-DNA integration: a mode of illegitimate recombination in plants. EMBO J. 10, 697–704Google Scholar
  37. Miller, J.H., ed. (1972) Experiments in molecular genetics. Cold spring Harbor Lab. Press, Cold Spring Harbor, New YorkGoogle Scholar
  38. Meyerowitz, E.M. (1987) Arabidopsis thaliana. Annu. Rev. Genet. 21, 93–111PubMedGoogle Scholar
  39. Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497Google Scholar
  40. Nagata, Z., Okada, K., Takebe, I. (1986) Strong dependency of the transformation of plant protoplasts on cell cycle. In: Fallen Leaf Lake conference on Agrobacterium and crown gall. pp. 9, Kado, C. ed., University of California, Davis, U.S.A.Google Scholar
  41. Neff, N.T., Binns, A.N., Brandt, C. (1987) Inhibitory effects of a pectin-enriched tomato cell wall fraction on Agrobacterium tumefaciens binding and tumor formation. Plant Physiol. 83, 525–528Google Scholar
  42. Nitsch, J.P., Nitsch, C. (1965) Néoformation de fleurs in vitro chez une espèce de jours courts Plumbago indica L. Ann. Physiol. Vég. 7, 251–258Google Scholar
  43. Ow, D.W., Wood, K.V., Deluca, M., Dewet, J.R., Helinski, D.R., Howell, S.H. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234, 856–859Google Scholar
  44. Potrykus, I. (1990) Gene transfer to cereals: An assessment. Biotechnology 8, 535–542Google Scholar
  45. Rayle, D.L., Cleland, R. (1977) Control of plant cell enlargement by hydrogen ions. Curr. Top. Devel. Biol. 11, 187–214Google Scholar
  46. Robinette, D., Matthysse, A.G. (1990) Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phoseolicota. J. Bacteriol. 172, 5742–5749Google Scholar
  47. Sangwan, R.S., Sangwan-Norreel, B.S. (1987) Biochemical cytology of pollen embryogenesis. Int. Rev. Cytol. 107, 221–272Google Scholar
  48. Sangwan, R.S., Bourgeois, Y., Sangwan-Norreel, B.S. (1991) Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency. Mol. Gen. Genet. 230, 475–485Google Scholar
  49. Schell, J. (1987) Transgenic plants as tool to study the molecular organization of plant genes. Science 237, 1176–1183Google Scholar
  50. Schmidt, R., Willmitzer, L. (1988) High efficiency Agrobacterium tumefaciens-mediated transformatin of Arabidopsis thaliana leaf and cotyledons explants. Plant Cell Rep. 7, 583–586Google Scholar
  51. Sgorbati, S., Sparvoli, E., Levi, M., Chiatante, D. (1989) Bivariate cytofluorimetric analysis of nuclear protein and DNA relative to cell kinetics during germination of Pisum sativum seed. Physiol. Plant. 75, 479–484Google Scholar
  52. Sheikholeslam, S.N., Weeks, D.P. (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol. Biol. 8, 291–298Google Scholar
  53. Silcock, D.J., Francis, D., Bryant, J.A., Hughes, S.G. (1990) Changes in nuclear DNA content, cell and nuclear size, and frequency of cell division in the cotyledons of Brassica napus L. during embryogenesis. J. Exp. Bot. 41, 401–407Google Scholar
  54. Somerville, C. (1989) Arabidopsis blooms. Plant Cell. 1, 1131–1135Google Scholar
  55. Stachel, S.E., Messens, E., Van Montagu, M., Zambryski, P. (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318, 624–629Google Scholar
  56. Stachel, S.E., Zambryski, P.C. (1989) Generic trans-kingdom sex. Nature 340, 190–191Google Scholar
  57. Teeri, T.H., Lehväslaiko, H., Franck, M., Uotila, J., Heino, P., Palva, E.T., Van Montagu, M., Herrera-Estrella, L. (1989) Gene fusion to lac Z reveals new expression patterns of chimeric genes in transgenic plants. EMBO J. 8, 343–350Google Scholar
  58. Thiéry, J.P. (1967) Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. J. Microsc. 6, 987–1018Google Scholar
  59. Thomashow, M.F., Karlinsky, J.E., Marks, J.R., Hurlbert, R.E. (1987) Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment. J. Bacteriol. 169, 3209–3216Google Scholar
  60. Valvekens, D., Van Montagu, M., Van Lijebettens, M. (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536–5540Google Scholar
  61. Vancanneyt, G., Schmidt, R., O'Connor-Sanchez, A., Willmitzer, L., Rocha-Sosa, M. (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220, 245–250Google Scholar
  62. Velten, J., Schell, J. (1985) Selection-expression plasmid for use in genetic transformation of higher plants. Nucleic Acid Res. 13, 6981–6998Google Scholar
  63. Weising, K., Schell, J., Kahl, G. (1988) Foreign genes in plants: transfer, structure, expression, and application. Annu. Rev. Genet. 22, 421–477Google Scholar
  64. Zambryski, P. (1988) Basic process underlying Agrobacterium- mediated DNA transfer to plants cells. Annu. Rev. Genet. 22, 1–30Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Rajbir S. Sangwan
    • 1
  • Yvan Bourgeois
    • 1
  • Spencer Brown
    • 2
  • Gérard Vasseur
    • 1
  • Brigitte Sangwan-Norreel
    • 1
  1. 1.Laboratoire Androgenèse et Biotechnologie, Université de Picardie Jules VerneAmiens CédexFrance
  2. 2.Institut des Sciences Végétales, Service de Cytométrie, C.N.R.S.Gif-sur-YvetteFrance

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