Production of Hairy Root Cultures and Transgenic Plants by Agrobacterium rhizogenes-Mediated Transformation

  • Mary C. Christey
  • Robert H. Braun
Part of the Methods in Molecular Biology™ book series (MIMB, volume 286)


Agrobacterium rhizogenes-mediated transformation results in the development of hairy roots at the site of infection. The production of hairy roots involves cocultivation of explants with A. rhizogenes and the subsequent selection of hairy roots on hormone-free medium. Hairy roots have many applications for research including secondary product production and for the study of biochemical pathways. In addition, transgenic plants regenerated from hairy roots often show an altered phenotype due to the presence of the rol genes. In this chapter we describe how to produce and grow hairy root cultures, how to regenerate shoots from these hairy roots, and how to conduct molecular analysis of these cultures.

Key Words

Agrobacterium rhizogenes hairy roots Ri phenotype rol genes transformation 


  1. 1.
    Christey, M. C. (2001) Use of Ri-mediated transformation for production of transgenic plants. In Vitro Cell Dev. Biol. Plant 37, 687–700.CrossRefGoogle Scholar
  2. 2.
    Quandt, H.-J., Phler, A., and Broer, I. (1993) Transgenic root nodules of Vicia hirsuta: a fast and efficient system for the study of gene expression in indeterminate-type nodules. Mol. Plant Microbe Interact. 6, 699–706.CrossRefGoogle Scholar
  3. 3.
    Uozumi, N. and Kobayashi, T. (1997) Artificial seed production through hairy root regeneration, in Hairy Roots: Culture and Applications (Doran, P. M., ed.), Harwood Academic, Amsterdam, The Netherlands, pp. 113–122.Google Scholar
  4. 4.
    Hamill, J. D. and Lidgett A. J. (1997) Hairy root cultures opportunities and key protocols for studies in metabolic engineering, in Hairy Roots: Culture and Applications (Doran, P. M., ed.), Harwood Academic, Amsterdam, The Netherlands, pp. 1–30.Google Scholar
  5. 5.
    Braun, R. H., Eady, C., Christey, M. C., Shaw, M., Pither-Joyce, M., and McCallum, J. (2002) The use of hairy root cultures for the study of sulfur metabolism in plants, in Microbes and Molecules 2002, A combined meeting of the NZSBMB, NZSPP and NZMS, November 26–29, 2002, University of Canterbury, Christchurch, New Zealand, p. 208.Google Scholar
  6. 6.
    Downs, C. G., Christey, M. C., Davies, K. M., King, G. A., Sinclair B. K., and Stevenson, D. G. (1994) Hairy roots of Brassica napus: II. Glutamine synthetase overexpression alters ammonia assimilation and the response to phosphinothricin. Plant Cell Rep. 14, 41–46.Google Scholar
  7. 7.
    Mugnier, J. (1997) Mycorrhizal interactions and the effects of fungicides, nematicides and herbicides on hairy root cultures, in Hairy Roots Culture and Applications (Doran, P. M., ed.), Harwood Academic, Amsterdam, The Netherlands, pp. 123–132.Google Scholar
  8. 8.
    Kifle, S., Shao, M., Jung, C., and Cai, D. (1999) An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.). Plant Cell Rep. 18, 514–519.CrossRefGoogle Scholar
  9. 9.
    Bais, H. P., Loyola-Vargas, V. M., Flores, H. E., and Vivanco, J. M. (2001) Root-specific metabolism: the biology and biochemistry of underground organs. In Vitro Cell. Dev. Biol. Plant 37, 730–741.CrossRefGoogle Scholar
  10. 10.
    Wongsamuth, R. and Doran, P. M. (1997) Production of monoclonal antibodies by tobacco hairy roots. Biotech. Bioeng. 54, 401–415.CrossRefGoogle Scholar
  11. 11.
    Hellens, R. and Mullineaux, P. (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451.PubMedCrossRefGoogle Scholar
  12. 12.
    Gleave, A. P. (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207.PubMedCrossRefGoogle Scholar
  13. 13.
    Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473–497.CrossRefGoogle Scholar
  14. 14.
    Christey, M. C., Sinclair, B. K., Braun, R. H., and Wyke, L. (1997) Regeneration of transgenic vegetable Brassicas (Brassica oleracea and B. campestris) via Ri-mediated transformation. Plant Cell Rep. 16, 587–593.CrossRefGoogle Scholar
  15. 15.
    Puddephat, I. J., Robinson, H. T., Fenning, T. M., Barbara, D. J., Morton, A., and Pink, D. A. C. (2001) Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay. Mol. Breed. 7, 229–242.CrossRefGoogle Scholar
  16. 16.
    Dessaux, Y., Petit, A., and Tempe, J. (1993) Chemistry and biochemistry of opines, chemical mediators of parasitism. Phytochemistry 34, 31–38.CrossRefGoogle Scholar
  17. 17.
    Christey, M. C. and Braun, R. H. (2004) Production of transgenic vegetable Brassicas, in Biotechnology in Agriculture and Forestry, Brassica Biotechnology (Pua, E. C. and Douglas C. J., eds), Springer-Verlag, Berlin, Germany.Google Scholar
  18. 18.
    Cogan, N. O. I., Lynn, J. R., King, G. J., Kearsey, M. J., Newbury, H. J., and Puddephat, I. J. (2002) Identification of genetic factors controlling the efficiency of Agrobacterium rhizogenes-mediated transformation in Brassica oleracea by QTL analysis. Theor. Appl. Genet. 105, 568–576.PubMedCrossRefGoogle Scholar
  19. 19.
    Cogan, N., Harvey, E., Robinson, H., et al. (2001) The effects of anther culture and plant genetic background on Agrobacterium rhizogenes-mediated transformation of commercial cultivars and derived doubled-haploid Brassica oleracea. Plant Cell Rep. 20, 755–762.CrossRefGoogle Scholar
  20. 20.
    Henzi, M. X., Christey, M. C., and McNeil, D. L. (2000) Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica). Plant Cell Rep. 19, 994–999.CrossRefGoogle Scholar
  21. 21.
    Doran, P. M., ed. (1997) Hairy Roots: Culture and Applications, Harwood Academic, Amsterdam, The Netherlands.Google Scholar
  22. 22.
    Coles, G. D., Abernethy, D. J., Christey, M. C., Conner, A. J., and Sinclair, B. K. (1991) Monitoring hairy root growth by image analysis. Plant Mol. Biol. Rep. 9, 11–18.CrossRefGoogle Scholar
  23. 23.
    Christey, M. C., Braun, R. H., and Reader, J. K. (1999) Field performance of transgenic vegetable brassicas (Brassica oleracea and B. rapa) transformed with Agrobacterium rhizogenes. SABRAO J. Breed. Genet. 31, 93–108.Google Scholar
  24. 24.
    Christey, M. C. (1997) Transgenic crop plants using Agrobacterium rhizogenes-mediated transformation, in Hairy Roots: Culture and Applications (Doran, P. M., ed.), Harwood Academic, Amsterdam, The Netherlands, pp. 99–111.Google Scholar
  25. 25.
    Edwards, K., Johnstone, C., and Thompson, C. (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19, 1349.PubMedCrossRefGoogle Scholar
  26. 26.
    Scorza, R., Zimmerman, T. W., Cordts, J. M., Footen, K. J., and Ravelonandro, M. (1994) Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes. J. Am. Soc. Horticult. Sci. 119, 1091–1098.Google Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Mary C. Christey
    • 1
  • Robert H. Braun
    • 1
  1. 1.New Zealand Institute for Crop & Food ResearchChristchurchNew Zealand

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