High-Frequency and Efficient Agrobacterium-Mediated Transformation ofArabidopsis thaliana Ecotypes “C24” and “Landsberg erecta” Using Agrobacterium tumefaciens

  • Mehdi Barghchi
Part of the Methods in Molecular Biology™ book series (MIMB, volume 44)


Arabidopsis thaliana has been widely used in studies on basic plant physiology and biochemistry as well as in plant molecular genetic manipulations and developmental biology research because of its small genome, low chromosome number, short regeneration time (4–6 wk), availability of many mutants and genetic maps, sexual self-compatibility, and prolific seed production. More extensive use of Arabidopsis has been hampered because of difficulties in efficient and rapid regeneration and transformation procedures. Several methods for plant regeneration have been reported (1, 2, 3, 4, 5, 6, 7). Transformed plants have been recovered from various explants, such as leaf (8), stem (9), callus tissue (10), germinating seeds (11), root (12), and by using direct gene transfer to protoplasts (13). Despite these reports, the frequency of regeneration of transgenic plants was still low and took at least a few months to produce transgenic plants. Also the long period of in vitro incubation during shoot regeneration in these methods may increase the possibility of somaclonal variation and increases in ploidy level. Many reports have indicated the high regeneration potential of cotyledon explants at various stages of development in maturing embryos or after seed germination (14, 15, 16, 17). This chapter presents a new procedure for rapid and prolific regeneration of shoots from cotyledon explants of Arabidopsis in ecotypes “Landsberg erecta” and “C24.” Furthermore, this regeneration procedure is developed establishing a method for rapid production of transgenic Arabidopsis shoots using disarmed Agrobacterium tumefaciens within 2–3 wk (18, 19, 20). A transformation procedure using root explants is also presented as a separate method.


Shoot Regeneration Somaclonal Variation Cotyledon Explants Root Explants Immature Cotyledon 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Negrutiu, I. and Jacobs, M. (1978) Factors which enhance in vitro morphogenesis of Arabidopsis thaliana. Z. Pflanzenphysiol. 90, 423–430.Google Scholar
  2. 2.
    Negrutiu, I., Jacobs, M., and de Gree, F. W. (1978) In vitro morphogenesis of Arabidopsis thaliana: the origin of the explant. Z. Pflanzenphysiol. 90, 363–372.Google Scholar
  3. 3.
    Goto, N. (1979) In vitro organogenesis from leaf explants of some dwarf mutants of Arabidopsis thaliana (L) Heynh. Jpn. J. Genet. 54, 303–306.CrossRefGoogle Scholar
  4. 4.
    Huang, B. C. and Yeoman, M. M. (1984) Callus proliferation and morphogenesis in tissue culture of Arabidopsis thaliana. Plant Sci. Letters, 33, 353–363.CrossRefGoogle Scholar
  5. 5.
    Acedo, G. N. (1986) Regeneration of Arabidopsis callus in vitro. Plant Cell Tissue Org. Cult. 6, 109–114.CrossRefGoogle Scholar
  6. 6.
    Feldman, K. A. and Marks, M. D. (1986) Rapid and efficient regeneration of plants from explants of Arabidopsis thaliana. Plant Sci. 47, 63–69.CrossRefGoogle Scholar
  7. 7.
    Gleddie, S. (1989) Plant regeneration from cell suspension cultures of Arabidopsis thaliana heynh. Plant Cell Rep. 8, 1–8.CrossRefGoogle Scholar
  8. 8.
    Lloyd, A. M., Barnason, A. R., Rogers, S. G., Byrne, M. C., Fraley, R. T., and Horsch, R. B. (1986) Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens. Science 234, 464–466.PubMedCrossRefGoogle Scholar
  9. 9.
    An, G., Watson, B. D., and Chiang, C. C. (1986) Transformation of tobacco, potato, and Arabidopsis using a binary Ti vector system. Plant Physiol. 81, 301–305.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang, H. and Somerville, C. R. (1987) Transfer of the maize transposable element MU1 into Arabidopsis thaliana. Plant Sci. 48, 165–173.CrossRefGoogle Scholar
  11. 11.
    Feldman, K. A. and Marks, M. D. (1987) Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol. Gen. Genet. 208 1–9.CrossRefGoogle Scholar
  12. 12.
    Valvekens, D., Van Montagu, M., and Van Lijsebettens, M. (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explant using kanamycin selection. Proc. Nat. Acad. Sci. USA 85, 5536–5540.PubMedCrossRefGoogle Scholar
  13. 13.
    Damm, B., Schmidt, R., and Willmitzer, L. (1989) Efficient transformations of Arabidopsis thaliana direct gene transfer to protoplasts. Mol. Gen. Genet. 217, 6–12.PubMedCrossRefGoogle Scholar
  14. 14.
    Ozcan, S., Barghchi, M., and Draper, J. (1992) High-frequency adventitious shoot regeneration from immature cotyledons of pea (Pisum sativum L). Plant Cell Rep. 11, 44–47.CrossRefGoogle Scholar
  15. 15.
    Duncan, D. R., Williams, M. E., Zehr, B. E., and Widholm, J. M. (1985) The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes. Planta 165, 322–332.CrossRefGoogle Scholar
  16. 16.
    Ranch, J. P., Oglesby, L., and Zielinski, A. C. (1985) In vitro plant regeneration from embryo-derived tissue cultures of soybeans. In Vitro Cell Dev. Biol. 21, 653–658.CrossRefGoogle Scholar
  17. 17.
    Patton, D. and Meinke, D. (1988) High-frequency plant regeneration from cultured cotyledons of Arabidopsis thaliana. Plant Cell Rep. 7, 233–237.CrossRefGoogle Scholar
  18. 18.
    Barghchi, M., Turgut, K., Paul, W., Hodge, R., Griffiths, N., Draper, J., and Scott, R. (1990) Genetic Engineering of Arabidopsis thaliana, Abstracts VIIth International Congress on Plant Tissue Culture, Amsterdam, p. 46Google Scholar
  19. 19.
    Barghchi, M., Turgut, K., Griffiths, N., and Draper, J. (1991) Transformation of Arabidopsis by A. tumefaciens. In Vitro Cell Dev. Biol. 27, 3, 150.Google Scholar
  20. 20.
    Barghchi, M., Turgut, K., Scott, R., and Draper, J. (1994) High-frequency Agrobacterium-mediated transformation of Arabidopsis thaliana ecotypes “C24” and “Landsberg erecta.” J. Plant Growth Reg. 14, 61–67.CrossRefGoogle Scholar
  21. 21.
    Murashige, T. and Skoog, F. (1962) A revised medium for raped growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497.CrossRefGoogle Scholar
  22. 22.
    Jefferson, R. A., Kavanagh. T. A., and Bevan, M. W. (1987) GUS fusions. β-glucuronidase as a sensitive and versatile gene fusion marke in higher plants. EMBO J. 6, 3301–3307.Google Scholar
  23. 23.
    Barghchi, M. and Alderson, P. G. (1989) Pistachio (Pistacia vera L.), in Biotechnology in Agriculture and Forestry, vol. II (Bajaj, Y. P. S. ed.), Springer Verlag, Berlin, pp 68–98.Google Scholar
  24. 24.
    Kouider, M., Korban, S. S., Skirvin, R. M., and Chu, C. M. (1984) Influence of embryonic dominance and polarity on adventitious shoot formation from apple (Malus domestica cultivar Delicious) cotyledons in vitro. Amer. Soc. Hort. Sci. 109, 383–385.Google Scholar
  25. 25.
    Mante, S., Scorza, R., and Cordts, J. (1989) A simple, rapid protocol for adventitious shoot development from mature cotyledons of Glycine max cv Bragg. In Vitro Cell Dev. Biol. 25, 385–388.CrossRefGoogle Scholar
  26. 26.
    Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  27. 27.
    Ozcan, S., Barghchi, M., Firek, S., and Draper, J. (1993) Efficient adventitious shoot regeneration and somatic embryogenesis in pea. Plant Cell Tissue Org. Cult. 34, 271–277.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1995

Authors and Affiliations

  • Mehdi Barghchi
    • 1
  1. 1.Department of Applied Biology and BiotechnologyDe Montfort UniversityScraptoft, LeicesterUK

Personalised recommendations