Agrobacterium-Mediated Transformation of Ruta graveolens L

  • Karine LièvreEmail author
  • Thi Lê Minh Tran
  • Sébastien Doerper
  • Alain Hehn
  • Paul Lacoste
  • Brigitte Thomasset
  • Frédéric Bourgaud
  • Eric Gontier
Part of the Methods in Molecular Biology book series (MIMB, volume 547)


Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- –to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1RifR containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and β-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding β-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

Key words

Agrobacterium tumefaciens Ruta graveolens Hypocotyl Direct regeneration Transgenic plant Gus nptII 



The authors would like to thank Andrew Saunders for help in improving the English quality of this manuscript.


  1. 1.
    Bourgaud. F, Hehn A., Larbat R., Doerper S.,Gontier E., Kellner S. and Matern U. (2006). Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450. Phytochemistry Reviews, 5: 293–308.CrossRefGoogle Scholar
  2. 2.
    Berenbaum M. R. (2002). Postgenomic chemical ecology: from genetic code to ecological interactions. Journal of Chemical Ecology 28: 873–896.CrossRefPubMedGoogle Scholar
  3. 3.
    Massot B., Milesi S., Gontier E., Bourgaud F. and Guckert A. (2000). Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens. Plant Cell, Tissue and Organ Culture 62: 11–19.CrossRefGoogle Scholar
  4. 4.
    Milési S., Massot B., Gontier E., Bourgaud F. and Guckert A. (2001). Ruta graveolens L.: a promising species for the production of furanocoumarins. Plant Science 161: 189–199CrossRefGoogle Scholar
  5. 5.
    Lièvre K., Hehn A., Tran T. L. M., Thomasset B., Bourgaud F. and Gontier E. (2005) Genetic transformation of the medicinal plant Ruta graveolens L. by an Agrobacterium tumefaciens-mediated method. Plant Science, 168: 883–888.CrossRefGoogle Scholar
  6. 6.
    Eilert, U. (1989). Elicitor Induction of Secondary Metabolism in Dedifferentiated in Vitro Systems of Ruta graveolens in W. G. W. Kurz, ed. Primary and Secondary Metabolism of Plant.Google Scholar
  7. 7.
    Porter, J. R. (1991). Host range and implications of plant infection by Agrobacterium rhizogenes. Critical Reviews inP lant Sciences 10: 387–421.CrossRefGoogle Scholar
  8. 8.
    Murashige, T. and Skoog F. (1962). A revised medium for rapid growth and bio assay with tobacco tissues cultures. Physiologica Plantarum 15: 473–497.CrossRefGoogle Scholar
  9. 9.
    Jefferson RA, Burgess SM, Hirsh D (1986). β-glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci 83: 8447–8451CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Karine Lièvre
    • 1
    Email author
  • Thi Lê Minh Tran
  • Sébastien Doerper
  • Alain Hehn
  • Paul Lacoste
  • Brigitte Thomasset
  • Frédéric Bourgaud
  • Eric Gontier
  1. 1.UMR Nancy-Université-INRA Agronomie et Environnement Nancy-ColmarVandoeuvre-Lès-NancyFrance

Personalised recommendations