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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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 547)

Summary

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 

Notes

Acknowledgments

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

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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

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