Wheat (Triticum aestivum L.)

  • Yuechun Wan
  • Jeanne Layton
Part of the Methods in Molecular Biology book series (MIMB, volume 343)

Summary

This chapter describes a procedure for Agrobacterium-mediated wheat transformation. Freshly isolated immature embryos, precultured immature embryos, or embryogenic calli are inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the β-glucuronidase (GUS) gene with an intron, and a selectable marker, neomycinphosphotranferase (NPT) II gene. The inoculated explants are selected on callus induction medium with the selective agent G418 for approximately 2 wk. The resistant callus pieces that develop are then transferred onto medium with the selective agent and reduced plant growth regulators for plant regeneration and further selection. It takes approximately 2.5 to 3 mo from inoculation to the establishment of R0 plants in soil. All the transformants should be morphologically normal and set seeds. Approximately 35% of the transgenic plants have a single copy of the transgene based on data obtained from previous experiments.

Key Words

Agrobacterium tumefaciens wheat Triticum aestivum L. plant transformation monocotyledon 

References

  1. 1.
    Hansen, G., Shillito, R. D., and Chilton, M. D. (1997) T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes. Proc. Natl. Acad. Sci. USA 94, 11726–11730.PubMedCrossRefGoogle Scholar
  2. 2.
    Hiei, Y., Komari, O. S., and Kumashiro, T. (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282.PubMedCrossRefGoogle Scholar
  3. 3.
    Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T., and Kumashiro, T. (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat. Biotechnol. 14, 745–750.PubMedCrossRefGoogle Scholar
  4. 4.
    Cheng, M., Fry, J. E., Pang, S., et al. (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115, 971–980.PubMedGoogle Scholar
  5. 5.
    Cheng, M., Hu, T., Layton, J., Liu, C. N., and Fry, J. E. (2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cell. Dev. Biol. Plant 39, 595–604.CrossRefGoogle Scholar
  6. 6.
    Hu, T., Metz, S., Chay, C., et al. (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep. 21, 1010–1019.PubMedCrossRefGoogle Scholar
  7. 7.
    Jefferson, R. A. (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387–405.CrossRefGoogle Scholar
  8. 8.
    Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Yuechun Wan
  • Jeanne Layton

There are no affiliations available

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