Agrobacterium Protocols

Volume 343 of the series Methods in Molecular Biology pp 171-184

Barley (Hordeum vulgare L.)

  • John Jacobsen
  • , Ingrid Venables
  • , Ming-Bo Wang
  • , Peter Matthews
  • , Michael Ayliffe
  • , Frank Gubler

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Crop improvement is limited by the availability of valuable traits in sexually compatible species. Access to new characters using genetic engineering would be of great value. Barley has been transformed using microprojectile bombardment and by direct gene transfer to protoplasts, but neither method has been able to produce fertile transformants in large numbers with simple transgene integration characteristics. Agrobacterium-mediated transformation was first achieved in 1997, and it has become the method of choice. Using immature embryos of the barley variety Golden Promise as the target organ, the binary vector pWBVec8 containing the intron-interrupted hygromycin resistance gene hph as the selectable marker, and selection of transformed cells on hygromycin, the Agrobacterium method is efficient, and the transgene insertion characteristics are superior to other methods. However, the procedure is strongly genotype dependent. In this report, we describe a transformation protocol giving details of plant culture, embryo isolation and preparation, vector details, Agrobacterium culture, infection methods, subsequent procedures for callus generation and plantlet production, and analysis of transgenic plants.

Key Words

Barley transformation barley embryos Golden Promise Agrobacterium pWBVec8