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A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21

Nature Protocols volume 4, pages 638–649 (2009)Cite this article

Abstract

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process—from wild-type seeds to transgenic T1 seeds—takes ∼8 months to complete.

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Figure 1: Binary vector pVec8-GFP used to transform B. distachyon genotype Bd21.
Figure 2: Agrobacterium-mediated transformation protocol for B. distachyon genotype Bd21.
Figure 3: Selection and screening of transgenic calli and plantlets using either combined chemical selection and visual screening or chemical selection alone.

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Acknowledgements

This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) and through a Short-Term Marie Curie EST fellowship.

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Authors and Affiliations

  1. Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney Lane, NR4 7UH, Norwich, UK

    Sílvia C Alves, Barbara Worland, Vera Thole, John W Snape & Philippe Vain

  2. Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, NR4 7UH, Norwich, UK

    Michael W Bevan

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  1. Sílvia C Alves
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  2. Barbara Worland
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  3. Vera Thole
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  4. John W Snape
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  6. Philippe Vain
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Correspondence to Philippe Vain.

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Alves, S., Worland, B., Thole, V. et al. A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21. Nat Protoc 4, 638–649 (2009). https://doi.org/10.1038/nprot.2009.30

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