Advertisement

Barley pp 115-126 | Cite as

Agrobacterium-Mediated Transformation of Barley Immature Embryos

  • Alison Hinchliffe
  • Wendy A. HarwoodEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1900)

Abstract

Barley transformation is an essential tool for a range of functional genomics studies as well as for future crop improvement. The demand for efficient crop transformation systems continues to grow, with new genome editing technologies adding to that demand. Here we describe an efficient and routine transformation protocol for the spring barley Golden Promise, based on Agrobacterium-mediated inoculation of immature embryos. This protocol has been widely used for overexpression and RNAi applications and more recently for CRISPR/Cas9 mediated genome editing. Average transformation efficiencies of 25% can be easily achieved.

Key words

Barley transformation Agrobacterium tumefaciens Immature embryos Genome editing Genome engineering 

Notes

Acknowledgments

We acknowledge support from the Biotechnology and Biological Sciences Research Council (BBSRC) via grant [BB/N019466/1] and grant [BB/P013511/1] to the John Innes Centre.

References

  1. 1.
    Lawrenson T, Shorinola O, Stacey N, Liu C, Østergaard L, Patron N, Uauy C, Harwood W (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol 16:258CrossRefGoogle Scholar
  2. 2.
    Ricroch A, Clairand P, Harwood W (2017) Use of CRISPR systems in plant genome editing: towards new opportunities in agriculture. Emerging Topics Life Sci 1(2):169–182CrossRefGoogle Scholar
  3. 3.
    Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104:37–48CrossRefGoogle Scholar
  4. 4.
    Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376CrossRefGoogle Scholar
  5. 5.
    Bartlett JG, Alves SC, Smedley M, Snape JW, Harwood WA (2008) High-throughput Agrobacterium-mediated barley transformation. Plant Methods 4:22CrossRefGoogle Scholar
  6. 6.
    Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789CrossRefGoogle Scholar
  7. 7.
    Volpi e Silva N, Patron N (2017) CRISPR-based tools for plant genome engineering. Emerging Topics Life Sci 1:135–149CrossRefGoogle Scholar
  8. 8.
    Harwood W, Smedley M (2009) Barley transformation using biolistic techniques. In: Jones H, Shewry P (eds) Transgenic wheat, barley and oats. Methods in molecular biology™ (Methods and Protocols), vol 478, Humana Press, pp 125–136Google Scholar
  9. 9.
    Garfinkel M, Nester EW (1980) Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol 144:732–743PubMedPubMedCentralGoogle Scholar
  10. 10.
    Smedley MA, Harwood WA (2014) Gateway-compatible plant transformation vectors. Methods Mol Biol 1223:3–16CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Crop Transformation Group, Department of Crop GeneticsJohn Innes CentreNorwichUK

Personalised recommendations