Summary
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wang, M. B., Abbott, D. C., and Waterhouse, P. M. (2000) A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol. Plant Pathol. 1, 347–356.
Wang, M. B., Abbott, D. C., Upadhyaya, N. M., Jacobsen, J. V., and Waterhouse, P. M. (2001) Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes. Aust. J. Plant Physiol. 28, 149–156.
Jacobsen, J., Matthews, P., Abbott, D., Wang, M., and Waterhouse, P. M. (2000) Transgenic barley, in Transgenic Cereals (O'Brien, L. and Henry, R. J., eds.), American Association of Cereal Chemists, St. Paul, MN, pp. 88–114.
Schunmann, P., Coia, G., and Waterhouse, P. (2002) Biopharming the SimpliRED diagnostic reagent in barley, potato and tobacco. Mol. Breed 9, 113–121.
Horvath, H., Huang, J., Wong, O., et al. (2000) The production of recombinant proteins in transgenic barley grains. Proc. Natl. Acad. Sci. USA 97, 1914–1919.
Jensen, L. G., Politz, O., Olsen, O., Thomsen, K. K., and von Wettstein, D. (1998) Inheritance of a codon-optimised transgene expressing heat-stable (1,3-1,4)-β-glucanase in scutellum and aleurone of germinating barley. Hereditas 129, 215–225.
Patel, M., Johnson, J., Brettell, R., Jacobsen, J., and Xue, G. (2000) Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains. Mol. Breeding 6, 113–123.
Matthews, P. R., Thornton, S., Gubler, F., White, R., and Jacobsen, J. V. (2002) Use of the green fluorescent protein to locate α-amylase expression in barley grains. Func. Plant Biol. 29, 1037–1043.
Ahlandsberg, S., Sathish, P., Sun, C., and Jannson, C. (1999) Green fluorescent protein as a reporter system in the transformation of barley cultivars. Physiol. Plantarum 107, 194–200.
Wan, Y. and Lemaux, P. G. (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104, 37–48.
Funatsuki, H., Kuroda, H., Kihara, M., et al. (1995) Fertile transgenic barley generated by direct DNA transfer to protoplasts. Theor. Appl. Genet. 91, 702–712.
Tingay, S., McElroy, D., Kalla, R., et al. (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J. 11, 1369–1376.
Kohli, A., Gahakwa, D., Vain, P., Laurie, D. A., and Christou, P. (1999) Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing. Planta 208, 88–97.
Matthews, P., Wang, M., Waterhouse, P., et al. (2001) Marker gene elimination from transgenic barley using co-transformation with adjacent “twin T-DNAs” on a standard Agrobacterium transformation vector. Mol. Breeding 7, 195–202.
Murray, F., Brettell, R., Matthews, P., Bishop, D., and Jacobsen, J. (2004) Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes Plant Cell Rep. 22, 397–402.
Trifonova, A., Madsen, S., and Olesen, A. (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci. 161, 871–880.
Wang, M. B., Matthews, P. R., Upadhyaya, N. M., and Waterhouse, P. M. (1998) Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants. Acta Hortic. 461, 401–407.
Wang, M., Upadhyaya, N. M., Brettell, R. I. S., and Waterhouse, P. M. (1997) Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J. Genet. Breed. 51, 325–334.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Jacobsen, J., Venables, I., Wang, MB., Matthews, P., Ayliffe, M., Gubler, F. (2006). Barley (Hordeum vulgare L.). In: Wang, K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 343. Humana Press. https://doi.org/10.1385/1-59745-130-4:171
Download citation
DOI: https://doi.org/10.1385/1-59745-130-4:171
Publisher Name: Humana Press
Print ISBN: 978-1-58829-536-1
Online ISBN: 978-1-59745-130-7
eBook Packages: Springer Protocols