Summary
Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important forage legume, a population of red clover with a high potential for regeneration in tissue culture has been developed. Here we provide a detailed procedure for Agrobacterium-mediated transformation of genotypes derived from this regenerable population. We have successfully used this methodology to express a β-glucuronidase (GUS) reporter gene and to silence an endogenous polyphenol oxidase gene in red clover.
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References
Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987) Gus fusions-β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907.
Sullivan, M. L., Hatfield, R. D., Thoma, S. L., and Samac, D. A. (2004) Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa. Plant Physiol. 136, 3234–3244.
Quesenberry, K. H., Wofford, D. S., Smith, R. L., Krottje, P. A., and Tcacenco, F. (1996) Production of red clover transgenic for neomycin phosphotransferase II using Agrobacterium. Crop Sci. 36, 1045–1048.
Wofford, D. S., Baltensperger, D. D., and Quesenberry, K. H. (1992) In vitro culture responses of alyceclover genotypes on four media systems. Crop Sci. 32, 261–265.
Wan, Y., Sorensen, E. L., and Liang, G. H. (1988) Genetic control of in vitro regeneration in alfalfa (Medicago sativa L). Euphytica 39, 3–9.
Quesenberry, K. H. and Smith, R. R. (1993) Recurrent selection for plant regeneration from red clover tissue culture. Crop Sci. 33, 585–589.
Smith, R. R. and Quesenberry, K. H. (1995) Registration of NewRC red clover germplasm. Crop Sci. 35, 295–295.
Gleave, A. P. (1992) A versatile binary vector system with a T-DNA organizational-structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207.
Rogers, S. G., Klee, H. J., Horsch, R. B., and Fraley, R. T. (1987) Improved vectors for plant transformation-expression cassette vectors and new selectable markers. Methods Enzymol. 153, 253–277.
Hellens, R., Mullineaux, P., and Klee, H. (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451.
Gamborg, O. L., Miller, R. A., and Ojima, K. (1968) Nutrient requirement of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158.
Moore, G. A., Jacono, C. C., Neidigh, J. L., Lawrence, S. D., and Cline, K. (1992) Agrobacterium-mediated transformation of citrus stem segments and regeneration of transgenic plants. Plant Cell Rep. 11, 238–242.
Rogers, S. G., Horsch, R. B., and Fraley, R. T. (1986) Gene transfer in plants—production of transformed plants using Ti plasmid vectors. Methods Enzymol. 118, 627–640.
Taylor, N. L. (1980) Red clover, in Hybridization of Crop Plants (Fehr, W. R. and Hadley, H. H., eds.), American Society of Agronomy and Crop Science Society of America, Madison, WI, pp. 261–272.
Adair, D., Irwin, R., and Traynor, P. L. (2001) A Practical Guide to Containment. Information Systems for Biotechnology, Blacksburg, VA.
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Sullivan, M.L., Quesenberry, K.H. (2006). Red Clover (Trifolium pratense). In: Wang, K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 343. Humana Press. https://doi.org/10.1385/1-59745-130-4:369
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DOI: https://doi.org/10.1385/1-59745-130-4:369
Publisher Name: Humana Press
Print ISBN: 978-1-58829-536-1
Online ISBN: 978-1-59745-130-7
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