Summary
Agrobacterium-mediated transformation of Nicotiana species, namely, Nicotiana tobaccum and Nicotiana benthamiana, using leaf disks as the target explant has provided the plant community with a valuable tool for rapid evaluation of transgenes in higher plants. This protocol has a number of desirable attributes: readily available explant material, high efficiency, and a relatively quick turnaround time. The in vitro regeneration scheme of the leaf disks is prolific and follows an indirect organogenic differentiation. N. tobaccum and N. benthamiana are highly susceptible to a variety of disarmed A. tumefaciens strains, and the transformation system is amenable to a number of selection agents. This protocol has proved to be easily transferable to a point at which a single investigator with minimal training can generate thousand of events per year. Moreover, seed size and quantity per event permit monitoring of segregation in Petri plates, and sufficient biomass can be accrued from an individual plant, which can be either clonally propagated or allowed to self-pollinate or easily outcrossed.
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References
Dasgupta, R., Garcia, B. H.2nd, and Goodman, R. M. (2001) Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes. Proc. Natl. Acad. Sci. USA 98, 4910–4915.
Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., and Fraley, R. T. (1985) A simple and general method for transferring genes into plants. Science 227, 1229–1231.
Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949–956.
Bendahmane, A., Querci, M., Kanyuka, K., and Baulcombe, D. C. (2000) Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. Plant J. 21, 73–81.
Bechtold, N., Ellis, J., and Pelletier, G. (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris 316, 1194–1199.
Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.
Fraley, R. T., Rogers, S. G., Horsch, R. B., et al. (1983) Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA 80, 4803–4807.
Gritz, L. and Davies, J. (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccaromyces cerevisiae. Gene 26, 179–188.
Thompson, C. J., Movva, N. R., Tizard, R., et al. (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J. 6, 2519–2523.
Joersbo, M. and Okkels, F. T. (1996) A novel principle for selection of transgenic plant cells: positive selection. Plant Cell Rep. 16, 219–221.
Cerutti, H. (2003) RNA interference: traveling in the cell and gaining functions? Trends Genet. 19, 39–46.
Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J., and Schilperoort, R. (1983) A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303, 179–180.
Koncz, C. and Schell, J. (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383–396.
Hood, E. E., Helmer, G. L., Fraley, R. T., and Chilton, M.-D. (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168, 1291–1301.
Hood, E. E., Gelvin, S. B., Melchers, L. S., and Hoekema, A. (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2, 208–218.
Luo, Z.-Q., Clemente, T., and Farrand, S. K. (2001) Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance. Mol. Plant Microbiol. Int. 14, 98–103.
Hajdukiewicz, P., Svab, Z., and Maliga, P. (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.
Hellens, R., Mullineaux, P., and Klee, H. (2000) Technical focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497.
Gamborg, O. L., Miller, R. A., and Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 152–158.
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Clemente, T. (2006). Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana). In: Wang, K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 343. Humana Press. https://doi.org/10.1385/1-59745-130-4:143
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DOI: https://doi.org/10.1385/1-59745-130-4:143
Publisher Name: Humana Press
Print ISBN: 978-1-58829-536-1
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