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Unformatted text preview: than the PCR detection method. After co-culture, leaf discs were assayed for GUS activity (using 0.5 mg ml 2 1 X-glcA) or transferred to regeneration media containing antibiotics. Rice ( Oryza sativa L. cv. Millin) was transformed using seed-derived callus based on the protocol in ref. 28, with modifications (see Supplementary Information). Arabidopsis thaliana L. cv. C24 was transformed using a floral-dip protocol 15 with bacteria resuspended in infiltration medium containing Murashige and Skoog Basal Medium, 5.0% (w/v) sucrose, 0.05% (v/v) Silwet L-77 and 0.1% (w/v) MES, pH 5.7. The modified media contained Murashige and Skoog Basal Medium, 1.0% (w/v) sucrose, 0.02% (v/v) Silwet L-77 and 0.1% (w/v) MES, pH 7.0. Analysis of transformed plants Genomic DNA was extracted from the regenerated plants using DNAzol (Invitrogen), digested with Eco RI and used for Southern blotting with a 32 P-labelled GUSPlus probe. Plant DNA sequences flanking the T-DNA insertion site(s) were determined by restriction digest/adaptor ligation 29 . Received 26 November; accepted 29 December 2004; doi:10.1038/nature03309. 1. Gelvin, S. B. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67, 16–37 (2003). 2. Roa-Rodrı ´guez, C. & Nottenburg, C. Agrobacterium-mediated transformation of plants. CAMBIA technology landscape paper k http://www.bios.net/Agrobacterium l (2003). 3. Van Montagu, M. Jeff Schell (1935–2003): steering Agrobacterium-mediated plant gene engineering. Trends Plant Sci. 8, 353–354 (2003). 4. Wood, D. W. et al. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294, 2317–2323 (2001). 5. Goodner, B. et al. Genome sequencing of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294, 2323–2328 (2001). 6. Klein, D. T. & Klein, R. M. Transmittance of tumor-inducing ability to avirulent crown-gall and related bacteria. J. Bacteriol. 66, 220–228 (1953). 7. Hooykaas, P. J. J., Klapwijk, P. M., Nuti, M. P., Schilperoort, R. A. & Ro ¨rsch, A. Transfer of the Agrobacterium tumefaciens Ti plasmid to avirulent Agrobacteria and to Rhizobium ex planta . J. Gen. Microbiol. 98, 477–484 (1977). 8. van Veen, R. J. M., den Dulk-Ras, H., Bisseling, T., Schilperoort, R. A. & Hooykaas, P. J. J. Crown gall tumor and root nodule formation by the bacterium Phyllobacterium myrsinacearum after the introduction of an Agrobacterium Ti plasmid or a Rhizobium Sym plasmid. Mol. Plant Microbe Interact. 1, 231–234 (1988). 9. van Veen, R. J. M., den Dulk-Ras, H., Schilperoort, R. A. & Hooykaas, P. J. J. Ti plasmid containing Rhizobium meliloti are non-tumorigenic on plants, despite proper virulence gene induction and T- strand formation. Arch. Microbiol. 153, 85–89 (1989)....
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