Abstract
With the accelerating advances in genetics and genomics research in Arabidopsis and Brassica, transformation technologies are now routinely being exploited to elucidate gene function as well as contributing to the development of novel enhanced crops. When a researcher’s desired goal is simply to modify or introduce candidate genes into a Brassica, the availability of easy-to-follow protocols and knowledge of readily transformable genotypes becomes a valuable resource. In this chapter we outline a basic A. tumefaciens-mediated transformation method, using 4-day-old cotyledonary explants, that has been successfully applied to a range of different B. oleracea and B. napus genotypes. For demonstration purposes, we focus primarily on the diploid species B. oleracea using a model doubled haploid genotype, AG DH1012. After only 3–4 weeks on kanamycin selection the first transgenic shoots can be isolated. Transformation efficiencies are typically in the region of 15–25 % (based on 15–25 PCR-positive independent shoots from 100 inoculated explants). Most explants will produce multiple shoots (1–3+ per explant) and so the total number of transgenic shoots produced will exceed 15–25 per 100 explant experiment. The protocol is also applicable to B. napus and modifications specific to this species are highlighted accordingly. For researchers wishing to use their own plant genotype, tissue culture phenotypes that are conducive to efficient transformation are also highlighted within this chapter.
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Acknowledgements
The authors acknowledge the support of the Biotechnology and Biological Science Research Council (BBSRC) grant BB/J004588/1 for funding and the John Innes Foundation.
Further illustrative photographs of the transformation of AG DH1012 can be seen on the Biotechnology Resources for Arable Crop Transformation (BRACT) website, www.bract.org. BRACT was originally established funded by Defra (UK) and now operates on a full cost recovery basis as a transformation resource for the research community.
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Sparrow, P.A.C.H.n., Irwin, J.A. (2015). Brassica oleracea and B. napus . In: Wang, K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 1223. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1695-5_23
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DOI: https://doi.org/10.1007/978-1-4939-1695-5_23
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