Summary
A better understanding of the genetic basis underlying the genotype dependence of Brassica oleracea transformation is enabling researchers to distinguish between recalcitrant and successful candidate genotypes for routine transformation. In this chapter we outline an A. tumefaciens-mediated transformation method for B. oleracea using 4-d-old cotyledonary explants and a model B. oleracea doubled haploid genotype, AG DH1012. After only 3 wk on kanamycin selection, the first transgenic shoots can be isolated. Transformation efficiencies in the region of 10 to 25% (based on 50-125 PCR-positive independent shoots from 500 infected explants) are typically achieved. For researchers wishing to use their own plant genotype, we highlight the tissue culture phenotypes that are conducive to efficient transformation.
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
Srivastava, V., Reddy, A. S., and Guha-Mukherjee, S. (1988) Transformation of Brassica oleracea mediated by oncogenic Agrobacterium tumefaciens. Plant Cell Rep. 7, 504–507.
De Block, M., Tenning, P., and De Brouwer, D. (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol. 91, 694–701.
Metz, T. D., Dixit, R., and Earle, E. D. (1995) Agrobacterium tumefaciens mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata). Plant Cell Rep. 15, 287–292.
Pius, P. K. and Achar, P. N. (2000) Agrobacterium-mediated transformation and plant regeneration of Brassica oleracea var. capitata. Plant Cell Rep. 19, 888–892.
Kuginuki, Y. and Tsukazaki, H. (2001) Agrobacterium-mediated transformation of Brassica vegetables. J. Jpn. Soc. Hortic. Sci. 70, 682–690.
Tsukazaki, H., Kuginuki, Y., Aida, R., and Suzuki, T. (2002) Agrobacterium-mediated transformation of a doubled haploid line of cabbage. Plant Cell Rep. 21, 257–262.
Sparrow, P. A. C., Dale, P. J., and Irwin, J. A. (2004) The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation. Plant Cell Rep. 23, 64–70.
Sparrow, P. A. C., Snape, J. W., Dale, P. J., and Irwin, J. A. (2005) The rapid identification of B. napus genotypes, for high-throughput transformation, using phenotypic tissue culture markers. Acta Horticulturae. Paper presented at the ‘Joint Meeting of the 14th Crucifer Genetics Workshop and the 4th ISHS Symposium on Brassicas’ held at Chungnam National University Daejeon, S. Korea, October 2004.
Sparrow, P. A. C., Townsend, T., Arthur, A. E., Dale, P. J., and Irwin, J. A. (2004) Genetic analysis of Agrobacterium tumefaciens susceptibility in Brassica oleracea. Theoret. Appl. Genet. 108, 664–650.
Sparrow, P. A. C., Townsend, T., Morgan, C. L., Arthur, A. E., Dale, P. J., and Irwin, J. A. (2004) Genetic analysis of in vitro shoot regeneration from cotyledonary petioles of Brassica oleracea. Theoret. Appl. Genet. 108, 1249–1255.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays and tobacco tissue culture. Physiol. Plant 15, 437–497.
Gamborg, O. L., Miller, R. B., and Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158.
Bohuon, E. J. R. (1995) A genetic analysis of Brassica oleracea. PhD submitted to the University of Birmingham.
Bouhuon, E. J. R., Keith, D. J., Parkin, I. A. P., Sharpe, A. G., and Lydiate, D. J. (1996) Alignment of the conserved C genomes of Brassica oleracea and Brassica napus. Theoret. Appl. Genet. 93, 833–839.
Moloney, M. M., Walker, J. M., and Sharma, K. K. (1989) High-efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep. 8, 238–242.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Sparrow, P.A.C., Dale, P.J., Irwin, J.A. (2006). Brassica oleracea. In: Wang, K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 343. Humana Press. https://doi.org/10.1385/1-59745-130-4:417
Download citation
DOI: https://doi.org/10.1385/1-59745-130-4:417
Publisher Name: Humana Press
Print ISBN: 978-1-58829-536-1
Online ISBN: 978-1-59745-130-7
eBook Packages: Springer Protocols