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The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

  • Genetic Transformation and Hybridization
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Abstract

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.

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Acknowledgements

The authors would like to thank Dr. M. Christey and Mr. R. Braun (Crop and Food Research, Christchurch, New Zealand) and Dr. M. Perry (John Innes Centre, Norwich, UK) for testing the efficacy of DH AG1012. Our thanks are further extended to Dr. R. Richardson and Mr. N. Bird for their assistance in the laboratory, and to Drs. W. Harwood, M. Perry and M. Smoker for their useful comments on this manuscript. The authors also thank the Department of Environment, Food and Rural Affairs (Defra) and the Biotechnology and Biological Science Research Council (BBSRC) for their support. This work was funded by Defra project HH0909SFV as part of an investigation of the genetic basis of transformation in horticultural brassicas. An extended protocol, including a timetable to aid experimental planning, has been posted on the Biotechnology Resources for Arable Crop Transformation (BRACT) website, http://www.bract.org. BRACT is funded by Defra.

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  1. John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK

    P. A. C. Sparrow, P. J. Dale & J. A. Irwin

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  1. P. A. C. Sparrow
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  2. P. J. Dale
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  3. J. A. Irwin
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Correspondence to P. A. C. Sparrow.

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Communicated by H. Lörz

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Sparrow, P.A.C., Dale, P.J. & Irwin, J.A. The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation. Plant Cell Rep 23, 64–70 (2004). https://doi.org/10.1007/s00299-004-0818-7

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  • DOI: https://doi.org/10.1007/s00299-004-0818-7

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