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Factors influencing successful Agrobacterium-mediated genetic transformation of wheat

  • Genetic Transformation and Hybridization
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Abstract

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (β-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.

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Acknowledgements

We are grateful to Alan Todd (Biomathematics unit, AEN, Rothamsted) for statistical assistance and to Alison Harvey (John Innes Centre) for supplying plasmids pAL156 and pAL154. Rothamsted receives grant-aided support from the Biotechnological and Biological Sciences Research Council UK. HW was sponsored by the UK Ministry of Agriculture, Fisheries and Food (now DEFRA). BKA was a Rothamsted International Fellow.

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  1. CPI Division, Rothamsted, Harpenden, AL5 2JQ, UK

    H. Wu, C. Sparks, B. Amoah & H. D. Jones

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  1. H. Wu
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  2. C. Sparks
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  3. B. Amoah
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  4. H. D. Jones
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Correspondence to H. D. Jones.

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Communicated by W. Harwood

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Wu, H., Sparks, C., Amoah, B. et al. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21, 659–668 (2003). https://doi.org/10.1007/s00299-002-0564-7

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  • DOI: https://doi.org/10.1007/s00299-002-0564-7

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