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Transformation of Plants with Multiple Cassettes Generates Simple Transgene Integration Patterns and High Expression Levels

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Abstract

We transformed rice (Oryza sativa L.) simultaneously with five minimal cassettes, each containing a promoter, coding region and polyadenylation site but no vector backbone. We found that multi-transgene cotransformation was achieved with high efficiency using multiple cassettes, with all transgenic plants we generated containing at least two transgenes and 16% containing all five. About 75% of the plants had simple transgene integration patterns with a predominance of single-copy insertions. The expression levels for all transgenes, and the overall coexpression frequencies, were much higher than previously reported in whole plasmid transformants. Four of five lines analyzed for transgene expression stability in subsequent generations showed stable and high expression levels over generations. A simple model is proposed, which accounts for differences in the molecular make-up and the expression profile of transgenic plants generated using whole plasmid or minimal cassettes. We conclude that gene transfer using minimal cassettes is an efficient and rapid method for the production of transgenic plants containing and stably expressing several different transgenes. Our results facilitate effective manipulation of multi-gene pathways in plants in a single transformation step.

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Authors and Affiliations

  1. Central Rice Research Institute, Cuttack, 753006, Orissa, India

    Pawan K. Agrawal

  2. Institute for Research on Environment and Sustainability, School of Biology, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE1 7RU, UK

    Ajay Kohli

  3. Department of Biology, University of York, Heslington, York, YO10 5DD, UK

    Richard M. Twyman

  4. Department de Produccio Vegetal i Ciencia Forestal, ICREA, Universitat de Lleida, Av. Alcalde Rovira Roure, 191, E-25198, LLEIDA, Spain

    Paul Christou

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  1. Pawan K. Agrawal
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  2. Ajay Kohli
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  3. Richard M. Twyman
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  4. Paul Christou
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Correspondence to Paul Christou.

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Agrawal, P.K., Kohli, A., Twyman, R.M. et al. Transformation of Plants with Multiple Cassettes Generates Simple Transgene Integration Patterns and High Expression Levels. Mol Breeding 16, 247–260 (2005). https://doi.org/10.1007/s11032-005-0239-5

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