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Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava

Nature Protocols volume 4, pages 1845–1854 (2009)Cite this article

Abstract

Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking ∼6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

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Figure 1
Figure 2: Procedure for producing transgenic cassava plants.

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Acknowledgements

This work was partially funded by the Bill & Melinda Gates Foundation (BioCassava Plus program). J.A.O. received a PhD fellowship from the Rockefeller Foundation. We thank Kim Schlegel, Simona Pedrussio and Noemi Peter (ETH Zurich) for valuable technical assistance. We also thank Nigel Taylor (Donald Danforth Plant Science Center) and Peng Zhang (Shanghai Institute for Plant Physiology and Ecology) for discussions on the cassava transformation protocol. Christof Sautter, Samuel C. Zeeman (ETH Zurich) and Ingo Potrykus are acknowledged for their support.

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Authors and Affiliations

  1. Department of Biology, Plant Biotechnology, ETH Zurich, Zurich, Switzerland

    S E Bull, J A Owiti, M Niklaus, W Gruissem & H Vanderschuren

  2. Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath, UK

    S E Bull & J R Beeching

Authors
  1. S E Bull
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  2. J A Owiti
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  3. M Niklaus
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  4. J R Beeching
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  5. W Gruissem
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  6. H Vanderschuren
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Contributions

S.E.B. and H.V. designed the experiments and prepared the paper; S.E.B. and J.A.O. undertook experimental work with technical support from M.N.; and H.V., J.R.B and W.G. supervised the project.

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Correspondence to S E Bull or H Vanderschuren.

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Bull, S., Owiti, J., Niklaus, M. et al. Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava. Nat Protoc 4, 1845–1854 (2009). https://doi.org/10.1038/nprot.2009.208

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