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Mesophyll Protoplasts and PEG-Mediated Transfections: Transient Assays and Generation of Stable Transgenic Canola Plants

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Transgenic Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1864))

Abstract

Plant protoplasts are derived by controlled enzymatic digestion that removes the plant cell wall without damaging the cell membrane. Protoplasts represent a true single-cell system and are useful for various biochemical and physiological studies. Protoplasts from several agriculturally important crop species can be regenerated into a fertile whole plant, extending the utility of protoplasts from transient expression assays to the generation of stable transformation events. Here we describe procedures for transient and stable transformation of leaf mesophyll protoplasts obtained from axenic shoot cultures of canola (Brassica napus). Key steps including enzymatic digestion for protoplast release, density gradient-based protoplast purification, PEG-mediated transfection, bead-type culturing (sea-plaque agarose and sodium alginate), and the recovery of putative transgenic canola plants are described. This method has been used for double-stranded DNA break-mediated genome editing and for the routine generation of stable transgenic canola events at commercial scale.

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Author information

Authors and Affiliations

  1. Department of Economic Development, Jobs, Transport and Resources (DEDJTR), Agribio, Bundoora, VIC, Australia

    Sareena Sahab, Matthew J. Hayden, John Mason & German Spangenberg

  2. School of Applied Systems Biology, La Trobe University, Agribio, Bundoora, VIC, Australia

    Matthew J. Hayden, John Mason & German Spangenberg

Authors
  1. Sareena Sahab
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  2. Matthew J. Hayden
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  3. John Mason
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  4. German Spangenberg
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Corresponding author

Correspondence to Sareena Sahab .

Editor information

Editors and Affiliations

  1. Corteva Agriscienceâ„¢, Agriculture Division of DowDuPontâ„¢, Johnston, IA, USA

    Sandeep Kumar

  2. Corteva Agriscienceâ„¢, Agriculture Division of DowDuPontâ„¢, Johnston, IA, USA

    Pierluigi Barone

  3. Corteva Agriscienceâ„¢, Agriculture Division of DowDuPontâ„¢, Johnston, IA, USA

    Michelle Smith

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Sahab, S., Hayden, M.J., Mason, J., Spangenberg, G. (2019). Mesophyll Protoplasts and PEG-Mediated Transfections: Transient Assays and Generation of Stable Transgenic Canola Plants. In: Kumar, S., Barone, P., Smith, M. (eds) Transgenic Plants. Methods in Molecular Biology, vol 1864. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8778-8_10

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  • DOI: https://doi.org/10.1007/978-1-4939-8778-8_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8777-1

  • Online ISBN: 978-1-4939-8778-8

  • eBook Packages: Springer Protocols

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