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Synchronization, Transformation, and Cryopreservation of Suspension-Cultured Cells

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Arabidopsis Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 323))

Abstract

We have recently described the selection of rapidly dividing Arabidopsis cell suspension cultures MM1 and MM2d that provide a powerful platform for plant cell-cycle research. Here we provide detailed protocols and procedures to achieve high levels of synchronization, either by starving the cell cultures of sucrose or by applying the toxin aphidicolin. Cell-cycle activity during cell-cycle reentry (starvation-induced synchrony) or further cell-cycle progression (aphidicolin-induced synchrony) can be conveniently followed by using various validation procedures, such as determination of labeling index and metaphase/anaphase index or flow cytometry. We also describe a procedure that allows clonal transformed cell-suspension lines to be produced using Agrobacterium-mediated transformation, and an optimized and straightforward method for the cryopreservation and recovery of both parental and transformed lines which is applicable both to Arabidopsis and the tobacco BY2 cell lines. Cell-cycle synchronization capacity of the parental lines is maintained after both transformation and recovery from cryopreservation. The techniques described here require no specialized equipment and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures. The ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for future high-throughput cell-cycle analysis.

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Author information

Authors and Affiliations

  1. Institute of Biotechnology, University of Cambridge, Cambridge, UK

    Margit Menges & James A. H. Murray

Authors
  1. Margit Menges
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  2. James A. H. Murray
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Editor information

Editors and Affiliations

  1. Dept. of Biotechnology, Instituto Nacional de Investigacion y TecnologĂ­a Agraria y Alimentaria (INIA), Madrid, Spain

    Julio Salinas PhD

  2. Dept. de Genetica Molecular de Plantas, Centro Nacional de BiotecnologĂ­a-CSIC, Campus de Cantoblanco, Madrid, Spain

    Jose J. Sanchez-Serrano PhD

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© 2006 Humana Press Inc.

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Menges, M., Murray, J.A.H. (2006). Synchronization, Transformation, and Cryopreservation of Suspension-Cultured Cells. In: Salinas, J., Sanchez-Serrano, J.J. (eds) Arabidopsis Protocols. Methods in Molecular Biology™, vol 323. Humana Press. https://doi.org/10.1385/1-59745-003-0:45

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  • DOI: https://doi.org/10.1385/1-59745-003-0:45

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-395-4

  • Online ISBN: 978-1-59745-003-4

  • eBook Packages: Springer Protocols

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