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Synchronization of Medicago sativa Cell Suspension Culture

  • Ferhan Ayaydin
  • Edit Kotogány
  • Edit Ábrahám
  • Gábor V. HorváthEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 761)

Abstract

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide the largest amount of biological sample for further analysis. Here we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division and also the application and removal of hydroxyurea blocking agent. A novel method is used for the estimation of cell portion that enters S phase during the assay. The protocol can be used in the case of other species as well.

Key words

Medicago sativa suspension culture cell cycle synchronization hydroxyurea 5-ethynyl-2′-deoxyuridine staining fluorescence microscopy 

Notes

Acknowledgment

The authors are grateful to Katalin Török for excellent technical assistance. This work was funded by OTKA grant no. NK 69227. Edit Ábrahám was supported by the János Bolyai Fellowship of the Hungarian Academy of Sciences.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ferhan Ayaydin
    • 1
  • Edit Kotogány
    • 1
  • Edit Ábrahám
    • 3
  • Gábor V. Horváth
    • 2
    Email author
  1. 1.Cellular Imaging LaboratoryBiological Research Centre, Hungarian Academy of SciencesSzegedHungary
  2. 2.Institute of Plant Biology, Biological Research Centre, Hungarian Academy of SciencesSzegedHungary
  3. 3.Cellular Imaging LaboratoryInstitute of Plant Biology, Biological Research Centre, Hungarian Academy of SciencesSzegedHungary

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