Synchronization of Medicago sativa Cell Suspension Culture

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


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 



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.


  1. 1.
    Bergounioux, C., Perennes, C., Brown, S. C., and Gadal, P. (1988) Cytometric analysis of growth regulator-dependent transcription and cell cycle progression in P. hybrida protoplast cultures. Planta 175, 500–505.CrossRefGoogle Scholar
  2. 2.
    Kapros, T., Bögre, L., Németh, K., Bakó, L., Györgyey, J., Wu, S. C., and Dudits, D. (1992) Differential expression of histone H3 gene variants during cell cycle and somatic embryogenesis in alfalfa. Plant Physiol. 98, 621–625.PubMedCrossRefGoogle Scholar
  3. 3.
    Carle, S. A., Bates, G. W., and Shannon, T. A. (1998) Hormonal control of gene expression during reactivation of the cell cycle in tobacco mesophyll protoplasts. J. Plant Growth Regul. 17, 221–230.PubMedCrossRefGoogle Scholar
  4. 4.
    Doležel, J., Číhalíková, J., and Lucretti, S. (1992) A high yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. Planta 188, 93–98.CrossRefGoogle Scholar
  5. 5.
    Doležel, J., Číhalíková, J., Weiserová, J., and Lucretti, S. (1999) Cell cycle synchronization in plant root meristems. Methods Cell Sci. 21, 95–107.PubMedCrossRefGoogle Scholar
  6. 6.
    Menges, M., and Murray, J. A. H. (2002) Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity. Plant J. 30, 203–212.PubMedCrossRefGoogle Scholar
  7. 7.
    Ghosh, S., Sen, J., Kalia, S., and Guha-Mukherjee, S. (1999) Establishment of synchronization in carrot cell suspension culture and studies on stage specific activation of glyoxalase I. Methods Cell Sci. 21, 141–148.PubMedCrossRefGoogle Scholar
  8. 8.
    Arumuganathan, K., Slattery, J. P., Tanksley, S. D., and Earle, E. D. (1991) Preparation and flow cytometric analysis of metaphase chromosomes of tomato. Theor. Appl. Genet. 82, 101–111.CrossRefGoogle Scholar
  9. 9.
    Wang, M. L., Leitch, A. R., Schwarzacher, T., Heslop-Harrison, J. S., and Moore, G. (1992) Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes. Nucleic Acids Res. 20, 1897–1901.PubMedCrossRefGoogle Scholar
  10. 10.
    Kumagai-Sano, F., Hayashi, T., Sano, T., and Hasezawa, S. (2006) Cell cycle synchronization of tobacco BY-2 cells. Nat. Protoc. 1, 2621–2627.PubMedCrossRefGoogle Scholar
  11. 11.
    Young, C. W., and Hodas, S. (1964) Hydroxyurea: inhibitory effect on DNA metabolism. Science 146, 1172.PubMedCrossRefGoogle Scholar
  12. 12.
    Watson, P. A., Hanauske-Abel, H. H., Flint, A., and Lalande, M. (1991) Mimosine reversibly arrests cell cycle progression at the G1-S phase border. Cytometry 12, 242–246.PubMedCrossRefGoogle Scholar
  13. 13.
    Kotogány, E., Dudits, D., Horváth, V. G., and Ayaydin, F. (2010) A rapid and robust assay for detection of S phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods 6, 1–15.CrossRefGoogle Scholar
  14. 14.
    Mészáros, T., Miskolczi, P., Ayaydin, F., Pettkó-Szandtner, A., Peres, A., Magyar, Z., Horváth, V. G., Bakó, L., Fehér, A., and Dudits, D. (2000) Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells. Plant Mol. Biol. 43, 595–605.PubMedCrossRefGoogle Scholar
  15. 15.
    Hirano, H., Harashima, H., Shinmyo, A., and Sekine M. (2008) Arabidopsis retinoblastoma-related protein 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation. Plant Mol. Biol. 66, 259–275.PubMedCrossRefGoogle Scholar
  16. 16.
    Nias, A. H. W., and Fox, M. (1971) Synchronization of mammalian cells with respect to the mitotic cycle. Cell Tissue Kinet. 4, 375–398.PubMedGoogle Scholar

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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