Synchronization of Medicago sativa Cell Suspension Culture
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 wordsMedicago 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.
- 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