Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized 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 large amount of biological sample for further analyses. 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. A novel 5-ethynyl-2′-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.
Key wordsMedicago sativa suspension culture Cell cycle synchronization Hydroxyurea Retinoblastoma-related protein phosphorylation 5-Ethynyl-2′-deoxyuridine staining Fluorescence microscopy