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 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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bergounioux C, Perennes C, Brown SC, Gadal P (1988) Cytometric analysis of growth regulator-dependent transcription and cell cycle progression in P. hybrida protoplast cultures. Planta 175:500–505
Kapros T, Bögre L, Németh K, Bakó L, Györgyey J, Wu SC, Dudits D (1992) Differential expression of histone H3 gene variants during cell cycle and somatic embryogenesis in alfalfa. Plant Physiol 98:621–625
Carle SA, Bates GW, Shannon TA (1998) Hormonal control of gene expression during reactivation of the cell cycle in tobacco mesophyll protoplasts. J Plant Growth Regul 17:221–230
Doležel J, Číhalíková J, Lucretti S (1992) A high yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. Planta 188:93–98
Doležel J, Číhalíková J, Weiserová J, Lucretti S (1999) Cell cycle synchronization in plant root meristems. Methods Cell Sci 21:95–107
Menges M, Murray JAH (2002) Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity. Plant J 30:203–212
Ghosh S, Sen J, Kalia S, 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
Arumuganathan K, Slattery JP, Tanksley SD, Earle ED (1991) Preparation and flow cytometric analysis of metaphase chromosomes of tomato. Theor Appl Genet 82:101–111
Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS, Moore G (1992) Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes. Nucleic Acids Res 20:1897–1901
Kumagai-Sano F, Hayashi T, Sano T, Hasezawa S (2006) Cell cycle synchronization of tobacco BY-2 cells. Nat Protocols 1:2621–2627
Young CW, Hodas S (1964) Hydroxyurea: inhibitory effect on DNA metabolism. Science 146:1172
Watson PA, Hanauske-Abel HH, Flint A, Lalande M (1991) Mimosine reversibly arrests cell cycle progression at the G1-S phase border. Cytometry 12:242–246
Ábrahám E, Miskolczi P, Ayaydin F, Yu P, Kotogány E, Bakó L, Ötvös K, Horváth VG, Dudits D (2011) Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells. J Exp Bot 62:2155–2168
Kotogány E, Dudits D, Horváth VG, 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
Mészáros T, Miskolczi P, Ayaydin F, Pettkó-Szandtner A, Peres A, Magyar Z, Horváth VG, Bakó L, Fehér A, Dudits D (2000) Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells. Plant Mol Biol 43:595–605
Hirano H, Harashima H, Shinmyo A, 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
Nias AHW, Fox M (1971) Synchronization of mammalian cells with respect to the mitotic cycle. Cell Tissue Kinet 4:375–398
Acknowledgement
The authors are grateful to Katalin Török for excellent technical assistance. This work was funded by OTKA grant no. NK 69227 (G.V.H.) and National Research, Development and Innovation Office, NKFIH grant no. K116318 (F.A.). Edit Ábrahám was supported by the János Bolyai Fellowship of the Hungarian Academy of Sciences.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ayaydin, F., Kotogány, E., Ábrahám, E., Horváth, G.V. (2017). Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture. In: Banfalvi, G. (eds) Cell Cycle Synchronization. Methods in Molecular Biology, vol 1524. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6603-5_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6603-5_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6602-8
Online ISBN: 978-1-4939-6603-5
eBook Packages: Springer Protocols