Abstract
Synchronization of root cells through chemical treatment can generate a large number of cells blocked in specific cell cycle phases. In plants, this approach can be employed for cell suspension cultures and plant seedlings. To identify plant cells in the course of the cell cycle, especially during mitosis in meristematic tissues, chemical inhibitors can be used to block cell cycle progression. Herein, we present a simplified and easy-to-apply protocol to visualize mitotic figures, nuclei morphology, and organization in whole Arabidopsis root apexes. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with DAPI. The protocol allows carrying out bulk analysis of nuclei and cell cycle phases in root cells and will be valuable to investigate mutants like overexpressing lines of genes disturbing the plant cell cycle.
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References
Fiskesjö G (1985) The Allium test as a standard in environmental monitoring. Hereditas 102:99–112
Planchais S, Glab N, Inzé D, Bergounioux C (2000) Chemical inhibitors: a tool for plant cell cycle studies. FEBS Lett 476:78–83
Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 121:1–30
Planchais S, Glab N, Trehin C, Perennes C, Bureau J-M, Meijer L, Bergounioux C (1997) Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension. Plant J 12:191–202
Tyburski J, Dunajska-Ordak K, Skorupa M, Tretyn A (2012) Role of ascorbate in the regulation of the Arabidopsis thaliana root growth by phosphate availability. J Bot. doi:10.1155/2012/580342
Bauwens S, Katsanis K, Van Montagu M, Van Oostveldt P, Engler G (1994) Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana. Plant J 6:123–131
Boisnard-Lorig C, Colon-Carmona A, Bauch M, Hodge S, Doerner P, Bancharel E, Dumas C, Haseloff J, Berger F (2001) Dynamic analyses of the expression of the HISTONE::YFP fusion protein in Arabidopsis show that syncytial endosperm is divided in mitotic domains. Plant Cell 13:495–509
Willemse J, Kulikova O, de Jong H, Bisseling T (2008) A new whole-mount DNA quantification method and the analysis of nuclear DNA content in the stem-cell niche of Arabidopsis roots. Plant J 55:886–894
Vieira P, Engler G, de Almeida-Engler J (2012) Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis. New Phytologist 195:488–496
Acknowledgments
This research was supported by grants from the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), of a PVE Science without Frontiers of the National Council for Science and Technology (CNPq) and by the CAPES/COFECUB program.
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de Souza Junior, J.D.A., de Sa, M.F.G., Engler, G., de Almeida Engler, J. (2016). Imaging Nuclear Morphology and Organization in Cleared Plant Tissues Treated with Cell Cycle Inhibitors. In: Caillaud, MC. (eds) Plant Cell Division. Methods in Molecular Biology, vol 1370. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3142-2_5
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DOI: https://doi.org/10.1007/978-1-4939-3142-2_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3141-5
Online ISBN: 978-1-4939-3142-2
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