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Estimation of nuclear DNA content in plants using flow cytometry

Nature Protocols volume 2, pages 2233–2244 (2007)Cite this article

Abstract

Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.

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Figure 1: Histograms of relative fluorescence intensities (relative nuclear DNA contents) obtained after the analysis of isolated plant nuclei.
Figure 2: Flow cytometric ploidy analysis.
Figure 3: Estimation of nuclear DNA content in absolute units.

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Acknowledgements

J.D. was partially supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant awards ME 884 and LC06004), and J.S. by the Ministry of Education, Youth and Sports of the Czech Republic (project no. MSM 0021620828), and the Academy of Sciences of the Czech Republic (no. AV0Z60050516). We thank David W. Galbraith (Tucson, AZ) for revision of the text, and an anonymous reviewer for useful comments.

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Authors and Affiliations

  1. Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, Olomouc, CZ-77200, Czech Republic

    Jaroslav Doležel

  2. Department of Cell Biology and Genetics, Palacký University, Šlechtitelů 11, Olomouc, CZ-78371, Czech Republic

    Jaroslav Doležel

  3. Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, Rennweg 14, Vienna, A-1030, Austria

    Johann Greilhuber

  4. Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, Prague, CZ-12801, Czech Republic

    Jan Suda

  5. Institute of Botany, Academy of Sciences of the Czech Republic, Průhonice 1, CZ-25243, Czech Republic

    Jan Suda

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Correspondence to Jaroslav Doležel.

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Doležel, J., Greilhuber, J. & Suda, J. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2, 2233–2244 (2007). https://doi.org/10.1038/nprot.2007.310

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