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Glycerol-treated nuclear suspensions—an efficient preservation method for flow cytometric analysis of plant samples

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Abstract

Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.

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Abbreviations

CV:

Coefficient of variation

DAPI:

4',6-diamidino-2-phenylindole

FCM:

Flow cytometry/flow cytometric

PI:

Propidium iodide

SD:

Standard deviation

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Acknowledgements

Jana Krejčíková and Pavel Trávníček helped us with the flow cytometric analyses and Jan Lepš, Kateřina Štajerová and Kenneth Molem kindly assisted with the collection and determination of the tropical species. The project was supported by projects 206/08/H049 and P506/10/0704 from the Czech Science Foundation. JT was supported by project 138/2010/P. Additional support was provided by the Academy of Science of the Czech Republic within the Institutional Research Programme AV0Z60050516 and the Ministry of Education, Youth and Sports of the Czech Republic (projects MSM0021620828 and MSM6007665801).

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Authors and Affiliations

  1. Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, 128 01, Prague, Czech Republic

    Filip Kolář, Magdalena Lučanová & Jan Suda

  2. Institute of Botany, Academy of Sciences of the Czech Republic, Zámek 1, 252 43, Průhonice, Czech Republic

    Filip Kolář, Magdalena Lučanová & Jan Suda

  3. Department of Botany, Faculty of Science, University of South Bohemia, Branišovská 31, 370 05, České Budějovice, Czech Republic

    Jakub Těšitel

  4. Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, P.O. Box 3046, 3001-455, Coimbra, Portugal

    João Loureiro

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  1. Filip Kolář
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  2. Magdalena Lučanová
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  3. Jakub Těšitel
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  4. João Loureiro
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  5. Jan Suda
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Correspondence to Filip Kolář.

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Responsible Editor: Hans de Jong.

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Kolář, F., Lučanová, M., Těšitel, J. et al. Glycerol-treated nuclear suspensions—an efficient preservation method for flow cytometric analysis of plant samples. Chromosome Res 20, 303–315 (2012). https://doi.org/10.1007/s10577-012-9277-0

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  • DOI: https://doi.org/10.1007/s10577-012-9277-0

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