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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.

References

  1. Doležel, J., Greilhuber, J. & Suda, J. Flow cytometry with plants: an overview. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 41–65 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  2. Loureiro, J., Suda, J., Doležel, J. & Santos, C. FLOWER: a plant DNA flow cytometry database. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 423–438 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  3. Greilhuber, J., Doležel, J., Lysák, M.A. & Bennett, M.D. The origin, evolution and proposed stabilization of the terms 'genome size' and 'C-value' to describe nuclear DNA contents. Ann. Bot. (Lond.) 95, 255–260 (2005).

    Article  CAS  Google Scholar 

  4. Greilhuber, J. et al. Smallest angiosperm genomes found in Lentibulariaceae, with chromosomes of bacterial size. Plant Biol. (Stuttg). 8, 770–777 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Leitch, I.J., Soltis, D.E., Soltis, P.S. & Bennett, M.D. Evolution of DNA amounts across land plants (Embryophyta). Ann. Bot. (Lond.) 95, 207–217 (2005).

    Article  CAS  Google Scholar 

  6. Gregory, T.R. Coincidence, co-evolution or causation? DNA content, cell size and the C-value enigma. Biol. Rev. Camb. Philos. Soc. 76, 65–101 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Bennett, M.D. Nuclear DNA content and minimum generation time in herbaceous plants. Proc. R. Soc. Lond. B. Biol. Sci. 181, 109–135 (1972).

    Article  CAS  PubMed  Google Scholar 

  8. Leitch, I.J. & Bennett, M.D. Genome size and its uses: the impact of flow cytometry. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 153–176 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  9. Kron, P., Suda, J. & Husband, B.C. Applications of flow cytometry to evolutionary and population biology. Annu. Rev. Ecol. Evol. Syst. 38, 847–876 (2007).

    Article  Google Scholar 

  10. Rabinowicz, P.D. & Bennetzen, J.L. The maize genome as a model for efficient sequence analysis of large plant genomes. Curr. Opin. Plant Biol. 9, 149–156 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Suda, J., Kron, P., Husband, B.C. & Trávníč ek, P. Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 103–130 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  12. Matzk, F. Reproduction mode screening. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 131–152 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  13. Barow, M. & Meister, A. Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size. Plant Cell Environ. 26, 571–584 (2003).

    Article  Google Scholar 

  14. Barow, M. & Jovtchev, G. Endopolyploidy in plants and its analysis by flow cytometry. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 349–372 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  15. Barow, M. & Meister, A. Lack of correlation between AT frequency and genome size in higher plants and the effect of nonrandomness of base sequences on dye binding. Cytometry 47, 1–7 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Meister, A. & Barow, M. DNA base composition of plant genomes. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 177–215 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  17. Pfosser, M., Magyar, Z. & Bögre, L. Cell cycle analysis in plants. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 323–347 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  18. Sgorbati, S., Sparvoli, E., Levi, M., Chiatante, D. & Giordano, P. Bivariate cytofluorometric analysis of DNA and nuclear protein content. Protoplasma 144, 180–184 (1988).

    Article  CAS  Google Scholar 

  19. Zhang, C.Q., Gong, F.C., Lambert, G.M. & Galbraith, D.W. Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant Methods 1, 7 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Darzynkiewicz, Z., Bedner, E., Li, X., Gorzyca, W. & Melamed, M.R. Laser-scanning cytometry: a new instrumentation with many applications. Exp. Cell Res. 249, 1–12 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Ulrich, I. & Ulrich, W. High-resolution flow cytometry of nuclear DNA in higher plants. Protoplasma 165, 212–215 (1991).

    Article  Google Scholar 

  22. Galbraith, D.W. et al. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051 (1983).

    Article  CAS  PubMed  Google Scholar 

  23. Suda, J. & Trávníček, P. Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry—new prospects for plant research. Cytometry A 69, 273–280 (2006).

    Article  PubMed  Google Scholar 

  24. Śliwińska, E., Zielińska, E. & Jedrzejczik, I. Are seeds suitable for flow cytometric estimation of plant genome size? Cytometry A 64, 72–79 (2005).

    Article  PubMed  Google Scholar 

  25. Nsabimana, A. & van Staden, J. Ploidy investigation of bananas (Musa spp.) from the National Banana Germplasm Collection at Rubona-Rwanda by flow cytometry. S. Afr. J. Bot. 72, 302–305 (2006).

    Article  Google Scholar 

  26. Heller, F.O. DNS-Bestimmung an Keimwurzeln von Vicia faba L. mit Hilfe der Impulscytophotometrie. Ber. Deutsch. Bot. Ges. 86, 437–441 (1973).

    CAS  Google Scholar 

  27. Sgorbati, S., Levi, M., Sparvoli, E., Trezzi, F. & Lucchini, G. Cytometry and flow cytometry of 4′,6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants. Physiol. Plantarum 68, 471–476 (1986).

    Article  CAS  Google Scholar 

  28. Levi, M., Sparvoli, E., Sgorbati, S. & Chiatante, D. Rapid immunofluorescent identification of cells in the S phase in pea root meristems: an alternative to autoradiography. Physiol. Plantarum 71, 68–72 (1987).

    Article  CAS  Google Scholar 

  29. Jarret, R.L. et al. DNA contents in Paspalum spp. determined by flow-cytometry. Genet. Resour. Crop Evol. 42, 237–242 (1995).

    Article  Google Scholar 

  30. Esteban, J.M. et al. Effects of various fixatives and fixation conditions on DNA ploidy analysis—a need for strict internal DNA standards. Am. J. Clin. Pathol. 95, 460–466 (1991).

    Article  CAS  PubMed  Google Scholar 

  31. Shapiro, H. Practical Flow Cytometry 4th edn. (Wiley-Liss, New York, 2003).

    Book  Google Scholar 

  32. Doležel, J., Sgorbati, S. & Lucretti, S. Comparison of three DNA fluorochromes for flow-cytometric estimation of nuclear DNA content in plants. Physiol. Plantarum 85, 625–631 (1992).

    Article  Google Scholar 

  33. Doležel, J. et al. Plant genome size estimation by flow cytometry: inter-laboratory comparison. Ann. Bot. (Lond.) 82 (Suppl A), 17–26 (1998).

    Article  Google Scholar 

  34. Greilhuber, J., Temsch, E.M. & Loureiro, J.C.M. Nuclear DNA content measurement. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J., Suda, J.) 67–101 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  35. Roux, N., Toloza, A., Radecki, Z., Zapata-Arias, F.J. & Doležel, J. Rapid detection of aneuploidy in Musa using flow cytometry. Plant Cell Rep. 21, 483–490 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Suda, J., Krahulcová, A., Trávníček, P. & Krahulec, F. Ploidy level versus DNA ploidy level: an appeal for consistent terminology. Taxon 55, 447–450 (2006).

    Article  Google Scholar 

  37. Loureiro, J., Rodriguez, E., Doležel, J. & Santos, C. Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Ann. Bot. (Lond.) 98, 679–689 (2006).

    Article  CAS  Google Scholar 

  38. Loureiro, J., Rodriguez, E., Doležel, J. & Santos, C. Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. Ann. Bot. (Lond.) 98, 515–527 (2006).

    Article  CAS  Google Scholar 

  39. Suda, J. & Trávníček, P. Estimation of relative nuclear DNA content in dehydrated plant tissues by flow cytometry. in Current Protocols in Cytometry (eds. Robinson, J.P. et al.) 7.30.1–7.30.14 (John Wiley & Sons, New York, 2006).

    Google Scholar 

  40. Śliwińska, E. Nuclear DNA content analysis of plant seeds by flow cytometry. in Current Protocols in Cytometry (ed. Robinson, J.P. et al.) 7.29.1–7.29.13 (John Wiley & Sons, New York, 2006).

    Google Scholar 

  41. Galbraith, D.W. Protoplast analysis using flow cytometry and sorting. in Flow Cytometry with Plant Cells (eds. Doležel, J., Greilhuber, J. & Suda, J.) 231–250 (Wiley-VCH, Weinheim, Germany, 2007).

    Chapter  Google Scholar 

  42. Doležel, J. & Göhde, W. Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry. Cytometry 19, 103–106 (1995).

    Article  PubMed  Google Scholar 

  43. Doležel, J. & Bartoš, J. Plant DNA flow cytometry and estimation of nuclear genome size. Ann. Bot. (Lond.) 95, 99–110 (2005).

    Article  Google Scholar 

  44. Johnston, J.S., Bennett, M.D., Rayburn, A.L., Galbraith, D.W. & Price, H.J. Reference standards for determination of DNA content of plant nuclei. Am. J. Bot. 86, 609 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Doležel, J., Bartoš, J., Voglmayr, H. & Greilhuber, J. Nuclear DNA content and genome size of trout and human. Cytometry A 51, 127–128 (2003).

    Article  PubMed  Google Scholar 

  46. Doležel, J., Doleželová, M. & Novák, F.J. Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biol. Plantarum 36, 351–357 (1994).

    Article  Google Scholar 

  47. Lysák, M.A. & Doležel, J. Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 52, 123–132 (1998).

    Article  Google Scholar 

  48. Price, H.J., Hodnett, G. & Johnston, J.S. Sunflower (Helianthus annuus) leaves contain compounds that reduce nuclear propidium iodide fluorescence. Ann. Bot. (Lond.) 86, 929–934 (2000).

    Article  CAS  Google Scholar 

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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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  1. Jaroslav Doležel
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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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