Estimation of Relative Nuclear DNA Content in Dehydrated Plant Tissues by Flow Cytometry

Jan Suda1, Pavel Trávníček1

1 Charles University in Prague and Academy of Sciences of the Czech Republic, Prague, null
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.30
DOI:  10.1002/0471142956.cy0730s38
Online Posting Date:  November, 2006
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Abstract

The power of flow cytometry in field plant research may be greatly enhanced by analysis of nonfresh tissues. Previous attempts to use chemical fixatives, however, received only little attention because of protocol complexity and limited time, after which successful assays were achieved. This unit describes simple and rapid procedures for relative nuclear DNA content estimation in dehydrated tissues of vascular plants by DAPI flow cytometry. Histograms with reasonable resolution can be obtained in several-year-old specimens. The approach here is particularly suitable for reliable determination of DNA ploidy level, although detection of small variation in nuclear DNA content is also possible in many cases. Retrospective ploidy inference in silica-dry or herbarium vouchers and simplified transport of plant material from remote areas are among the principal benefits for plant biosystematics, ecology, and population biology.

Keywords: DAPI; DNA ploidy level; desiccation; flow cytometry; genome size; herbarium vouchers; silica-dry material; tissue preservation; vascular plants

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Two-Step Procedure Without Centrifugation
  • Alternate Protocol: Two-Step Procedure with Centrifugation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Two-Step Procedure Without Centrifugation

 Materials
  • Cleaning solution for flow systems (Partec; see Partec GmbH, http://www.partec.de)
  • Calibration particles (e.g., DNA Control UV, Partec; see unit 1.3) or appropriate living plant standard(s)
  • Herbarium vouchers, silica-dry material, or other rapidly desiccated plant tissue for analysis
  • Otto I isolation buffer (see recipe), ice cold
  • Otto II staining buffer (see recipe)
  • Reference (internal) standard: living plant(s) with appropriate genome size
  • Flow cytometer with UV excitation (e.g., mercury arc lamp or laser tuned to 340 to 380 nm), and appropriate software for histogram evaluation (e.g., Partec FloMax)
  • 5.5-cm plastic petri dishes
  • Razor blades (double-edged)
  • 30- to 50-µm nylon mesh or CellTrics filters (green or yellow; Partec GmbH)
  • 3.5-ml flow cytometer sample tubes and appropriate tube holder

Alternate Protocol: Two-Step Procedure with Centrifugation

 Additional Materials (also see Basic Protocol)
  • Tabletop centrifuge equipped with a rotor suitable for 3.5-ml flow cytometer sample tubes
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Figures

  •  FigureFigure 7.30.1 Flow cytometric histogram documenting detection of small variation in nuclear DNA content between two tetraploid individuals of Senecio carniolicus. Nuclei were isolated from silica-dry samples stored 7 months at room temperature and treated simultaneously according to the Basic Protocol. The difference in DNA content amounts to 5.7%.
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  •  FigureFigure 7.30.2 Flow cytometric analysis of DAPI-stained nuclei isolated from 3-year-old herbarium vouchers of hexaploid Vaccinium oxycoccus (prepared according to the Alternate Protocol). (A) Material stored at room temperature, CV = 6.54%. (B) Material stored in deep freezer, CV = 2.96%. Note marked difference in peak quality.
    View Image
  •  FigureFigure 7.30.3 Flow cytometric analysis of DAPI-stained nuclei prepared according to the Basic Protocol. Panels A and B represent analyses with fresh tissue and panels C and D represent analyses with dry tissue (herbarium vouchers) of the investigated plant species. Fresh Lycopersicon esculentum cv. Stupické polní tykové rané (2C = 1.96 pg) was used as internal standard in all runs. (A) Fresh Pachysandra terminalis, peak ratio 1.691. (B) Fresh Stachys germanica, peak ratio 1.221. (C) Dry Pachysandra stored 20 months at room temperature, peak ratio 1.535 (fluorescence loss 10.2%). (D) Dry Stachys stored 20 months at room temperature, peak ratio 1.244 (fluorescence loss 1.9%). Peak designations: L = Lycopersicon esculentum, P = Pachysandra terminalis, S = Stachys germanica.
    View Image
  •  FigureFigure 7.30.4 Flow cytometric analysis of DAPI-stained nuclei isolated from herbarium vouchers of diploid (2x), tetraploid (4x), pentaploid (5x), and hexaploid (6x) individuals of Vaccinium subg. Oxycoccus stored 5 months at room temperature. Sample was prepared according to the Alternate Protocol. Peak ratios are 1:2.023:2.513:3.005, which corresponds perfectly to ratios obtained in fresh tissue runs.
    View Image
  •  FigureFigure 7.30.5 Simultaneous flow cytometric analysis of DAPI-stained nuclei isolated from fresh Zea mays CE-777 and herbarium voucher of Coreopsis verticillata stored 20 months at room temperature (prepared according to the Alternate Protocol). The storage period was beyond the dry sample lifetime, resulting in histogram with unacceptably low resolution. Peak designations: Z = Zea mays, C = Coreopsis verticillata.
    View Image

Videos

Literature Cited

Literature Cited
    Alsos, I.G., Suda, J., Engelskjøn, T., Gielly, L., Taberlet, P., and Brochmann, C. Polyploid evolution and morphology in cpDNA lineages of Vaccinium uliginosum coll. (Ericaceae). Manuscript in preparation.
    Dart, S., Kron, P., and Mable, B.K. 2004. Characterizing polyploidy in Arabidopsis lyrata using chromosome counts and flow cytometry. Can. J. Bot. 82:185-197.
    Doleel, J. and Bartoš, J. 2005. Plant DNA flow cytometry and estimation of nuclear genome size. Ann. Bot. 95:99-110.
    Doleel, J. and Göhde, W. 1995. Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry. Cytometry 19:103-106.
    Doleel, J., Greilhuber, J., Lucretti, S., Meister, A., Lysák, M.A., Nardi, L., and Obermayer, R. 1998. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Ann. Bot. 82:17-26.
    Hopping, M.E. 1993. Preparation and preservation of nuclei from plant-tissues for quantitative DNA analysis by flow cytometry. N. Z. J. Bot. 31:391-401.
    Hülgenhof, E., Weidhase, R.A., Schlegel, R., and Tewes, A. 1988. Flow cytometric determination of DNA content in isolated nuclei of cereals. Genome 30:565-569.
    Jarret, R.L., Oziasakins, P., Phatak, S., Nadimpalli, R., Duncan, R., and Hiliard, S. 1995. DNA contents in Paspalum spp. determined by flow-cytometry. Genet. Resour. Crop Evol. 42:237-242.
    Johnston, J.S., Bennett, M.D., Rayburn, A.L., Galbraith, D.W., and Price, H.J. 1999. Reference standards for determination of DNA content of plant nuclei. Am. J. Bot. 86:609-613.
    Otto, F. 1990. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In Methods in Cell Biology, Vol. 33 (H.A. Crissman and Z. Darzynkiewicz, eds.) pp. 105-110. Academic Press, New York.
    Popp, M., Gizaw, A., Nemomissa, S., Suda, J., and Brochmann, C. Colonization and diversification in the afro-alpine “sky islands” by Eurasian Lychnis L. (Caryophyllaceae). Submitted for publication.
    Schönswetter, P., Suda, J., Popp, M., Weiss-Schneeweiss, H., and Brochmann, C. 2006. Circumpolar phylogeography of Juncus biglumis (Juncaceae) inferred from AFLP fingerprints, cpDNA sequences, nuclear DNA content and chromosome numbers. Mol. Phylogenet. Evol. (In press).
    Sgorbati, S., Levi, M., Sparvoli, E., Trezzi, F., and Lucchini, G. 1986. Cytometry and flow-cytometry of 4¢,6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants. Physiol. Plant. 68:471-476.
    Sliwinska, E., Zielinska, E., and Jedrzejczyk, I. 2005. Are seeds suitable for flow cytometric estimation of plant genome size? Cytometry 64:72-79.
    Šmarda, P. and Staník, D. 2006. Ploidy level variability in South American fescues (Festuca L., Poaceae): Use of flow cytometry in up to 5½-year-old caryopses and herbarium specimens. Plant Biol. 8:73-80.
    Šmarda, P., Müller, J., Vrána, J., and Koí, K. 2005. Ploidy level variability of some Central European fescues (Festuca subg. Festuca, Poaceae). Biologia (Bratisl) 60:25-36.
    Suda, J. 2004. An employment of flow cytometry into plant biosystematics. PhD thesis, Department of Botany, Faculty of Science, Charles University, Prague. http://www.ibot.cas.cz/fcm/suda/presentation/disertation.pdf.
    Suda, J. and Trávníek, P. 2006. Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry: New prospects for plant research. Cytometry 69:273-280.
    Suda, J., Wei-Schneewei, H., Tribsch, A., Schneewei, G., Trávníek, P., and Schönswetter, P. Complex distribution patterns of di-, tetra- and hexaploid cytotypes in the mountain plant Senecio carniolicus (Asteraceae, Asteroideae) from the Eastern European Alps. Manuscript in preparation.
    Ulrich, I. and Ulrich, W. 1991. High-resolution flow cytometry of nuclear DNA in higher plants. Protoplasma 165:212-215.
    Voglmayr, H. 2000. Nuclear DNA amounts in mosses (Musci). Ann. Bot. 85:531-546.
 Internet Resources
    http://www.ibot.cas.cz/fcm

Home page of the Laboratory of Flow Cytometry, Institute of Botany, Academy of Sciences of the Czech Republic, focused on the applications of FCM in plant biosystematics, ecology, and population biology.

    http://www.ueb.cas.cz/Olomouc1

Home page of the Olomouc Research Centre (with Laboratory of Flow Cytometry), Institute of Experimental Botany, Academy of Sciences of the Czech Republic, containing useful information on FCM methodology (protocols, standards, etc.).

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