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
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.30
DOI:  10.1002/0471142956.cy0730s38
Online Posting Date:  November, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Two‐Step Procedure Without Centrifugation
  • Alternate Protocol 1: Two‐Step Procedure with Centrifugation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: 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 1: Two‐Step Procedure with Centrifugation

  • Tabletop centrifuge equipped with a rotor suitable for 3.5‐ml flow cytometer sample tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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.
   Doležel, J. and Bartoš, J. 2005. Plant DNA flow cytometry and estimation of nuclear genome size. Ann. Bot. 95:99‐110.
   Doležel, J. and Göhde, W. 1995. Sex determination in dioecious plants Melandrium album and M. rubrum using high‐resolution flow cytometry. Cytometry 19:103‐106.
   Doležel, 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 Stančík, 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 Kočí, 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 Trávníček, 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., Weiβ‐Schneeweiβ, H., Tribsch, A., Schneeweiβ, G., Trávníček, 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.).
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library