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Analysis of Nuclear DNA Content and Ploidy in Higher Plants

David W. Galbraith1,  Georgina M. Lambert1,  Jiri Macas1,  Jaroslav Dolezel2

1University of Arizona, Tucson, Arizona
2De Montfort University Norman Borlaug Centre for Plant Science, Olomouc, Czech Republic


Publication Name: 
Current Protocols in Cytometry
Unit Number: 
UNIT 7.6
DOI: 
10.1002/0471142956.cy0706s02
Online Posting Date: 
May, 2001
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Abstract

This is the first of a series of units discussing the application of cytometry to plant material. Techniques commonly used for mammalian nuclei evaluation need considerable modification to be successful with plant material. David Galbraith and his colleagues bring together many years of knowledge in plant cytometry. Their unit provides detailed protocols on measuring DNA content, ploidy, and cell cycle status of plant tissue using both conventional laser based instruments as well as arc lamp cytometers. This unit provides an excellent starting point for those interested in doing cytometry with plants.

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Analysis of Somatic DNA Content, Ploidy, and Cell-Cycle Status of Plant Tissues Using a Laser-Based Flow Cytometer
  • Analysis of Somatic DNA Content in Plant Tissues Using an Arc Lamp–Based Flow Cytometer
  • Alternate Protocol 1: Nuclear DNA Content Analysis Using LB01 Buffer or Partec Buffer and Arc Lamp–Based Flow Cytometer
  • Alternate Protocol 2: Two-Step Nuclear DNA Content Analysis Using Arc Lamp-Based Flow Cytometer
  • Alternate Protocol 3: DNA Content Analysis of Fixed Protoplasts
  • Alternate Protocol 4: DNA Content analysis of Formaldehyde-Fixed Tissues and Cells
  • Alternate Protocol 5: DNA Content Analysis of Fixed, Intact Cells
  • Alternate Protocol 6: Bulk Ploidy Screening
  • Support Protocol 1: Preparation of Unfixed Chicken Red Blood Cells for Use as Internal Standards in Genome-Size Measurements
  • Support Protocol 2: Preparation of Fixed Chicken Red Blood Cell Nuclei for Instrument Alignment
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
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Materials

Basic Protocol: Analysis of Somatic DNA Content, Ploidy, and Cell-Cycle Status of Plant Tissues Using a Laser-Based Flow Cytometer

 Materials
  • Plant material for analysis: intact plant tissues, plant tissue culture or callus, or plant protoplasts
  • Internal standards: e.g., chicken red blood cells (unfixed CRBCs; see Support Protocol 1) or plants with known nuclear DNA content (see Critical Parameters)
  • Homogenization buffer (see recipe)
  • 1 mg/ml propidium iodide (PI) stock solution (see recipe)
  • 1 mg/ml RNase stock solution (see recipe)
  • 0.1 mg/ml mithramycin (MI) stock solution (see recipe)
  • Appropriate sheath fluid
  • Fluorescent microspheres (DNACheck, Coulter)
  • 5.5-cm plastic petri dishes
  • New single-edged razor blades
  • 15-µm pore-size nylon mesh
  • Flow cytometer with 488 nm (PI) or 457 nm (MI) light source
  • 0.22-µm Millipore GSWP 047 filters
  • Cell-cycle analysis software (optional; Phoenix Flow Systems, Verity Software, or Eric Martz at http://www.bio.umass.edu/mcbfacs/flowcat.html)

NOTE: All steps must be carried out on ice; it is also recommended that procedures be performed in a walk-in cold room.

Alternate Protocol 1: Nuclear DNA Content Analysis Using LB01 Buffer or Partec Buffer and Arc Lamp–Based Flow Cytometer

 Additional Materials (also see Basic Protocol)
  • Lysis buffer LB01 or Partec buffer (see recipes) with fluorochrome, ice-cold
  • 1 mg/ml propidium iodide (PI) stock solution (see recipe) or 0.1 mg/ml 4¢,6-diamidino-2-phenylindole (DAPI) stock solution (see recipe) and 1 mg/ml ribonuclease (RNase) stock solution (see recipe)
  • Particles for instrument alignment: e.g., microspheres or fixed chicken red blood cells (CRBCs; see Support Protocol 2) stained with the fluorochrome used (also see unit 1.3)
  • Glass petri dish
  • 42-µm pore-size nylon mesh
  • Flow cytometer equipped with high-pressure mercury-arc lamp
  • Filters and dichroic mirror appropriate for fluorochrome used

Alternate Protocol 2: Two-Step Nuclear DNA Content Analysis Using Arc Lamp-Based Flow Cytometer

 Additional Materials (also see Basic Protocol)
  • Otto I buffer (see recipe) and Otto II buffer (with fluorochrome; see recipe)
  • 1 mg/ml propidium iodide (PI) stock solution (see recipe) or 0.1 mg/ml 4¢,6-diamidino-2-phenylindole (DAPI) stock solution (see recipe) and 1 mg/ml ribonuclease (RNase) stock solution (see recipe)
  • Glass petri dish
  • 42-µm pore-size nylon mesh
  • Tabletop centrifuge
  • Flow cytometer equipped with high-pressure mercury-arc lamp
  • Additional reagents and equipment for analysis with an arc lamp–based flow cytometer (see Alternate Protocol 1, steps to )

Alternate Protocol 3: DNA Content Analysis of Fixed Protoplasts

 Additional Materials (also see Basic Protocol)
  • AES fixative (see recipe), ice-cold
  • 70% ethanol
  • Homogenization buffer (see recipe)
  • Tabletop centrifuge
  • 60-µm pore-size nylon mesh

Alternate Protocol 4: DNA Content analysis of Formaldehyde-Fixed Tissues and Cells

 Additional Materials (also see Basic Protocol and Alternate Protocol 1)
  • Formaldehyde fixative (see recipe), 4°C
  • Tris buffer (see recipe), 4°C
  • Lysis buffer LB01 (see recipe) without fluorochrome, 4°C
  • 0.1 mg/ml DAPI stock solution (see recipe).
  • Tabletop centrifuge
  • Glass petri dish
  • 42-µm pore size nylon mesh

Alternate Protocol 5: DNA Content Analysis of Fixed, Intact Cells

 Additional Materials (also see Basic Protocol or Alternate Protocol 1)
  • Ethanol/acetic acid fixative (see recipe), ice-cold
  • 70% ethanol, ice-cold
  • Citrate buffer (see recipe), 4°C
  • Enzyme solution (see recipe)
  • 0.1 mg/ml DAPI stock solution (see recipe).
  • Tabletop centrifuge

Support Protocol 1: Preparation of Unfixed Chicken Red Blood Cells for Use as Internal Standards in Genome-Size Measurements

 Materials
  • Live chicken
  • Acid citrate dextrose (ACD) buffer (see recipe)
  • Dimethylsulfoxide (DMSO)
  • 21-G needle
  • Heparinized blood collection tube
  • Tabletop centrifuge
  • Polypropylene cryotubes (e.g., Nunc)
  • Liquid nitrogen

Support Protocol 2: Preparation of Fixed Chicken Red Blood Cell Nuclei for Instrument Alignment

 Materials
  • Fresh chicken blood (collected in heparinized tube to prevent coagulation)
  • CRBC buffers I, II, and III (see recipes)
  • Ethanol/acetic acid fixative (see recipe), ice-cold
  • 70% ethanol, ice-cold
  • Tabletop centrifuge
  • 15-ml polypropylene centrifuge tubes
  • 30-G needle and syringe
  • 42-µm pore-size nylon mesh
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 7.6.1
    (A) Flow cytometric analysis of tobacco leaf homogenates stained with propidium iodide. (B) Same analysis with inclusion of chicken red blood cells (CRBC) as an internal standard for genome size measurement. In leaf tissue the G1 nuclei, which have a 2C DNA content, greatly outnumber those in S phase and in G2, which have a 4C DNA content.

    View Image
  • Figure 7.6.2
    Analysis of the genome size of Alstroemeria caryophyllacea using N. tabacum as the internal standard and PI as the fluorochrome. (A) Employing linear amplification, only the G1 (2C) peak for A. caryophyllacea is on scale. (B) Employing logarithmic amplification, G1 and G2 nuclei for both species are observed (from Bharathan et al., 1994).

    View Image
  • Figure 7.6.3
    Flow cytometric analysis of nuclear DNA contents in young Arabidopsis thaliana leaf tissues. (A) Use of mithramycin as the fluorochrome and CRBCs as the internal standard to provide a measure of the genome size (insert represents the same sample under logarithmic amplification); from Galbraith et al., 1991. (B) Use of fluorochrome DAPI, Partec buffer, and the Partec PAS II flow cytometer. This gives particularly well separated and discrete peaks of fluorescence; the extensive somatic endoreduplication is readily observed and accurately measured.

    View Image
  • Figure 7.6.4
    Flow cytometric analysis of nuclear DNA content distribution in maize endosperm (28 days after pollination) using PI as the fluorochrome (from Galbraith and Lambert, 1995).

    View Image

Literature Cited

Literature Cited
    Arumuganathan, K. and Earle, E.D. 1991. Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 9:208-218.
    Baranyi, M. and Greilhuber, J. 1996. Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum. Theor. Appl. Genet. 92:297-307.
    Bennett, M.D. and Leitch, I.J. 1995. Nuclear DNA amounts in angiosperms. Ann. Bot. 76:113-176.
    Bharathan, G., Lambert, G.M., and Galbraith, D.W. 1994. Nuclear DNA contents of monocotyledons and related taxa. Am. J. Bot. 81:381-386.
    Burton, K. 1968. Determination of DNA concentration with diphenylamine. Methods Enzymol. 12:163-168.
    de Laat, A.M.M., Göhde, W., and Vogelzang, J.D.C. 1987. Determination of ploidy of single plants and plant populations by flow cytometry. Plant Breed. 99:303-307.
    DeRocher, E.J., Harkins, K.R., Galbraith, D.W., and Bohnert, H.J. 1990. Developmentally-regulated systemic endopolyploidy in succulents with small genomes. Science 250:99-101.
    Dolezel, J. and Göhde, W. 1995. Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry. Cytometry 19:103-106.
    Dolezel, J., Binarova, P., and Lucretti, S. 1989. Analysis of nuclear DNA content in plant cells by flow cytometry. Biol. Plant. 31:113-120.
    Dolezel, J., Sgorbati, S., and Lucretti, S. 1992. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol. Plant. 85:625-631.
    Dolezel, J., Dolezelova, M., and Novak, F.J. 1994. Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biol. Plant. 36:351-357.
    Galbraith, D.W. 1990. Flow cytometric analysis of plant genomes. Methods Cell Biol. 33:549-562.
    Galbraith, D. W. and Lambert, G. M. 1995. Advances in the flow cytometric characterization of plant cells and tissues. In Flow Cytometric Applications in Cell Culture (M. Al-Rubeai and A. N. Emery, eds.) pp. 311-326. Marcel Dekker, New York
    Galbraith, D.W. and Shields, B.A. 1982. The effects of inhibitors of cell wall synthesis on tobacco protoplast development. Physiol. Plant. 55:25-30.
    Galbraith, D. W., Harkins, K. R., Maddox, J. R., Ayres, N. M., Sharma, D. P., and Firoozabady, E. 1983. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049-1051.
    Galbraith, D.W., Harkins, K.R., and Knapp, S. 1991. Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiol. 96:985-989.
    Godelle, B., Cartier, D., Marie, D., Brown, S.C., and Siljak-Yakovlev, S. 1993. A heterochromatin study demonstrating the non-linearity of fluorometry useful for calculating genomic base composition. Cytometry 14:618-626.
    Greilhuber, J. and Obermayer, R. 1997. Genome size and maturity group in Glycine max. Heredity 78:547-551.
    Harkins, K.R., Jefferson, R.A., Kavanaugh, T.A., Bevan, M.W., and Galbraith, D.W. 1990. Expression of photosynthetic-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. Proc. Natl. Acad. Sci. U.S.A. 87:816-820.
    Kowles, R.V., Srienc, F., and Phillips, R.L. 1990. Endoreduplication of nuclear DNA in the development of maize endosperm. Dev. Genet. 11:125-132.
    Marie, D. and Brown, S.C. 1993. A cytometric exercise in plant DNA histograms, with 2C values for 70 species. Biol. Cell 78:41-51.
    Nakamura, D., Tiersch, T.R., Douglass, M., and Chandler, R.W. 1990. Rapid identification of sex in birds by flow cytometry. Cytogenet. Cell Genet. 53:201-205.
    O'Brien, I. E. W., Baguley, B. C., Morris, B. A. M., Murray, B. G., and Ferguson, I. B. 1996. Chromatin condensation followed by nDNA fragmentation indicative of Programmed Cell Death occurs in plants. Cytometry I.S.A.C. Congress XVIII Abstract BD21.
    Otto, F. 1990. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In Methods in Cell Biology, Vol. 33. (Z. Darzynkiewicz and H.A. Crissman eds.) pp. 105-110, Academic Press, New York
    Overton, W.R. and McCoy, J.P. 1994. Reversing the effect of formalin on the binding of propidium iodide to DNA. Cytometry 16:351-356.
    Pfosser, M. 1989. Improved method for critical comparison of cell cycle data of asynchronously dividing and synchronized cell cultures of Nicotiana tabacum. J. Plant Physiol. 134:741-745.
    Pfosser, M., Amon, A., Lelley, T., and Heberle-Bors, E. 1995. Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry 21:387-393.
    Rayburn, A.L., Auger, J.A., and McMurphy, L.M. 1992. Estimating percentage constitutive heterochromatin by flow cytometry. Exp. Cell. Res. 198:175-178.
    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.
 Key Reference
    Galbraith, D. W., Harkins, K. R., Maddox, J. R., Ayres, N. M., Sharma, D. P., and Firoozabady, E. 1983. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049-1051.

Outlines the basic procedures for rapid analysis of plant nuclei using flow cytometry which, with various modifications, are now employed worldwide for a variety of different applications.

 Internet Resources
    Bennett, M.D., Cox, A.V., and Leitch, I.J. 1997. Angiosperm DNA C-values database. http://www.rbgkew.org.uk/cval/database1.html.
     
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