Nuclear DNA Content Analysis of Plant Seeds by Flow Cytometry

Elwira Sliwinska1

1 University of Technology and Agriculture, Bydgoszcz
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.29
DOI:  10.1002/0471142956.cy0729s35
Online Posting Date:  February, 2006
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Abstract

Procedures describing the utilization of seeds or their parts for flow cytometric determination of plant ploidy and endopolyploidy, genome size, and cell cycle activity are presented. The methods have been developed for a single‐fluorescence‐parameter flow cytometer, equipped with light sources for 488‐nm and UV‐light illumination. The procedures presented in this unit utilize the two most widely used fluorochromes for plant DNA content analysis, propidium iodide (PI) and 4′,6‐diamidino‐2‐phenylindole (DAPI). These methods provide an alternative to estimation of DNA content based on the fluorescence of DNA in cell nuclei isolated from plant leaves. In some instances seeds are more suitable for analysis than leaves, e.g., when plant material must be transported for a long distances or stored for prolonged periods before flow cytometric analysis, or when leaves contain fluorochrome‐staining inhibitors. In addition, flow cytometric determination of nuclear replication stages in seeds gives information about their physiological status (e.g., maturity, advancement of germination), which is valuable to seed producers and technologists.

Keywords: flow cytometry; ploidy; genome size; endoreplication; cell cycle; seed; embryo; endosperm

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Ploidy, Cell Cycle Activity, and Endoreplication in Seeds
  • Basic Protocol 2: Estimation of Absolute Nuclear DNA Content
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of Ploidy, Cell Cycle Activity, and Endoreplication in Seeds

  Materials
  • Seeds for analysis
  • Nuclei isolation/staining buffer with 2 µg/ml DAPI (ice cold; see recipe)
  • External standard (seeds or leaves of a diploid plant of the studied species; not required for cell‐cycle and endoreplication analyses)
  • Sheath fluid: distilled deionized water with one drop of Tween 20 per 10 liters of water
  • Preparation needle (optional)
  • Magnifying glass with light (optional)
  • 5.5‐cm plastic petri dishes
  • 100‐ to 1000‐µl variable‐volume automatic pipet with appropriate tips or glass Pasteur pipets
  • Razor blades
  • 2‐ to 3.5‐ml plastic cytometer sample tubes and appropriate holders
  • 30‐ or 50‐µm nylon mesh or CellTrics (green or yellow; Partec GmbH, http://www.partec.com)
  • Flow cytometer with UV excitation source (e.g., mercury arc lamp or laser tuned to 340 to 380 nm)
  • Calibration particles (see unit 1.3)
  • Software for histogram evaluation (e.g., Partec DPAC, http://www.partec.com; not required for ploidy estimations)

Basic Protocol 2: Estimation of Absolute Nuclear DNA Content

  Materials
  • Seeds for analysis
  • Internal standard tissue (e.g., seeds or leaves of a plant species or blood cells, with known nuclear DNA content; see unit 1.3& UNIT )
  • Nuclei isolation/staining buffer with 50 µg/ml PI and 50 µg/ml RNase (ice cold; see recipe)
  • Sheath fluid: distilled, deionized water with one drop of Tween 20 per 10 liters of water
  • Razor blades
  • Preparation needle (optional)
  • Magnifying glass with light (optional)
  • 5.5‐cm plastic petri dishes
  • 100‐ to 1000‐µl variable‐volume automatic pipet with appropriate tips or glass Pasteur pipets
  • 2‐ to 3.5‐ml plastic cytometer sample tubes and appropriate holders
  • 30‐ or 50‐µm nylon mesh or CellTrics (green or yellow; Partec GmbH, http://www.partec.com)
  • Flow cytometer with 488‐nm excitation and filter set for detection of PI emission
  • Calibration particles (see unit 1.3)
  • Software for histogram evaluation (e.g., Partec DPAC, http://www.partec.com)
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Figures

Videos

Literature Cited

   Bewley, J.D. and Black, M. 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York and London.
   Bino, R.J., Lanteri, S., Verhoeven, H.A., and Kraak, H.L. 1993. Flow cytometric determination of nuclear replication stages in seed tissues. Ann. Bot. 72:181‐187.
   Doležel, J. 1998. Flow cytometry, its application and potential for plant breeding. In Current Topics in Plant Cytogenetics Related to Plant Improvement (T. Lelley, ed.) pp. 80‐90. Universitätsverlag, Vienna.
   Finch‐Savage, W.E., Bergervoet, J.H.W., Bino, R.J., Clay, H.A., and Groot, S.P.C. 1998. Nuclear replication activity during seed development, dormancy breakage and germination in three tree species: Norway maple (Acer platanoides L.), sycamore (Acer pseudoplatanus L. and cherry (Prunus avium L.). Ann. Bot. 81:519‐526.
   Górnik, K., de Castro, R., Liu, Y., Bino, R.J., and Groot, S.P.C. 1997. Inhibition of cell division during cabbage (Brassica oleracea L.) seed germination. Seed Sci. Res. 7:333‐340.
   Greilhuber, J. 1988. “Self‐tanning” ‐ a new and important source of stoichiometric error in cytophotometric determination of nuclear DNA content in plants. Plant Syst. Evol. 158:87‐96.
   Greilhuber, J., Dolezel, J., Lysak, M.A., andBennett, M.D. 2005. The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C‐value’ to describe nuclear DNA contents. Ann. Bot. 95:155‐260.
   Jing, H.C., van Lammeren, A.A.M., de Castro, R., Bino, R.J., Hilhorst, H.W.M., and Groot, S.P.C. 1999. β‐Tubulin accumulation and DNA synthesis are sequentially resumed in embryo organs of cucumber (Cucumis sativus L.) seeds during germination. Protoplasma 208:230‐239.
   Kowles, R.V., Yerk, G.L., Srienc, F., and Phillips, R.L. 1992. Maize endosperm tissue as an endoreduplication system. Genet. Engineering 14:65‐88.
   Kowles, R.V., Yerk, G.L., Haas, K.M., and Phillips, R.L. 1997. Maternal effects influencing DNA endoreduplication in developing endosperm of Zea mays. Genome 40:798‐805.
   Lanteri, S., Saracco, F., Kraak, H.L., and Bino, R.J. 1994. The effects of priming on nuclear replication activity and germination of pepper (Capsicum annuum) and tomato (Lycopersicon esculentum) seeds. Seed Sci. Res. 4:81‐87.
   Liu, Y., Hilhorst, H.W.M., Groot, S.P.C., and Bino, R.J. 1997. Amounts of nuclear DNA and internal morphology of gibberelin‐ and abscisic acid‐deficient tomato (Lycopersicon esculentum Mill.) seeds during maturation, imbibition and germination. Ann. Bot. 79:161‐168.
   Matzk, F., Meister, A., and Schubert, I. 2000. An efficient screen for reproductive pathways using mature seeds of monocots and dicots. Plant J. 21:97‐108.
   Price, H.J., Hodnett, G., and Johnston, J.S. 2000. Sunflower (Helianthus annuus) leaves contain compounds that reduce nuclear propidium iodide fluorescence. Ann. Bot. 86:929‐934.
   Sliwinska, E. 1997. Flow cytometric analysis of sugar beet seeds different in vigour. In Basic and Applied Aspects of Seed Biology (R.H. Ellis, M. Black, and T.D Hong, eds.) pp. 577‐584. Kluwer Academic Publishers, Dordrecht, The Netherlands.
   Sliwinska, E. 2000. Analysis of the cell cycle in sugarbeet seed during development, maturation and germination. In Seed Biology: Advances and Applications (M. Black, K.J. Bradford, and J. Vazquez‐Ramos, eds.) pp. 133‐139. CAB International, Wallingford, UK.
   Sliwinska, E. 2003. Cell cycle and germination of fresh, dried and deteriorated sugar‐beet seeds as indicators of optimal harvest time. Seed Sci. Res. 13:131‐138.
   Sliwinska, E. and Jendrzejczak, E. 2002. Sugar‐beet seed quality and DNA synthesis in the embryo in relation to hydration‐dehydration cycles. Seed Sci. Technol. 30:597‐608.
   Sliwinska, E., Zielinska, E., and Jedrzejczyk, I. 2005. Are seeds suitable for flow cytometric estimation of plant genome size? Cytometry 64A:72‐79.
Internet Resources
   http://www.rbgkew.org.uk/cval/database1.html
  Plant DNA C‐values database by M.D. Bennett and I.J. Leitch
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