Abstract
A one-step procedure is presented for simultaneous measurement of cell number and DNA content in cultured plant cells by flow cytometry. In order to obtain nuclei representative of the growth stadium of the culture and of all phases of the cell cycle, cells were carefully sampled and immediately fixed. Next, nuclei were isolated by enzymatic and mechanical maceration, and stained with a DNA-specific fluorescent dye. In the resultant preparation, cells can be counted at relative ease by means of a fluorescence microscope. However, flow-cytometric counting appeared to be superior to manual counting since the time needed for flow-cytometric counting was one-fourth that for manual counting and the variance between counts of the samples was significantly less. In addition, from the same routine, accurate DNA distributions were obtained as a second important parameter of the population dynamics.
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References
Amino S, Fujimura T & Komamine A (1983) Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus. Physiol. Plant. 59: 393–396
Bennett MD & Smith JB (1991) Nuclear DNA amounts in angiosperms. Phil. Trans. Roy. Soc. Lond. B 334: 309–345
Bergounioux C, Moyne AL, Thor V, Perennes C, Sarda C, Brown SC & Gadal P (1986) Cell cycle analysis and protoplast division. Biol. Cell 58 (suppl.), 2a
Berlin J (1988) Biotechnology in agriculture and forestry. In: Bajai YPS (Ed) Medicinal and Aromatic Plants, I, Vol 4 (pp 37–59). Springer-Verlag, Berlin, Heidelberg
Blom TJM, Sierra M, van Vliet TB, Franke-van Dijk MEI, de Koning P, van Iren F, Verpoorte R & Libbenga KR (1991) Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don. and its conversion into serpentine. Planta 183: 170–177
Boot KJM, van der Zaal BJ, Velterop J, Quint A, Mennes AM, Hooykaas PJJ & Libbenga KR (1993) Further characterization of expression of auxin-induced genes in tobacco (Nicotiana tabacum) cell-suspension cultures. Plant Physiol. 102: 513–520
Brown SC & Bergounioux C (1989) Plant flow cytometry. In: Yen A (Ed) Flow Cytometry: Advanced Research and Clinical Applications, Vol 2 (pp 195–220). CRC Press, Boca Raton, FL
Butcher DN & Street HE (1960) The effect of gibberellins on the growth of excised tomato roots. J. Exp. Bot. 11: 206–216
Collin HA (1987) Determinants of yield of secondary products in plant tissue cultures. Adv. Bot. Res. 13: 145–187
Conia J, Alexander RG, Wier ME, Richards KR, Rice ME & Jackson PJ (1990) Reversible accumulation of plant suspension cell cultures in G1 phase and subsequent synchronous traverse of the cell cycle. Plant Physiol. 94: 1568–1574
De Gunst (1989) A random model for plant cell population growth. CWI Tract 58, 1–152. Centre for Mathematics and Computer Science, Amsterdam.
De Gunst (1990) Modeling the growth of a batch culture of plant cells: a corpuscular approach. Enzyme Microb. Technol. 12: 61–71
De Laat AMM, Göhde W & Vogelzang MJDC (1987) Determination of ploidy of single plants and plant populations by flow cytometry. Plant Breed. 99: 303–307
Dickson EE, Arumuganathan K, Kresovich S & Doyle JJ (1992) Nuclear content variation within the Rosaceae. Am. J. Bot. 79: 1081–1086
Galbraith DW (1984) In: Vasil IK (Ed) Cell Culture and Somatic Cell Genetics of Plants (pp 765–777). Academic Press, New York
Galbraith DW (1989) Analysis of higher plants by flow cytometry and cell sorting. Int. Rev. Cytol. 116: 165–228
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP & Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220: 1094–1051
Gould AR (1984) Control of the cell cycle in cultured plant cells. CRC Crit. Rev. Plant Sci. 1: 315–344
Karlsson SB & Vasil IK (1986) Growth, cytology and flow cytometry of embryogenic cell suspension cultures of Panicum maximum Jacq. and Pennisetum purpureum Schum. J. Plant Physiol. 123: 211–227
Linsmaier EM & Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18: 100–127
Minocha R, Minocha SC, Komamine A & Shortle WC (1991) Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus cell suspension cultures. Plant Cell Rep. 10: 126–130
Nash DT & Davies ME (1972) Some aspects of growth and metabolism of Paul's scarlet rose cell suspension. J. Exp. Bot. 23: 75–91
Ormrod J & Francis D (1993) Molecular and Cell Biology of the Plant cell Cycle. Kluwer Academic Publishers, Dordrecht, The Netherlands
Pinder AC, Purdy CW, Poulter SAG & Clark DC (1990) Validation of flow cytometry for rapid enumeration of bacterial concentrations in pure cultures. J. Appl. Bacteriol. 69: 92–100
Puite KJ & Ten Broeke WRR (1983) DNA staining of fixed and nonfixed plant protoplasts for flow cytometry with Hoechst 33342 Plant Sci. Lett. 32: 79–88
Steen HB, Boye E, Skarstad K, Bloom B, Godal T & Mustafa S (1982) Applications of flow cytometry on bacteria: cell cycle kinetics, drug effects, and quantitation of antibody binding. Cytometry 2: 249–257
Thorpe TA (1990) The current status of plant tissue culture. In: Bhojwani SS (Ed) Plant Tissue Culture: Application and Limitations, Vol 19 (pp 1–33). Elsevier Publishers, Amsterdam
Ulrich I, Fritz B & Ulrich W (1988) Application of DNA fluorochroms for flow cytometric DNA analysis of plant protoplasts. Plant Sci. 55: 151–158
Ulrich I & Ulrich W (1991) High-resolution flow cytometry of nuclear DNA in higher plants. Protoplasma 165: 212–215
Van Gulik W (1990) Growth Kinetics of Plant Cells in Suspension Culture. PhD Thesis, Delft University of Technology, Delft, The Netherlands
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Nicoloso, F.T., Val, J., van der Keur, M. et al. Flow-cytometric cell counting and DNA estimation for the study of plant cell population dynamics. Plant Cell Tiss Organ Cult 39, 251–259 (1994). https://doi.org/10.1007/BF00035978
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DOI: https://doi.org/10.1007/BF00035978