Skip to main content
SpringerLink
Log in

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

  • Published:
Journal of Plant Growth Regulation Aims and scope Submit manuscript

Abstract

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G1 phase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BrdU:

5-bromo-2-deoxyuridine

DI:

deionized water

FITC:

fluorescein isothiocyanate

HU:

hydroxyurea

l-AOPP:

l-α-aminooxy-β-phenylpropionic acid

LI:

labeling index

PA:

polyamine

PI:

propidium iodide

References

  • Bayliss MW (1985) Regulation of the cell division cycle in cultured plant cells. In: Bryant JA, Francis D (eds) The cell division cycle in plants. Society for Experimental Biology: Seminar Series 26, Cambridge University Press, pp 157–177

  • Beisker W, Dolbeare F, Gray JW (1987) An improved immunocytochemical procedure for high sensitivity detection of incorporated bromodeoxyuridine. Cytometry 8:235–239

    Article  PubMed  CAS  Google Scholar 

  • Blaschke JR, Froche E, Neumann KH (1978) Investigations on the cell cycle of haploid and diploid tissue cultures of Datura innoxia and its synchronisation. Planta 144:7–12

    Article  CAS  Google Scholar 

  • Brown R, Rickless AP (1949) A new method for the study of cell division and cell extension with preliminary observations on the effect of temperature and nutrients. Proc R Soc Lond B 136:110–125

    PubMed  CAS  Google Scholar 

  • Burgess J, Watts JW, Fleming EN, King JM (1973) Plasmalemma fine structure in isolated tobacco mesophyll protoplasts. Planta 110:291–301

    Article  Google Scholar 

  • Cho S-C (1983) Effects of cytokinin and several inorganic cations on the polyamine content of lettuce cotyledons. Plant Cell Physiol 24:27–32

    CAS  Google Scholar 

  • Chu YE, Lark KG (1976) Cell-cycle parameters of soybean (Glycine max L.) cells growing in suspension culture: Suitability of the system for genetic studies. Planta 132:259–268

    Article  CAS  Google Scholar 

  • DelDuca S, Serafini-Fracassini D (1993) Polyamines and protein modification during the cell cycle. In: Ormrod JC, Francis D (eds) Molecular and cell biology of the plant cell cycle. Kluwer Academic Publishers, Dordrecht, pp 143–156

    Google Scholar 

  • Dudits D, Lazar G, Bajszar G (1979) Reversible inhibition of somatic embryo differentiation by bromodeoxyuridine in cultured cells of Daucus carota. Cell Differ 8:135–144

    Article  CAS  Google Scholar 

  • Fosket DE, Short KC (1973) The role of cytokinin in the regulation of growth, DNA synthesis and cell proliferation in cultured soybean tissues (Glycine max var. Biloxi). Physiol Plant 28:14–23

    Article  CAS  Google Scholar 

  • Francis D, Halford NG (1995) The plant cell cycle. Physiol Plant 93:365–374

    Article  CAS  Google Scholar 

  • Geber G, Schweizer D (1988) Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads. Plant Syst Evol 158:97–106

    Article  CAS  Google Scholar 

  • Gratzner HG (1982) Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: A new reagent for detection of DNA synthesis. Science 218:474–475

    Article  PubMed  CAS  Google Scholar 

  • Grosskopf DF, Kroiher M (1988) Gradients in plastid and mitochrondrial DNA-synthesis in Funaria protonemata visualization by bromodeoxyuridine immunohistochemistry. Protoplasma 147:1–4

    Article  Google Scholar 

  • Hammat N, Davey MR (1988) Isolation and culture of soybean hypocotyl protoplasts. In Vitro Cell Dev Biol 24:601–604

    Article  Google Scholar 

  • Hemanataranjan A, Vaishampayan A (1984) Effect of cytokinin and various inorganic cations on the polyamine content of french bean (Phaseolus vulgare L.) cotyledons. Biochem Physiol Pflanz 179:553–559

    Google Scholar 

  • Houssa C, Jacqmard A, Bernier G (1990) Activation of replicon origins as a possible target for cytokinins in shoot meristems of Sinapis. Planta 181:324–326

    Article  CAS  Google Scholar 

  • Jacqmard A, Houssa C, Bernier G (1994) Regulation of the cell cycle by cytokinins. In: Mok DWS, Mok MC (eds) Cytokinins: Chemistry, activity, and function. CRC Press, Boca Raton, FL, pp 197–215

    Google Scholar 

  • John PCL, Zhang K, Dong C (1993a) A p34cdc2-based cell cycle: Its significance in monocotyledonous, dicotyledonous and unicellular plants. In: Ormrod JC, Francis D (eds) Molecular and cell biology of the plant cell cycle. Kluwer Academic Publishers, Dordrecht, pp 9–34

    Google Scholar 

  • John PCL, Zhang K, Dong C, Diederich L, Wightman F (1993b) p34cdc2-Related proteins in control of cell cycle progression, the switch between division and differentiation in tissue development, and stimulation of division by auxin and cytokinin. Aust J Plant Physiol 20:503–526

    CAS  Google Scholar 

  • Johnson GD, deNogueira Araujo GM (1981) A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43:349–350

    Article  PubMed  CAS  Google Scholar 

  • Jones DH (1984) Phenylalanine ammonia-lyase: Regulation of its induction, and its role in plant development. Phytochemistry 23:1349–1359

    Article  CAS  Google Scholar 

  • Kwon YH, Hoch HC (1991) Temporal and spatial dynamics of appressorium formation in Uromyces appendiculatus I. Exp Mycol 15:116–131

    Article  Google Scholar 

  • Levi M, Sparvioli E, Sgorbati S, Chiattante D (1987) Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography. Physiol Plant 71:68–72

    Article  CAS  Google Scholar 

  • Magyar Z, Bakó L, Bögre L, Dedeoglu D, Kapros T, Dudits D (1993) Active cdc2 genes and cell cycle phase-specific cdc2-related kinase complexes in hormone-stimulated alfalfa cells. Plant J 4:151–161

    Article  CAS  Google Scholar 

  • Maki H, Ando S, Kodama H, Komamine A (1991) Polyamines and the cell cycle of Catharanthus reseus cells in culture. Plant Physiol 96:1008–1013

    Article  PubMed  CAS  Google Scholar 

  • Miller CO (1967) Cytokinins in Zea mays. Ann NY Acad Sci 144:251–257

    Article  CAS  Google Scholar 

  • Miller CO, Skoog F, vonSaltza MH, Strong FM (1955) Kinetin, a cell division factor from deoxyribonucleic acid. J Am Chem Soc 77:1392–1393

    Article  CAS  Google Scholar 

  • Navarrete MH, Perez-Villamil B, Lopez-Saez JF (1979) Analysis of the interphase accumulation induced by hydroxyurea on proliferating plant cells. Exp Cell Res 124:151–158

    Article  PubMed  CAS  Google Scholar 

  • Patau K, Das NK, Skoog F (1957) Induction of DNA synthesis by kinetin and indoleacetic acid in excised tobacco pith tissue. Physiol Plant 10:949–966

    Article  CAS  Google Scholar 

  • Pfosser M (1989) Improved method for critical comparison of cell cycle data of asynchronously dividing and synchronised cell cultures of Nicotiana tabacum. J Plant Physiol 134:741–745

    Google Scholar 

  • Pfosser M (1993) Free and conjugated polyamines during the initiation of the cell cycle of G0 arrested Medicago varia cell cultures. J Plant Physiol 142:605–609

    CAS  Google Scholar 

  • Pfosser M, Königshofer H, Kandeler R (1990) Free, conjugated and bound polyamines during the cell cycle of synchronized cell suspension cultures of Nicotiana tabacum. J Plant Physiol 136: 574–579

    CAS  Google Scholar 

  • Serafini-Fracassini D (1991) Cell cycle-dependent changes in plant polyamine metabolism. In: Slocum RD, Flores HE (eds) Biochemistry and physiology of polyamines in plants. CRC Press, Boca Raton, FL, pp 159–174

    Google Scholar 

  • Takegami MH, Ito M (1982) Effect of hydroxyurea on inhibition of mitotic and meiotic divisions in explanted lily Lilium longiflorum microsporocytes. Cell Struct Funct 7:29–38

    Article  CAS  Google Scholar 

  • Vyskot B, Bezdek M (1982) An autoradiographic study of DNA synthesis in cytokinin autotrophic tobacco cells. Z Pflanzenphysiol 106:431–446

    CAS  Google Scholar 

  • Walker MA, Roberts DR, Dumbroff EB (1988) Effects of cytokinins and light on polyamines during the greening response of cucumber cotyledons. Plant Cell Physiol 29:201–205

    CAS  Google Scholar 

  • Wang H, Cutler AJ, Saleem M, Fowke LC (1989a) Immunocytochemical detection of DNA synthesis in plant cells. J Plant Physiol 135:15–20

    CAS  Google Scholar 

  • Wang H, Cutler AJ, Saleem M, Fowke LC (1989b) DNA replication in maize leaf protoplasts. Plant Cell Tissue Organ Cult 18:33–46

    Article  Google Scholar 

  • Wang TL, Everett NP, Gould AR, Street HE (1981) Studies on the control of the cell cycle in cultured plant cells. III. The effect of cytokinin. Protoplasma 106:23–35

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Plant Sciences, University of Cambridge, Downing St., CB2 3EA, Cambridge, UK

    J. C. Mader & D. E. Hanke

Authors
  1. J. C. Mader
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. E. Hanke
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mader, J.C., Hanke, D.E. Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells. J Plant Growth Regul 15, 95–102 (1996). https://doi.org/10.1007/BF00192938

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00192938

Key Words

Search

Navigation