Skip to main content
SpringerLink
Log in

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

  • Published:
Theoretical and Applied Genetics Aims and scope Submit manuscript

Abstract

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with 2–3 × 106 chromosomes ml−1 was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin A3, univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes.

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

References

  • Arumuganathan K, Slattery JP, Tanksley SD, Earle ED (1991) Preparation and flow cytometric analysis of metaphase chromo-somes of tomato. Theor Appl Genet 82:101–111

    Article  Google Scholar 

  • Arumuganathan K, Martin GB, Telenius H, Tanksley SD, Earle ED (1993) Chromosome 2-specific DNA clones from flow-sorted chromosomes of tomato. Cytometry Suppl 6:71

    Google Scholar 

  • Bashir A, Auger JA, Rayburn LA (1993) Flow cytometric DNA analysis of wheat-rye addition lines. Cytometry 14:843–847

    Article  PubMed  CAS  Google Scholar 

  • Bennett MD, Smith JB (1976) Nuclear DNA amounts in the angiosperms. Phil Trans R Soc Lond B 274:227–274

    Article  CAS  Google Scholar 

  • Blumenthal AD, Dieden JD, Kapp LN, Sedat JW (1979) Rapid isolation of metaphase chromosomes containing high-molecular-weight DNA. J Cell Biol 81:255–259.

    Article  PubMed  CAS  Google Scholar 

  • Carter NP (1994a) Bivariate chromosome analysis using a commercial flow cytometer. Methods Mol Biol 29:187–204

    PubMed  CAS  Google Scholar 

  • Carter NP (1994b) Chromosome analysis by chromosome painting. Cytometry 18:2–10

    Article  PubMed  CAS  Google Scholar 

  • Carter NP, Ferguson-Smith MA, Perryman MT, Telenius H, Pelmear AH, Leversha MA, Glancy MT, Wood SL, Cook K, Dyson HM, Ferguson-Smith ME, Willatt LR (1992) Reverse chromosome painting: a method for the rapid analysis of aberrant chromosomes in clinical cytogenetics. J Med Genet 29:299–307

    Article  PubMed  CAS  Google Scholar 

  • Chen DF, Dale PJ, Heslop-Harrison JS, Snape JW, Harwood W, Bean S, Mullineaux PM (1994) Stability of transgenes and the presence of N6 methyladenine DNA in transformed wheat cells. Plant J 5:429–436

    Article  Google Scholar 

  • Conia J, Bergounioux C, Perennes C, Muller P, Brown S, Gadal P (1987) Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts. Cytometry 8:500–508

    Article  PubMed  CAS  Google Scholar 

  • Conia J, Muller P, Brown S, Bergounioux C, Gadal P (1989) Monoparametric models of flow cytometric karyotypes with spread-sheet software. Theor Appl Genet 77:295–303

    Article  Google Scholar 

  • Cotter F, Nasipuri, S, Lam G, Young BD (1989) Gene mapping by enzymatic amplification from flow-sorted chromosomes. Genomics 5:470–474

    Article  PubMed  CAS  Google Scholar 

  • Deaven LL, Van Dilla MA, Bartholdi MF, Carrano AV, Cram LS, Fuscoe JC, Gray JW, Hildebrand CE, Moyzis RK, Perlman J (1986) Construction of human chromosome-specific DNA libraries from flow-sorted chromosomes. Cold Spring Harbor Symp Quantit Biol LI: 159–167

    Google Scholar 

  • Devos KM, Atkinson MD, Chinoy CN, Koebner RMD, Francis HA, Harcourt RL, Liu CJ, Masojc P, Xie DX, Gale MD (1993) Chromosomal rearrangements in the rye genome relative to that of wheat. Theor Appl Genet 85:673–680

    Article  CAS  Google Scholar 

  • Dolezel J (1991) KARYOSTAR: microcomputer program for modelling of monoparametric flow karyotypes. Biologia 46:1059–1064

    Google Scholar 

  • Dolezel J, Lucretti S, Schubert I (1994) Plant chromosome analysis and sorting by flow cytometry. CRC Crit Rev Plant Sci 13: 275–309

    Article  Google Scholar 

  • Frearson EM, Power JB, Cocking EC (1973) The isolation, culture and regeneration of Petunia leaf protoplasts. Dev Biol 33: 130–137

    Article  PubMed  CAS  Google Scholar 

  • Fuchs J, Houben A, Brandes A, Schubert I (1996) Chromosome ‘painting’ in plants — a feasible technique? Chromosoma 104:315–320

    PubMed  CAS  Google Scholar 

  • Fukui K, Minezawa M, Kamisugi Y, Ishikawa M, Ohmido N, Yanagisawa T, Fujishita M, Sakai F (1992) Microdissection of plant chromosomes by argon-ion laser beam. Theor Appl Genet 84:787–791

    Article  Google Scholar 

  • Galasso, I, Schmidt T, Pignone D, Heslop-Harrison JS (1995) The molecular cytogenetics of Vigna unguiculata (L.) Walp: the physical organization and characterization of 18s–5.8s–25s rRNA genes, 5s rRNA genes, telomere-like sequences, and a family of centromeric repetitive DNA sequences. Theor Appl Genet 91:928–935

    Article  CAS  Google Scholar 

  • Gray JW, Cram LS (1990) Flow karyotyping and chromosome sorting. In: Melamed MR, Lindmo T, Mendelsohn ML (eds) Flow cytometry and sorting. Wiley-Liss, New York, pp 503–529

    Google Scholar 

  • Griesbach RG, Malmberg RL, Carlson PS (1982) An improved technique for the isolation of higher plant chromosomes. Plant Sci Lett 24:55–60

    Article  CAS  Google Scholar 

  • Gupta PK, Tsuchiya T (1991) Chromosome engineering in plants: genetics, breeding, evolution. Part A. Elsevier, Amsterdam

    Google Scholar 

  • Jung C, Claussen U, Horsthemke B, Fischer F, Herrmann RG (1992) A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes. Plant Mol Biol 20:503–511

    Article  PubMed  CAS  Google Scholar 

  • Kao KN (1977) Chromosomal behavior in somatic hybrids of soybean — Nicotiana glauca. Mol Gen Genet 150:225–230

    Article  Google Scholar 

  • Langford CF, Fischer PE, Binns MM, Holmes NG, Carter NP (1996) Chromosome-specific paints from a high-resolution flow karyotype of the dog. Chrom Res 4:115–123

    Article  PubMed  CAS  Google Scholar 

  • Laat AMM de, Blaas J (1984) Flow-cytometric characterization and sorting of plant chromosomes. Theor Appl Genet 67:463–467

    Article  Google Scholar 

  • Lee M, Phillips R (1988) The chromosomal basis of somaclonal variation. Annu Rev Plant Physiol Plant Mol Biol 39:413

    Article  Google Scholar 

  • Leitch AR, Schwarzacher T, Wang ML, Leitch IJ, Surlan-Momirovic G, Moore G, Heslop-Harrison JS (1993) Molecular cytogenetic analysis of repeated sequences in a long-term wheat-suspension culture. Plant Cell, Tissue Org Cul 33:287–296

    Article  CAS  Google Scholar 

  • Lucretti S, Dolezel J, Schubert I, Fuchs J (1993) Flow karyotyping and sorting of Vicia faba chromosomes. Theor Appl Genet 85:665–672

    Article  Google Scholar 

  • Macas J, Dolezel J, Lucretti S, Pich U, Meister A, Fuchs J, Schubert I (1993) Localization of seed-storage protein genes on flow-sorted field bean chromosomes. Chrom Res 1:107–115

    Article  PubMed  CAS  Google Scholar 

  • Pearce SR, Harrison G, Wilkinson M, Li D, Heslop-Harrison JS, Flavell AJ, Kumar A (1996) The Ty1-copia group retrotransposons in Vicia species: copy number, sequence heterogeneity and chromosomal localisation. Mol Gen Genet 250:305–315

    PubMed  CAS  Google Scholar 

  • Pfosser M, Amon A, Lelley T, 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

    Article  PubMed  CAS  Google Scholar 

  • Sandery MJ, Forster JW, Macadam SR, Blunden R, Jones RN, Brown SDM (1991) Isolation of a sequence common to A-and B-chromosomes of rye (Secale cereale) by microcloning. Plant Mol Biol Rep 9:21–30

    Article  CAS  Google Scholar 

  • Schmidt T, Kubis S, Heslop-Harrison JS (1995) Analysis and chromosomal localization of retrotransposons in sugar beet (Beta vulgaris L.): LINEs and Ty1-copia-like elements as major components of the genome. Chrom Res 3:335–345

    Article  PubMed  CAS  Google Scholar 

  • Schondelmaier J, Martin R, Jahoor A, Houben A, Graner A, Koop H-U, Herrmann RG, Jung C (1993) Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS. Theor Appl Genet 86:629–636

    Article  CAS  Google Scholar 

  • Sillar R, Young BD (1981) A new method for the preparation of metaphase chromosomes for flow analysis. J Histochem Cytochem 29:74–78

    PubMed  CAS  Google Scholar 

  • Trask B, van den Engh G, Nussbaum R, Schwartz C, Gray J (1990) Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping. Cytometry 11:184–195

    Article  PubMed  CAS  Google Scholar 

  • Verhoeven HA, Sree Ramulu K, Dijkhuis P (1990) A comparison of the effects of various spindle toxins on metaphase arrest and formation of micronuclei in cell-suspension cultures of Nicotiana plumbaginifolia. Planta 182:403–414

    Article  Google Scholar 

  • Vershinin A, Schwarzacher T, Heslop-Harrison JS (1995) The largescale genomic organization of repetitive DNA families at the telomeres of rye chromosomes. Plant Cell 7:1823–1833

    Article  PubMed  CAS  Google Scholar 

  • Veuskens J, Marie D, Brown SC, Jacobs M, Negrutiu I (1995) Flow sorting of the Y sex-chromosome in the dioecious plant Melandrium album. Cytometry 21:363–373

    Article  PubMed  CAS  Google Scholar 

  • Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS, Morre G (1992) Construction of a chromosome-enriched Hpa II library from flow-sorted wheat chromosomes. Nucleic Acids Res 20: 1897–1901

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. M. L. Wang

    Present address: Department of Molecular Biology, Massachusetts General Hospital, 02114, Boston, Massachusetts, USA

  2. A. R. Leitch

    Present address: Queen Mary and Westfield College, Mill End Road, El 4NS, London, UK

Authors and Affiliations

  1. John Innes Centre, Colney Lane, NR4 7UH, Norwich, UK

    T. Schwarzacher, M. L. Wang, A. R. Leitch, G. Moore & J. S. Heslop-Harrison

  2. Babraham Hall, Babraham Institute, CB2 4AT, Cambridge, UK

    N. Miller

Authors
  1. T. Schwarzacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. L. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. R. Leitch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Moore
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. S. Heslop-Harrison
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. N. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by I. Potrykus

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schwarzacher, T., Wang, M.L., Leitch, A.R. et al. Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line. Theoret. Appl. Genetics 94, 91–97 (1997). https://doi.org/10.1007/s001220050386

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s001220050386

Key words

Search

Navigation