biologia plantarum

International journal on Plant Life established by Bohumil Nìmec in 1959

Biologia plantarum 48:199-203, 2004 | DOI: 10.1023/B:BIOP.0000033445.15336.04

Isolation of Chromosomes from Picea abies and their Analysis by Flow Cytometry

I. Überall1,*, J. Vrána2, J. Barto¹2, J. ©merda1, J. Dole¾el2, L. Havel1
1 Institute of Botany and Plant Physiology, Mendel University of Agriculture and Forestry, Brno, Czech Republic
2 Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic

A high-yield method for preparation of suspensions of intact Norway spruce [Picea abies (L.) Karst.] chromosomes was developed for the first time. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatments with 0.625 mM hydroxyurea for 18 h and after 8 h recovery in distilled water with 0.05 % (m/v) colchicine for 8 h. These treatments resulted in 50 % metaphase indices. Synchronized root tips were fixed in 2 % formaldehyde for 10 min and chromosomes were released into a lysis buffer by mechanical homogenisation, producing 5 × 105 chromosomes from 50 root tips, at average. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes indicated a possibility to sort at least three different chromosome types.

Keywords: chromosome sorting; chromosome suspensions; flow karyotype; Norway spruce
Subjects: chromosome, isolation, sorting; chromosome, karyotype; colchicine; flow cytometry; Norway spruce, chromosome isolation; Picea abies

Published: June 1, 2004  Show citation

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Überall, I., Vrána, J., Barto¹, J., ©merda, J., Dole¾el, J., & Havel, L. (2004). Isolation of Chromosomes from Picea abies and their Analysis by Flow Cytometry. Biologia plantarum, 48(2), 199-203. doi: 10.1023/B:BIOP.0000033445.15336.04
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    References

    1. Arumuganathan, K., Martin, G.B., Telenius, H., Tanksley, S.D., Earle, E.D.: Chromosome 2-specific DNA clones from flow-sorted chromosomes of tomato.-Mol. gen. Genet. 242: 551-558, 1994. Go to original source...
    2. Binarová, P., Hause, B., Dole¼el, J., Dráber, P.: Assotiation of γ-tubulin with kinetochore-centromeric region of plant chromosomes.-Plant J. 14: 751-757, 1998. Go to original source...
    3. Binelli, G., Bucci, G.: A genetic-linkage map of Picea abies (L.) Karst, based on RAPD markers, as a tool in population genetics.-Theor. appl. Genet. 88: 283-288, 1994. Go to original source...
    4. Dole¼el, J.: KARYOSTAR: Microcomputer program for modelling of monoparametric flow karyotypes.-Biológia (Bratislava) 46: 1059-1064, 1991.
    5. Dole¼el, J., Binarová, P., Lucretti, S.: Analysis of nuclear DNA content in plant cells by flow cytometry.-Biol. Plant. 31: 113-120, 1989. Go to original source...
    6. Dole¼el, J., Èíhalíková, J., Lucretti, S.: A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L.-Planta 188: 93-98, 1992. Go to original source...
    7. Dole¼el, J., Lysák, M.A., Kubaláková, M., ©imková, H., Macas, J., Lucretti, S.: Sorting of plant chromosomes.-In: Darzynkiewicz, Z., Crissman, H.A., Robinson, J.P. (ed.): Methods in Cell Biology. Pp. 3-31. Academic Press, San Diego 2001. Go to original source...
    8. Dole¼el, J., Macas, J., Lucretti, S.: Flow analysis and sorting of plant chromosomes.-In: Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Dressler, G., Orfao, A., Rabinovitch, P.S., Stewart, C.C., Tanke, H.J., Wheeless, L.L.: Current Protocols in Cytometry Current Protocols in Cytometry. Pp. 531-533, John Wiley & Sons, New York 1999.
    9. Fuchs, J., Joos, S., Lichter, P., Schubert, I.: Localization of vicilin genes on field bean chromosome II by fluorescent in situ hybridization.-Heredity 85: 487-488. 1994. Go to original source...
    10. Geburek, T., Wuehlisch, G.: Linkage analysis of isozyme gene loci in Picea abies (L.) Karst.-Heredity 62: 185-191, 1989. Go to original source...
    11. Köhler, B., Guttenberger, H., Borzan, ¼.: Giemsa C-banding pattern of the chromosomes in the macrogametophyte of Norway spruce.-Silvae Genet. 45: 16-21, 1996.
    12. Kubaláková, M., Lysák, M.A., Vrána, J., ©imková, H., Èíhalíková, J., Dole¼el, J.: Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS.-Cytometry 41: 102-108, 2000. Go to original source...
    13. Kubaláková, M., Macas, J., Dole¼el, J.: Mapping of repeat DNA sequences in plant chromosomes by PRINS and C-PRINS.-Theor. appl. Genet. 94: 758-763, 1997. Go to original source...
    14. Lagercrantz, U., Ryman, N.: Genetic structure of Norway spruce (Picea abies): concordance of morphological and allozymic variations.-Evolution 44: 38-53, 1990.
    15. Lucretti, S., Dole¼el, J.: Cell cycle synchronization, chromosome isolation, and flow-sorting in plants.-In: Galbraith, D.W., Bourque, D.P., Bohnert, H.J. (ed.): Methods in Cell Biology. Vol. 50. Pp. 61-83. Academic Press, New York 1995. Go to original source...
    16. Macas, J., Dole¼el, J., Lucretti, S, Pich U., Meister, A. Fuchs, J., Schubert, I.: Localization of seed protein genes on flow-sorted field bean chromosomes.-Chromosome Res. 1: 107-115, 1993. Go to original source...
    17. Macas, J., Gualberti, G., Nouzová, M., Samec, P., Lucretti, S., Dole¼el, J.: Construction of chromosome-specific DNA libraries covering the whole genome of field bean (Vicia faba L.).-Chromosome Res. 4: 531-539, 1996. Go to original source...
    18. Neumann, P., Po¼árková, D., Vrána, J., Dole¼el, J., Macas, J.: Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L.).-Chromosome Res. 10: 63-71, 2002. Go to original source...
    19. Paglia, G.P., Olivieri, A.M., Morgante, M.: Towards second-generation STS (sequence-tagged sites) linkage maps in conifers: a genetic map of Norway spruce (Picea abies Karst.).-Mol. gen. Genet. 258: 466-478, 1998. Go to original source...
    20. Portugal, J., Waring, M.: Assignment of DNA-binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258): A comparative footprinting study.-Biochim. biophys. Acta 949: 158-168, 1988. Go to original source...
    21. Po¼árková, D., Koblí¼ková, A., Román, B., Torres, A.M., Lucretti, S., Lysák, M., Dole¼el, J., Macas, J.: Development and characterization of microsatellite markers from chromosome 1-specific DNA libraries of Vicia faba.-Biol. Plant. 45: 337-345, 2002. Go to original source...
    22. Schubert, I., Dole¼el, J., Houben, H., Schertan, H., Wanner, G.: Refined examination of plant metaphase chromosome and stability of wheat cell culture line.-Theor. appl. Genet. 94: 91-97, 1993.
    23. Siljak-Yakovlev, S., Cerbah, M., Couland, J., Stojan, V., Brown, S.C., Zoldos, V., Jelenic, S., Papes, D.: Nuclear DNA content, base composition, heterochromatin and rDNA in Picea omorica and Picea abies.-Theor. appl. Genet. 104: 505-512, 2002. Go to original source...
    24. Skov, E., Wellendorf, H.: A partial linkage map of Picea abies clone V6470 based on recombination of RAPD markers in haploid megagametophytes.-Silvae Genet. 47: 273-282, 1998.

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