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

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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.