Bivariate flow karyotyping in broad bean (Vicia faba)

In the present study, we report on the development of bivariate flow karyotyping in the legume broad bean (Vicia faba). We optimised chromosome staining with 4',6-diamidino-2-phenylindole and mithramycin A and analysed chromosome suspensions prepared from a line with standard (wild-type) karyotype and from six translocation lines with reconstructed karyotypes. Chromosomes were isolated from formaldehyde-fixed root tips after cell cycle synchronisation, and their fluorescence was analysed with dual-laser flow cytometry after the staining. High-resolution bivariate flow karyotypes were obtained in all broad bean lines analysed. Compared with univariate analysis, the bivariate analysis permitted discrimination of more chromosome types. However, peaks corresponding to newly resolved chromosomes were rather closely spaced, which could have compromised the purity of sorted fractions. With only a few exceptions, chromosome peaks were in a straight line, suggesting only minor differences in the AT:GC ratio among the chromosomes. These results indicate the limited potential of bivariate flow cytometric analysis and sorting in broad bean.