BIVARIATE FLOW KARYOTYPING IN BROAD BEAN (VICIA-FABA)

In the present study, we report on the development of bivariate flow k aryotyping in the legume broad bean (Vicia faba). We optimised chromos ome staining with 4',6-diamidino-2-phenylindole and mithramycin A and analysed chromosome suspensions prepared from a Line with standard (wi ld-type) karyotype and from six translocation Lines with reconstructed karyotypes. Chromosomes were isolated from formaldehyde-fixed root ti ps after cell cycle synchronisation, and their fluorescence was analys ed with dual-laser flow cytometry after the staining, High-resolution bivariate flow karyotypes were obtained in all broad bean Lines analys ed. Compared with univariate analysis, the bivariate analysis permitte d discrimination of more chromosome types. However peaks corresponding to newly resolved chromosomes mere rather closely spaced, which could have compromised the purity of sorted fractions. With only a few exce ptions, chromosome peaks mere in a straight Line, suggesting only mino r differences in the AT:GC ratio among the chromosomes. These results indicate the Limited potential of bivariate flow cytometric analysis a nd sorting in broad bean. (C) 1997 Wiley-Liss, Inc.