Fluorescent labeling of plant chromosomes in suspension by FISH

By optimizing the concentration and time of treatment with hydroxyurea (HU), a DNA synthesis inhibitor, and trifluralin, a microtubule inhibitor, a highly effective (over 60%) cell cycle synchronization method for rye and barley meristem cells was developed. Chromosome suspensions containing highly purified and morphologically intact rye and barley chromosomes were prepared from the meristems of their root tips by homogenization. Digoxigenin-labeled 5S rDNA was used as a probe in FISH for the rye chromosomes in the suspension, and biotin-labeled 17S rDNA and centromeric DNA were used in FISH for the rye and barley chromosome suspensions, respectively. Bright signals were detected at the specific regions of interest on the chromosomes. The results indicate that the method developed in this study is useful for selection and sorting of chromosomes that are not distinguishable by other means, using specific fluorescent labeling by FISH of the chromosomes in suspension.