Plant Genome IV Conference Plant Genome IV Conference
Metaphase chromosome suspensions suitable for flow cytometric analysis and sorting were prepared from synchronized root tips of rice, sorghum, maize, barley, oat, and wheat. For effective cell synchronization in root-tip meristems, combinations of an S-phase inhibitor (hydroxyurea) and several microtubule inhibitors (colchicine, amiprophos-methyl or trifluralin) were employed. Uniform germinations were obtained by first immersing the seeds in running tap water for 1-3 days and then by placing on moistened paper towels for 1-2 days. Seedlings with optimal-sized primary roots were exposed to hydroxyurea solutions of different concentrations (0.5 mM to 5.0 mM). Cell cycle progress of root tip meristems were monitored by flow cytometric analysis of nuclei suspension prepared form root tips at one hour intervals during hydroxyurea treatment. When majority of the root-tip cells reached late S-phase or early G2 phase, seedlings were exposed to microtubule inhibitors for 4-6 h for accumulation of large number of cells at metaphase. Suspensions of metaphase chromosomes were prepared from synchronized root tips by simple chopping in a chromosome stabilizing buffer, stained with propidium iodide and analyzed on FACScan analyzing flow cytometer. Peaks corresponding to chromosomes and nuclei were positively identified by examinations of sorted fractions under a fluorescent microscope.