ROOT-TIP CELL-CYCLE SYNCHRONIZATION AND METAPHASE-CHROMOSOME ISOLATION SUITABLE FOR FLOW SORTING IN COMMON WHEAT (TRITICUM-AESTIVUM L.)

An efficient procedure for cell-cycle synchronization in meristematic root tips was achieved in common wheat. Treatment parameters for synch ronizing the cell cycle of root tip meristem cells, such as time-cours e and applied concentrations of various chemicals, were systematically tested and optimized by flow cytometric analysis of isolated nuclei. High mitotic indices (69.5% in the root tip meristematic area) were ro utinely obtained by treating germinating seeds with 1.25 mM hydroxyure a for 16 h, followed by incubation in a hydroxyurea-free solution for 2 h, and treatment with 1 mu M trifluralin for 4 h. Uniform seed germi nation prior to treatment is very important for achieving consistently high metaphase indices in the root tips. Large numbers of metaphase c hromosomes, suitable for flow cytometric analysis and sorting, were is olated from synchronized root tip cells. Flow sorted wheat chromosomes , via univariate and bivariate analysis, showed four major chromosome peaks. Each discrete peak may represent wheat chromosome types with si milar DNA content. Bivariate flow karyotyping based on AT and GC conte nt did not improve the separation of wheat chromosomes.