CELL SYNCHRONIZATION AND ISOLATION OF METAPHASE CHROMOSOMES FROM MAIZE (ZEA-MAYS L) ROOT-TIPS FOR FLOW CYTOMETRIC ANALYSIS AND SORTING

Accumulation of cells containing metaphase chromosomes is an important step in cytological analyses and chromosome sorting procedures. The g oal of this research was to optimize treatment parameters to synchroni ze the cell cycle of maize root tip meristem cells. Levels of hydroxyu rea, a DNA synthesis inhibitor, were assessed for their utility in acc umulating cells at the G1 phase of the cell cycle. Trifluralin, amipro phos-methyl, and colchicine were used to accumulate cells containing m etaphase chromosomes upon release from hydroxyurea inhibition. Optimal mitotic indices were achieved by treating seedlings with 5 mM hydroxy urea for 18 h, incubating for 1 h without chemical treatment to releas e the hydroxyurea block, and then treating emerging roots with 1 mu M trifluralin for 4 h. The mitotic index of synchronized maize root tips was over 70%. Uniformity of synchronization depended upon selection o f seeds with emerging radicles that were similar in length at the time of treatment. Suspensions of intact chromosomes were prepared by a si mple slicing procedure. The chromosome preparations were found to be s uitable for flow cytometric characterization and sorting. Chromosome p eaks of the observed flow karyotype resembled the predicted flow karyo type calculated on the basis of maize chromosome size.