IMMUNOCYTOCHEMICAL STUDY OF CELL-CYCLE CONTROL BY CYTOKININ IN CULTURED SOYBEAN CELLS

An immunocytochemical method was used to determine the proportion of c ells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyl edonary origin, the stably cytokinin-dependent tissue used in the cyto kinin bioassay devised by Carlos O. Miller. A standard cell synchroniz ation protocol involving hydroxyurea was used to demonstrate the appli cability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell d ivision cycle was restarted by the addition of cytokinin. We have foll owed the pattern of resumption of S phase after the readdition of cyto kinin. This pattern reveals the existence of three subpopulations of c ells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, on e in S phase, and one in the G(1) phase.