TECHNIQUES TO STUDY THE CELL-CYCLE IN CONIFER SHOOT APICAL MERISTEMS

A method is presented to determine the mitotic index of conifer shoot apical meristems in a rapid and standardized manner using permanent Feulgen-stained squash preparations observed with a series of horizontal scans at fixed vertical intervals. Interpretation and application of mitotic index and its relationship to the cell cycle are discussed. Methods used to label nuclei within conifer apical meristems with tritiated thymidine and procedures that may improve incorporation of label and decrease cellular damage are discussed. The percentage of cells in S stage was determined using autoradiography after apices were immersed in a solution containing 203.5 kBq/mL tritiated thymidine in 1% DMSO for 2 h. With nuclei in S stage identified by autoradiography, G1 and G2 nuclei were visually identified based on Feulgen staining intensity and nuclear area. Using these techniques it was determined that loblolly pine (Pinus taeda L.) apical meristems in rapid neoformed needle initiation had 75% of their cells in G1, 14.8% in S, 4.7% in G2. and 4.6% in mitosis. The relationships between the size of nuclei, the initiation of DNA synthesis, and zonation in the apical dome are discussed.