Flow Cytometric Analysis of Partially Synchronized Suspension Cultures of Hemerocallis hybrida

Abstract

Cell division of suspension cultures of Hemerocallis hybrida was partially synchronized by treating with hydroxyurea (HU), a DNA synthesis inhibitor, followed by treatment with colchicine (COL), a spindle toxin, as the first step toward an efficient preparation of microprotoplasts. During COL treatment, suspension cells were analyzed for relative DNA contents using flow cytometry (FCM) as well as mitotic index (MI) under microscopy. MI reached a peak after 20h of COL treatment. In FCM analyses, histogram of asynchronous cultures showed three peaks (2C, 4C and 8C nuclei), and the numbers of both 4C and 8C nuclei gradually increased until 18h after the initiation of COL treatment. After 20h of COL treatment, the numbers of 4C and 8C nuclei transiently decreased, indicating that a large number of cells were at the prometaphase to metaphase stages. These results indicate that FCM analyses offer a simple and rapid means of preliminary examination for arresting cell-cycle at metaphase in partially synchronized suspension cultures of H. hybrida.