Isolation of Mitotic Chromosomes from Partially Synchronized Carrot (d. C a R O T a ) Cell Suspension Cultures*

Condensed mitotic chromosomes were isolated from cell suspension cultures of carrot, Daucus carota var. Danvers. Mitotic cells were obtained from partially synchronized cell cultures by adding colchicine 15 h after inoculation of 4-day-old cultures into fresh medium. From 12% to 18% of the cells were in mitosis 6 h after colchicine addition. Other methods of inducing synchrony and collecting mitotic cells, including using 5-fluorodeoxyuridine (Fudr) or hydroxyurea (HU), were less effective. Chromosomes were isolated by incubating the cells in cell wall degrading enzymes for 1 h, rupturing the cells and subjecting the extract to differential centrifugation to partially remove nuclei and cell debris. Paired mitotic chromosomes were identified in these extracts using phase contrast and fluorescence microscopy.