THE ROLE OF CALCIUM AND CALMODULIN IN CARROT SOMATIC EMBRYOGENESIS

The role of Ca2+ and calmodulin in carrot somatic embryo formation was examined. Embryogenic cell clumps were induced to form embryos in med ium containing 0-3 mM Ca2+. Embryo formation was not affected until th e concentration of Ca2+ was lower than 200 muM and after this threshol d was reached the percent of embryo formation decreased with lower Ca2 + concentrations. Treatment of developing embryos with either verapami l or nifedipine, Ca2+-channel blockers, or the Ca2+ ionophore A23187, inhibited embryo formation. These results suggest that exogenous Ca2or the maintenance of Ca2+ gradients are required for proper embryo de velopment. Analysis of membrane-associated Ca2+ and total membrane dis tribution using the fluorescent dyes chlorotetracycline and N-phenyl-1 -napthylamine, respectively, indicated changes in the distribution of membranes during embryogenesis without any significant alterations in the concentration of Ca2+ associated with the membranes. In heart- and torpedo-stage embryos, calmodulin-Ca2+ complexes were localized in re gions containing the developing meristems of both the cotyledon tips a nd rhizoid regions while total calmodulin protein appeared to be more uniformly distributed. Calmodulin mRNA levels increased slightly when cell clumps were induced to form embryos.