Explants obtained from young sporophytes of Saccharina japonica were cultured in an artificial medium with different concentrations of Ca 2+ (0-20 mM). The culture with 10 mM Ca 2+ promoted the formation of unpigmented filamentous callus-like cells in the cortical layer. In contrast, explants cultured with 5 mM Ca 2+ formed pigmented round callus-like cells in the epidermis at a high percentage. The thallus regeneration rate of explants in 5 mM Ca 2+ was ten times higher than those of explants cultured in 10 mM Ca 2+ . Ambient Ca 2+ concentrations also influenced the production of radical oxygen species (ROS) in explants. Explants cultured in 10 mM Ca 2+ produced higher ROS than did those cultured in 5 mM. The ROS production was histologically observed mainly in the plasma membrane of callus-like cells using 2′, 7′-dichlorodihydrofluorescein diacetate. Moreover, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride, inhibited the ROS production with propagation of filamentous callus-like cells. These results suggest that Ca 2+ concentration in medium influences the development of callus-like cells and thallus regeneration by affecting NADPH oxidase activity and ROS production in the plasma membrane of the callus-like cells. Therefore, the morphological form of callus-like cells and the development of thallus will be controlled by Ca 2+ concentration in the medium.
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