Yasutake Sugawara

Enhancement of Cell Division in Marchanda Protoplast Culture by Activated Charcoal

Summary Protoplasts isolated from callus culture of liverwort (Marchantia polymorpha L.) were cultured in the presence of activated charcoal. The rate of cell division of regenerated protoplasts was enhanced by an addition of activated charcoal to the culture medium. Partial but considerable enhancement of cell division was observed with frequent washing of the protoplasts and replacement of the medium during the initial stage of culture. These results suggest that a certain substance(s) inhibitory to cell division and absorbed by activated charcoal is released from protoplasts and/or regenerated protoplasts.

A simple and rapid method for determining cell survival in the cryopreserved shoot apex using luciferin–luciferase ATP assay

Abstract A new method was developed to estimate the survival rate of cells in the shoot apex using an ATP assay with luciferin–luciferase. Shoot apices isolated as uniformly in size as possible from germinated pea seedlings were frozen after treatment with cryoprotectant to temperatures as low as −50°C and then transferred to liquid nitrogen temperature (−196°C) by a two-step freezing method. The frozen-thawed or unfrozen control shoot apices were washed with fresh medium and an individual shoot apex was suspended in a small volume of distilled water and then heated for 1.5 min on a boiling water bath. To release ATP more completely, the heated shoot apex was rapidly frozen in liquid nitrogen and thawed at room temperature. The content of released ATP was determined with the luciferin-luciferase assay. The percentage of cells in the pea shoot apex which survived freezing and thawing under present conditions was found to be 10% as estimated by this method.