Research on Plant Cell Culture with Ultrasonic Permeabilization for the Release of Intracellular Product

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In this study, we reported a plant-cell culture system with ultrasonic permeabilization for release of useful secondary metabolite accumulated in the cells. Anthocyanin production by cultured strawberry (Fragaria ananassa cv. Shikinari) cells was investigated.(1)Factors affecting anthocyanin production by cultured strawberry cells.Time courses of anthocyanin formation by the cultured strawberry cells in batch cultivation were affected mainly by the dissolved oxygen concentration, the balance and the concentration of nitrogen sources.(2)Conditions for ultrasonic permeabilization.We investigated the effect of ultrasonication with the frequency of ca. 1 MHz on the release of vacuole-located pigment anthocyanin and the cell viability. The pigment was released from the cells mainly by the chemical action of radicals induced by ultrasonication, whereas the cell viability decreased with the physical action by ultrasonic cavitation. Configuration of sonication vessel was modified to increase the viability and the anthocyanin release.(3)Mechanism of ultrasonic permeabilization.The sensitivity of cells to the ultrasonication was indicated to relate a dependency on the fluidity of plasma membrane with the measurement of fluorescence depolarization using a probe molecule. For the estimation of effect of ultrasonic treatment on individual cells, a novel method using image analysis was developed to determine the intracellularly stored pigment quantitatively.These results have broad range of application on plant-cell culture system for the production of intracellularly accumulated metabolites.