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

在这项研究中，我们报道了一种超声波渗透的植物细胞培养系统，以释放积累在细胞中的有用的次生代谢物。栽培草莓花青素的生产。对Shikinari细胞进行了研究。(1)草莓细胞培养产生花青素的影响因素。分批培养草莓细胞花青素形成的时间进程主要受溶解氧浓度、平衡和氮源浓度的影响。(2)超声渗透条件。研究了频率约为1 MHz的超声对液泡定位色素花青素释放及细胞活力的影响。色素主要通过超声诱导自由基的化学作用从细胞中释放出来，而超声空化的物理作用则使细胞活力下降。通过对超声血管结构的改进，提高了超声血管的活力和花青素的释放。(3)超声渗透机理。细胞对超声的敏感性与质膜的流动性有关，并与使用探针分子测量荧光去极化有关。为了估计超声处理对单个细胞的影响，提出了一种利用图像分析定量测定细胞内储存色素的新方法。这些结果在细胞内积累代谢物的植物细胞培养系统中具有广泛的应用价值。

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.