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

植物细胞超声透化释放细胞内产物的研究

基本信息

批准号：
04453118
负责人：
FURUSAKI Shintaro
金额：
$ 4.54万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04453118/
关键词：
Cultured Plant Cell Bioreactor Permeabilization Anthocyanin Ultrasound Strawberry Image Analysis

项目摘要

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.

本文报道了一种利用超声波透化作用释放植物细胞中积累的有用次生代谢产物的培养系统。以草莓（Fragaria ananassa cv. Shikinari）细胞。（1）影响草莓培养细胞产生花青苷的因素：草莓培养细胞在分批培养中花青苷形成的时间进程主要受溶氧浓度、氮源平衡和氮源浓度的影响。（2）超声波渗透条件：研究了频率约为100Hz，超声波频率约为200Hz。1 MHz对液泡色素花青素的释放和细胞活力的影响。超声波作用引起的自由基化学作用主要导致细胞色素的释放，而超声波空化引起的物理作用则导致细胞活力下降。改变超声处理容器的结构以增加活力和花青素苷的释放。（3）超声波透化机理：细胞对超声波的敏感性与细胞膜流动性的依赖性和荧光去极化的检测有关。为了评价超声波处理对单个细胞的影响，本文发展了一种利用图像分析技术定量测定细胞内色素的新方法，该结果在植物细胞培养系统中产生细胞内积累的代谢产物方面具有广泛的应用前景。