The Fine Structure and Function of Associations of the Endoplasmic Reticulum, Chloroplasts and Plasmalemma in Cultured Protoplasts of Skimmia japonica Thunb

Works with isolated protoplasts have attracted much attention as ideal experi mental materials for the investigation of many problems in physiology, genetics and biochemistry. Since the mesophyll tissue provides an excellent source of plant protoplasts (Takebe et al. 1968, Otsuki and Takebe 1969) several workers have studied the ultrastructure of the cell wall regeneration around isolated protoplasts of mesophyll cells (reviewed by Willison 1976). Relatively little has been reported about the ultrastructural changes of their organelles, especially their chloroplasts occurring during the preparation of protoplasts and their subsequent culture. At the light microscope level their histological structure was described by Takebe et al. (1968), Nagata and Takebe (1970) and Gigot et al. (1975) for tobacco protoplasts. In a previous study Gigot et al. (1975) described some ultrastructural aspects of chloroplasts during tobacco protoplast preparation and culture. The purpose of the present investigation was to report more fully on the whole sequence of changes occurring in the chloroplasts of Skimmia japonica during proto plast preparation and culture. Furthermore close contacts are shown between the chloroplasts and the rough endoplasmic reticulum, also between rough endo plasmic reticulum and the plasmalemma. Since thin sectioning alone did not result in sufficient ultrastructural evidence we applied the freeze-etch method in order to supplement the electron-microscopic thin section technique and to obtain further information about the fine structure of the organelles. Furthermore artefact struc tures by this method are relatively scarce in comparison to those of the thin section technique, because the ultrastructure can be made visible in the still-latent living condition.