Freeze-substitution transmission electron microscopy of gentian shoot tips cryopreserved at ultra low temperatures

• Published in: Plant Biotechnology Vol. 35; no. 4; pp. 335 - 340
• Main Authors: Tanaka, Daisuke, Niino, Takao, Fujikawa, Seizo, Uemura, Matsuo
• Format: Journal Article
• Language: English; Japanese
• Published: Japan Japanese Society for Plant Cell and Molecular Biology 25.12.2018; Japan Science and Technology Agency
• Subjects: Cryopreservation; Crystals; Cytoplasm; Damage prevention; freeze substitution; Ice; Ice crystals; Ice formation; Leaves; Liquid nitrogen; Low temperature; Nitrogen; Original Paper; Plant growth; Plasmolysis; shoot tips; Slush; Substitutes; Tips; Transmission electron microscopy; Ultrastructure; Vitrification; transmission electron microscopy; freeze substitution; ultrastructure; shoot tips; cryopreservation
• ISSN: 1342-4580; 1347-6114
• DOI: 10.5511/plantbiotechnology.18.0718a

Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells. In addition, the cells of PVS2-treated shoot tips exhibited considerable plasmolysis and formation of small vesicles in cytoplasm. These results clearly demonstrate that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot tips.