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Methodology of freeze-substitution for study of freezing injury in plant cells by transmission electron microscopy
528
ABSTRACTS,
25th ANNUAL
by a grunt from the United States Department of Health and Human Services, National Institute of General Medical Sciences Grant 1 ROI GM37575-01). 56. An Ultrastructural Investigation into FreezingInduced Dehydration of Chlamydomonas reinhardtii CW15-k. S. ROBERTSAND B. W. W. GROUT (Cell Systems, Ltd., Cambridge Science Park, Cambridge, United Kingdom). Extracellular freezing in samples of C. reinhardtii CW15 + results in cell shrinkage accompanied by distortion. On thawing, cell death is evident as massive swelling followed by deflation or detachment of the surrounding membrane. This phenomenon has been investigated by electron microscopy using both chemical and freeze fixation techniques. The results of TEM indicate that membrane layering and fusion may be taking place within the cell during the freeze-thaw cycle. This would break down the compartmentalization of the cell and could provide extra membrane material at the cell surface to take part in the blebbing response. 57. Methodology of Freeze-Substitution for Study of Freezing Injury in Plant Cells by Transmission Electron Microscopy. D. M. R. HARVEY AND K. PIHAKASKI (Department of Biology, University of Turku, Turku, Finland). Examination of the structural bases of cold acclimation and freezing injury in plants requires examination of slowly frozen tissues prepared for electron microscopy within thawing or desiccation. Study of osmotically manipulated isolated protoplasts provides information only on dehydration effects in cells lacking walls. Freeze-substitution combined with osmium tetroxide fixation can produce transmission electron microscope sections in which the major organelles and membranes are clearly distinguishable. Six centimeter long leaf pieces for Secale cereale plants were placed on a moist filter paper on a Cu plate, cooled to - 3”C, and ice nucleated with slivers of solid CO,. They were then slowly cooled over 1 hr to the temperature of 0 or 50% survival (LT,,). Segments ~1 mm long were cut and loaded into Cu mesh baskets maintained at the correct temperature in wells in the Cu plate. The plate containing the baskets was brought under the specimen arm of an electromagnetically controlled plunge freezer (Kryovak Oy, Finland). The basket was held, the Cu plate was withdrawn, and the specimens were plunged at 3 m s -’ into 8% methylcyclohexane in 2methylbutane cooled to - 170°C by liquid nitrogen. The segments were freeze-substituted in anhydrous acetone containing 3.3% 0~0, for 2 days at - 80°C and 5 days at -20°C rinsed twice (8 and 16 hr) at -20°C with anhydrous acetone, and infiltrated with ERL 4206 and Spur-r’s resin, starting at - 20°C. Preliminary ex-
MEETING
periments showed that this tissue was not significantly fixed within 7 days by 0~0, at - 80 or - 50°C. The final procedure permits removal of ice by acetone before the temperature is raised for fixation, minimizing expansion of small ice crystals formed during rapid freezing and comparability of preparation for specimens slowly frozen to different temperatures. Many cells, particularly in the interior of tissue segments, showed visible ice crystal damage arising from rapid freezing: ice crystal boundaries about 60 nm apart were visible in both slowly frozen tissues and controls maintained at room temperatures. However, in areas which were free of such damage, differences in cell ultrastructure between slowly frozen cells and wellstructured control cells were observed. 58. Cryopreservation of Azolla Sporocarp, Seedling, andShoot Apex. W. D. SUN, H. Y. ZHU, T. C. HUA, AND P. J. Lu* (Cryobiological Engineering Laboratory, Shanghai Institute of Mechanical Engineering, Shanghai; and *National Azolla Resource Center Fuzhou, Fuijang, China). Azolla, a kind of aquatic fern, is a superior green manure and fodder. It attracts extraordinary attention of many biologists by its simbiotic alga, Anabaena azollue Strasburgue, in its leaf cavities, and is regarded as perfect material for research about photosynthesis, nitrogen fixation, and nitrogen emanation. The following results are obtained from our experimental studies. (1) Measurement of osmolalities of Azolla juices: The osmolalities of imbricata, pinnata, jiliculoides, caroliniarc, and microphylla are 127.7, 137.7, 155.3, 112.7, 118.0 mosm, respectively. (2) Toxic effect of DMSO on Azollafiliculoides: Put some isolated shoot apieces of Azolla filiculoides in DMSO solution at a concentration of 5, 10, and IS%, respectively, and keep them in solution for several hours; then move them into improved Yoshida culture medium for 5 days and check the survival. When the concentration of DMSO is 10% and the pretreated times are 1, 3, and 6 hr, the survival rates of Azolla filiculoides are 87.5,75, and 50%, respectively. (3) Dry Azollu sporocarps cooled rapidly to - 196°C can resuscitate with high survival after thawing. Moreover, the sporocarps cryopreserved could germinate 11% higher and 2 days earlier than the unfrozen ones. The survival of Azolla seedling cooled to - 30°C is 65-85%, but that of shoot apex is only 5.0%. 59. Electric Potential Variation in Cryoprotective Agent during Solidt$cation M. D. XIA, W. D. SUN, AND T. C. HUA* (Department of Physics and *Cyrobiological Engineering Laboratory, Shanghai Institute of Mechanical Engineering, Shanghai, China).
ABSTRACTS,
25th ANNUAL
by a grunt from the United States Department of Health and Human Services, National Institute of General Medical Sciences Grant 1 ROI GM37575-01). 56. An Ultrastructural Investigation into FreezingInduced Dehydration of Chlamydomonas reinhardtii CW15-k. S. ROBERTSAND B. W. W. GROUT (Cell Systems, Ltd., Cambridge Science Park, Cambridge, United Kingdom). Extracellular freezing in samples of C. reinhardtii CW15 + results in cell shrinkage accompanied by distortion. On thawing, cell death is evident as massive swelling followed by deflation or detachment of the surrounding membrane. This phenomenon has been investigated by electron microscopy using both chemical and freeze fixation techniques. The results of TEM indicate that membrane layering and fusion may be taking place within the cell during the freeze-thaw cycle. This would break down the compartmentalization of the cell and could provide extra membrane material at the cell surface to take part in the blebbing response. 57. Methodology of Freeze-Substitution for Study of Freezing Injury in Plant Cells by Transmission Electron Microscopy. D. M. R. HARVEY AND K. PIHAKASKI (Department of Biology, University of Turku, Turku, Finland). Examination of the structural bases of cold acclimation and freezing injury in plants requires examination of slowly frozen tissues prepared for electron microscopy within thawing or desiccation. Study of osmotically manipulated isolated protoplasts provides information only on dehydration effects in cells lacking walls. Freeze-substitution combined with osmium tetroxide fixation can produce transmission electron microscope sections in which the major organelles and membranes are clearly distinguishable. Six centimeter long leaf pieces for Secale cereale plants were placed on a moist filter paper on a Cu plate, cooled to - 3”C, and ice nucleated with slivers of solid CO,. They were then slowly cooled over 1 hr to the temperature of 0 or 50% survival (LT,,). Segments ~1 mm long were cut and loaded into Cu mesh baskets maintained at the correct temperature in wells in the Cu plate. The plate containing the baskets was brought under the specimen arm of an electromagnetically controlled plunge freezer (Kryovak Oy, Finland). The basket was held, the Cu plate was withdrawn, and the specimens were plunged at 3 m s -’ into 8% methylcyclohexane in 2methylbutane cooled to - 170°C by liquid nitrogen. The segments were freeze-substituted in anhydrous acetone containing 3.3% 0~0, for 2 days at - 80°C and 5 days at -20°C rinsed twice (8 and 16 hr) at -20°C with anhydrous acetone, and infiltrated with ERL 4206 and Spur-r’s resin, starting at - 20°C. Preliminary ex-
MEETING
periments showed that this tissue was not significantly fixed within 7 days by 0~0, at - 80 or - 50°C. The final procedure permits removal of ice by acetone before the temperature is raised for fixation, minimizing expansion of small ice crystals formed during rapid freezing and comparability of preparation for specimens slowly frozen to different temperatures. Many cells, particularly in the interior of tissue segments, showed visible ice crystal damage arising from rapid freezing: ice crystal boundaries about 60 nm apart were visible in both slowly frozen tissues and controls maintained at room temperatures. However, in areas which were free of such damage, differences in cell ultrastructure between slowly frozen cells and wellstructured control cells were observed. 58. Cryopreservation of Azolla Sporocarp, Seedling, andShoot Apex. W. D. SUN, H. Y. ZHU, T. C. HUA, AND P. J. Lu* (Cryobiological Engineering Laboratory, Shanghai Institute of Mechanical Engineering, Shanghai; and *National Azolla Resource Center Fuzhou, Fuijang, China). Azolla, a kind of aquatic fern, is a superior green manure and fodder. It attracts extraordinary attention of many biologists by its simbiotic alga, Anabaena azollue Strasburgue, in its leaf cavities, and is regarded as perfect material for research about photosynthesis, nitrogen fixation, and nitrogen emanation. The following results are obtained from our experimental studies. (1) Measurement of osmolalities of Azolla juices: The osmolalities of imbricata, pinnata, jiliculoides, caroliniarc, and microphylla are 127.7, 137.7, 155.3, 112.7, 118.0 mosm, respectively. (2) Toxic effect of DMSO on Azollafiliculoides: Put some isolated shoot apieces of Azolla filiculoides in DMSO solution at a concentration of 5, 10, and IS%, respectively, and keep them in solution for several hours; then move them into improved Yoshida culture medium for 5 days and check the survival. When the concentration of DMSO is 10% and the pretreated times are 1, 3, and 6 hr, the survival rates of Azolla filiculoides are 87.5,75, and 50%, respectively. (3) Dry Azollu sporocarps cooled rapidly to - 196°C can resuscitate with high survival after thawing. Moreover, the sporocarps cryopreserved could germinate 11% higher and 2 days earlier than the unfrozen ones. The survival of Azolla seedling cooled to - 30°C is 65-85%, but that of shoot apex is only 5.0%. 59. Electric Potential Variation in Cryoprotective Agent during Solidt$cation M. D. XIA, W. D. SUN, AND T. C. HUA* (Department of Physics and *Cyrobiological Engineering Laboratory, Shanghai Institute of Mechanical Engineering, Shanghai, China).