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Patterns of ice nucleation and lethal injury in floral organs of overwintered cauliflower
534
ABSTRACTS, 25th ANNUAL
exposure to 25% (v/v) (3 . 4 M) propane-l ,Zdiol for 20 min at 0 or - 5°C was consistently tolerated only when 2.5% (w/v) chondroitin sulphate was included in the Ringer solution. Exposure to 30% (v/v) (4 . 1 M) propane-l ,2-diol was harmful both at - 5 and - 10°C. On the other hand, endothelial function was retained after exposure to 25% (v/v) (3 . 4 M) glycerol for 20 min at -5°C. Exposure to 30% (v/v) (4 ~1M> glycerol for 10 min at - 10°C was also tolerated. However, exposure to 35% (v/v) (4 . 8 M) glycerol resulted in a poor endothelium with many large and misshapen cells. Neither propane-l ,2-diol nor glycerol alone are tolerated at sufficiently high concentrations to permit cornea vitrification. However, since a mixture of 20% (v/v) propane-l ,Zdiol plus 20% (v/v) glycerol will apparently vitrify, a combination of these two cryoprotectants could provide a suitable solution for corneal vitrification. SESSION X PLANT CELLS AND ULTRASTRUCTURAL INVESTIGATIONS 75. A Consecutive Cryo LmlCryo Sem Technique and Its Use in the Study of Freezing Injury in Chlamydomonas reinhardtii CWI5 + . S.
ROBERTS,*G. J. Moaars,t G. CoursoNt and B. GROUT* (*Plymouth Polytechnic, Plymouth, and tCel1 Systems, Ltd., Cambridge Science Park, Cambridge, United Kingdom). A technique has been developed whereby samples can be observed during cooling on a cryo light microscope stage, then transferred to the cold stage of a scanning electron microscope for further examination. Ultrastructural data from both LM and SEM can be obtained for any chosen stage in the freeze-thaw cycle by fixing the sample within a specially designed, transferable, specimen holder. The technique has been used reinto examine the response of Chlamydomonas hardtii CWl5 + to a slow cooling treatment. Light microscopy alone shows that extracellular freezing results in the formation of radially extending membrane extrusions, as described by Steponkus (1) during freezing-induced shrinkage of acclimated higher plant protoplasts. The combined LM/SEM technique reveals that in this case the formation of membrane extrusions may be an artifact. During thawing, cells are seen by LM to swell and to form large membranebound blisters. Cryo SEM allows observation of that membrane and has shown the surface texture to ditfer from that of controls or shrunken cell membranes. REFERENCE 1. Steponkus, P. L. Cryobiology
20, 448465 (1983).
76. Plant Cell Suspensions Resistant to and Recalcitrant to Cryopreservation Procedures. F. VAN
MEETING IREN,* J. SCHRIPSEMA,*E. W. M. SCHRIJNEMAKERS, R. VAN DER HEIJDEN,* R. VERPOORTE,*AND K. R. LIBBENGA (Departments of Plant Molecular Biology and *Pharmacognosy, Leiden University, Leiden, The Netherlands).
In many laboratories, plant cell suspension cultures are studied with respect to, e.g., production of secondary metabolites, transport phenomena, signal transduction, regeneration, and genetic manipulation. Such cultures are inheritantly unstable, maintenance is costly, and they may be lost after human or technical failures. Therefore, there is a need for stable storage procedures. Since the early eighties, several routine procedures for cryopreservation of cell suspensions have been published. Because these appear successful for somewhat more than half of the cultures tested in several laboratories (personal communications), including our own, further research is urgent. We sucessfully and reproducibly cryopreserved cultures of the following species: periwinkle, tobacco, petunia, rice, and Tabernaemontana divaricata. Growth and respiration parameters and alkaloid production patterns before and after cryopreservation were compared for one periwinkle culture and T. divaricata. They appeared essentially unchanged. Several other cultures, including Cinchona and another T. divaricata, did neither recover from the same procedures nor from procedures with several modifications of pretreatment, thawing, and recovery conditions. Resistant and recalcitrant cultures (especially the two T. divaricata lines) were compared with respect to a.o. uptake of cryoprotectants during pretreatment and loss of cell contents after thawing by means of NMR spectroscopy and other techniques. 77. Patterns Floral
of Ice Nucleation and Lethal Injury in Organs of Overwintered Cauliflower.
G. G. WHITE, B. W. W. GROUT, AND M. P. FULLER (Crop Improvement Group, Plymouth Polytechnic, Plymouth, United Kingdom). As part of a study to improve frost resistance in the overwintered cauliflower the mechanisms underlying freezing injury in the floral tissues have been studied. Within a single plant spontaneous ice nucleation will occur at multiple sites, at temperatures ranging between -2 and 12°C. Surface tissues, where the cells are small, less vacuolate, and bulk vascular solutions are absent, undercool more than vascular branches within the floral head. These branches are where the fast nucleations are sited and rapid ice travel can be monitored from these areas proceeding toward, and subsequently invading, surface tissues. Ice may travel both up and down in vascular tisue and in all available directions at branchpoints. Osmometry of vascular sap indicates a maximum freezing point depression of
ABSTRACTS,
25th ANNUAL
- 2°C within a single plant and a large seed population. The distribution pattern of primary ice nucleation within a single plant appears to be completely random and has no correlation with position or developmental stage within the floral tissues. Much of the material is, by maturity, suffering from nonsymptomatic infections of a range of Pseudomonai bacteria. The ice nucleation active species Pseudomonas syringiae is absent, and none of the species isolated has shown nucleation activity when tested in vitro. The pattern of nucleation is, therefore, unpredictable and apparently random, not being related to any obvious characteristic of the floral tissues. This frustrates attempts at selective screening and subsequent breeding for improved resistance by freezing avoidance in cooled floral tissues. 78. Localization of Fluid Microdomains in Frozen Populus Using Mechanical Analysis and Electron Microscopy. A. HIRSH, T. T. BENT, E.
ERBE, AND R. J. WILLIAMS (Jerome Holland Laboratory, American Red Cross, Rockville, Maryland). Winter-hardened Populus balsamifera variety virginiana can withstand freezing to liquid nitrogen temperatures at rates less than about 0.7”Clmin. Lethal intracellular freezing occurs at higher rates. We performed dynamic mechanical analysis (DMA) of twigs cooled at rates above and below the critical rate. Correlating the results with freeze+tch electron micrographs allowed us to postulate that the intracellular liquid-liquid phase separations implied by the DMA data took place largely in the cytoplasmic compartment. Studies of model solutions using differential scanning calorimetry allowed us to further characterize the fluid microdomains as being protein rich or sugar rich. This appears to be an example of spinodal decomposition in a living system. It is likely that these plants have evolved such that spinodal decomposition during slow freezing leads to minimal injury. Mechanisms might include localization of sugar-rich domains next to cellular membranes and stabilization of key proteins by control of subdomain composition. 79. Ultrastructure of Slowly Frozen Nonacclimated Rye Leaf Cells. K. PIHAKASKI, D. M. R. HARVEY, AND U. M. SUORANTA(Department
of Biology, University of Turku, Turku, Finland). Freeze substitution and quick freezing allow ultrastructure to be studied at subzero temperatures without prior melting or artificial dehydration, which is important when freezing injuries are to be studied. Seeds of winter rye (Secale cereale C.V. Voima) were germinated in vermiculite and grown in a greenhouse at 25°C with a photoperiod of 16 hr light for 10 days.
MEETING
535
Pieces of leaves were cooled slowly (ca. l”C/min) to -4°C (LQ or to - 12°C (LT,), equilibrated for 1 hr, and quickly frozen by plunging to 8% methylcyclohexane in 2-methylbutane at - 170°C. The leaves from room temperature served as controls. The specimens were then freeze substituted with OsO,-acetone, rinsed in acetone, infiltrated, and embedded in Spurr’s low viscosity resin. It was more difficult to preserve ultrastructure in nonacclimated leaf tissues than in acclimated ones, possibly because the acclimated cells contain more solutes acting as natural cryoprotectants. There were large variations from cell to cell even in the same piece of tissue. Ice crystals of different sizes (from 60 to approx 150 nm) were observed, probably arising from fast freezing. The cells which had the largest ice crystals were not contracted at all. In the best preserved controls the plasmalemma was firmly attached to the cell wall, and the tonoplast was also intact. The mitochondrial cristae were often more narrowly spaced than in the conventionally fixed specimens. Dictyosomes and endoplasmic reticulum strands had conventional appearances. Chloroplasts were generally the best preserved organelles. At - 12”C, the cells were severely distorted. Most of the cells, consisting of electron-dense cytoplasm, were contracted. Occasionally the cytoplasm had mostly pulled away from the cell wall. Chloroplasts were still recognizable and the lamellar system could be easily observed. Plastid and cytoplasmic ribosomes were in tight groups, the rest of the cytoplasm was poorly discernible. At -4°C there was a wide variety of cells with different kinds of cell contents. A portion of these were well structured. Their cytoplasm was slightly shrunken but the vacuole, small vesicles in abundance, and different organelles were visible. In some cells the plasmalemma was loosened from the cell wall and partly broken; cytoplasm was totally destroyed and had a granular appearance, also containing small membrane-bound vesicles and membrane fragments. 80. Ultrastructure of Slowly Frozen Cold-Acclimated Rye Leaf Cells. D. M. R. HARVEY, K. PIHAKASKI, AND U. M. SUORANTA (Depart-
ment of Biology, University of Turku, Turku, Finland). Plant of Secale cereale c.v. Voima were grown for I days at 25°C and were cold acclimated by further growth for 4 weeks at 5°C. LT,,, determined by measuring efflux of ninhydrin-positive substances from leaf pieces, was -8°C; there was 0% survival from temperatures of below - 15°C. Six-centimeter-long leaf pieces were ice nucleated at -3°C and were slowly frozen to - 8 or - 16°C. Specimens from these pieces or from plants maintained at 5°C were prepared for transmission electron microscopy by rapid freezing to - 170°Cfollowed by freeze-substitution-fixation using acetone-OsO,. In sections from plants maintained
ABSTRACTS, 25th ANNUAL
exposure to 25% (v/v) (3 . 4 M) propane-l ,Zdiol for 20 min at 0 or - 5°C was consistently tolerated only when 2.5% (w/v) chondroitin sulphate was included in the Ringer solution. Exposure to 30% (v/v) (4 . 1 M) propane-l ,2-diol was harmful both at - 5 and - 10°C. On the other hand, endothelial function was retained after exposure to 25% (v/v) (3 . 4 M) glycerol for 20 min at -5°C. Exposure to 30% (v/v) (4 ~1M> glycerol for 10 min at - 10°C was also tolerated. However, exposure to 35% (v/v) (4 . 8 M) glycerol resulted in a poor endothelium with many large and misshapen cells. Neither propane-l ,2-diol nor glycerol alone are tolerated at sufficiently high concentrations to permit cornea vitrification. However, since a mixture of 20% (v/v) propane-l ,Zdiol plus 20% (v/v) glycerol will apparently vitrify, a combination of these two cryoprotectants could provide a suitable solution for corneal vitrification. SESSION X PLANT CELLS AND ULTRASTRUCTURAL INVESTIGATIONS 75. A Consecutive Cryo LmlCryo Sem Technique and Its Use in the Study of Freezing Injury in Chlamydomonas reinhardtii CWI5 + . S.
ROBERTS,*G. J. Moaars,t G. CoursoNt and B. GROUT* (*Plymouth Polytechnic, Plymouth, and tCel1 Systems, Ltd., Cambridge Science Park, Cambridge, United Kingdom). A technique has been developed whereby samples can be observed during cooling on a cryo light microscope stage, then transferred to the cold stage of a scanning electron microscope for further examination. Ultrastructural data from both LM and SEM can be obtained for any chosen stage in the freeze-thaw cycle by fixing the sample within a specially designed, transferable, specimen holder. The technique has been used reinto examine the response of Chlamydomonas hardtii CWl5 + to a slow cooling treatment. Light microscopy alone shows that extracellular freezing results in the formation of radially extending membrane extrusions, as described by Steponkus (1) during freezing-induced shrinkage of acclimated higher plant protoplasts. The combined LM/SEM technique reveals that in this case the formation of membrane extrusions may be an artifact. During thawing, cells are seen by LM to swell and to form large membranebound blisters. Cryo SEM allows observation of that membrane and has shown the surface texture to ditfer from that of controls or shrunken cell membranes. REFERENCE 1. Steponkus, P. L. Cryobiology
20, 448465 (1983).
76. Plant Cell Suspensions Resistant to and Recalcitrant to Cryopreservation Procedures. F. VAN
MEETING IREN,* J. SCHRIPSEMA,*E. W. M. SCHRIJNEMAKERS, R. VAN DER HEIJDEN,* R. VERPOORTE,*AND K. R. LIBBENGA (Departments of Plant Molecular Biology and *Pharmacognosy, Leiden University, Leiden, The Netherlands).
In many laboratories, plant cell suspension cultures are studied with respect to, e.g., production of secondary metabolites, transport phenomena, signal transduction, regeneration, and genetic manipulation. Such cultures are inheritantly unstable, maintenance is costly, and they may be lost after human or technical failures. Therefore, there is a need for stable storage procedures. Since the early eighties, several routine procedures for cryopreservation of cell suspensions have been published. Because these appear successful for somewhat more than half of the cultures tested in several laboratories (personal communications), including our own, further research is urgent. We sucessfully and reproducibly cryopreserved cultures of the following species: periwinkle, tobacco, petunia, rice, and Tabernaemontana divaricata. Growth and respiration parameters and alkaloid production patterns before and after cryopreservation were compared for one periwinkle culture and T. divaricata. They appeared essentially unchanged. Several other cultures, including Cinchona and another T. divaricata, did neither recover from the same procedures nor from procedures with several modifications of pretreatment, thawing, and recovery conditions. Resistant and recalcitrant cultures (especially the two T. divaricata lines) were compared with respect to a.o. uptake of cryoprotectants during pretreatment and loss of cell contents after thawing by means of NMR spectroscopy and other techniques. 77. Patterns Floral
of Ice Nucleation and Lethal Injury in Organs of Overwintered Cauliflower.
G. G. WHITE, B. W. W. GROUT, AND M. P. FULLER (Crop Improvement Group, Plymouth Polytechnic, Plymouth, United Kingdom). As part of a study to improve frost resistance in the overwintered cauliflower the mechanisms underlying freezing injury in the floral tissues have been studied. Within a single plant spontaneous ice nucleation will occur at multiple sites, at temperatures ranging between -2 and 12°C. Surface tissues, where the cells are small, less vacuolate, and bulk vascular solutions are absent, undercool more than vascular branches within the floral head. These branches are where the fast nucleations are sited and rapid ice travel can be monitored from these areas proceeding toward, and subsequently invading, surface tissues. Ice may travel both up and down in vascular tisue and in all available directions at branchpoints. Osmometry of vascular sap indicates a maximum freezing point depression of
ABSTRACTS,
25th ANNUAL
- 2°C within a single plant and a large seed population. The distribution pattern of primary ice nucleation within a single plant appears to be completely random and has no correlation with position or developmental stage within the floral tissues. Much of the material is, by maturity, suffering from nonsymptomatic infections of a range of Pseudomonai bacteria. The ice nucleation active species Pseudomonas syringiae is absent, and none of the species isolated has shown nucleation activity when tested in vitro. The pattern of nucleation is, therefore, unpredictable and apparently random, not being related to any obvious characteristic of the floral tissues. This frustrates attempts at selective screening and subsequent breeding for improved resistance by freezing avoidance in cooled floral tissues. 78. Localization of Fluid Microdomains in Frozen Populus Using Mechanical Analysis and Electron Microscopy. A. HIRSH, T. T. BENT, E.
ERBE, AND R. J. WILLIAMS (Jerome Holland Laboratory, American Red Cross, Rockville, Maryland). Winter-hardened Populus balsamifera variety virginiana can withstand freezing to liquid nitrogen temperatures at rates less than about 0.7”Clmin. Lethal intracellular freezing occurs at higher rates. We performed dynamic mechanical analysis (DMA) of twigs cooled at rates above and below the critical rate. Correlating the results with freeze+tch electron micrographs allowed us to postulate that the intracellular liquid-liquid phase separations implied by the DMA data took place largely in the cytoplasmic compartment. Studies of model solutions using differential scanning calorimetry allowed us to further characterize the fluid microdomains as being protein rich or sugar rich. This appears to be an example of spinodal decomposition in a living system. It is likely that these plants have evolved such that spinodal decomposition during slow freezing leads to minimal injury. Mechanisms might include localization of sugar-rich domains next to cellular membranes and stabilization of key proteins by control of subdomain composition. 79. Ultrastructure of Slowly Frozen Nonacclimated Rye Leaf Cells. K. PIHAKASKI, D. M. R. HARVEY, AND U. M. SUORANTA(Department
of Biology, University of Turku, Turku, Finland). Freeze substitution and quick freezing allow ultrastructure to be studied at subzero temperatures without prior melting or artificial dehydration, which is important when freezing injuries are to be studied. Seeds of winter rye (Secale cereale C.V. Voima) were germinated in vermiculite and grown in a greenhouse at 25°C with a photoperiod of 16 hr light for 10 days.
MEETING
535
Pieces of leaves were cooled slowly (ca. l”C/min) to -4°C (LQ or to - 12°C (LT,), equilibrated for 1 hr, and quickly frozen by plunging to 8% methylcyclohexane in 2-methylbutane at - 170°C. The leaves from room temperature served as controls. The specimens were then freeze substituted with OsO,-acetone, rinsed in acetone, infiltrated, and embedded in Spurr’s low viscosity resin. It was more difficult to preserve ultrastructure in nonacclimated leaf tissues than in acclimated ones, possibly because the acclimated cells contain more solutes acting as natural cryoprotectants. There were large variations from cell to cell even in the same piece of tissue. Ice crystals of different sizes (from 60 to approx 150 nm) were observed, probably arising from fast freezing. The cells which had the largest ice crystals were not contracted at all. In the best preserved controls the plasmalemma was firmly attached to the cell wall, and the tonoplast was also intact. The mitochondrial cristae were often more narrowly spaced than in the conventionally fixed specimens. Dictyosomes and endoplasmic reticulum strands had conventional appearances. Chloroplasts were generally the best preserved organelles. At - 12”C, the cells were severely distorted. Most of the cells, consisting of electron-dense cytoplasm, were contracted. Occasionally the cytoplasm had mostly pulled away from the cell wall. Chloroplasts were still recognizable and the lamellar system could be easily observed. Plastid and cytoplasmic ribosomes were in tight groups, the rest of the cytoplasm was poorly discernible. At -4°C there was a wide variety of cells with different kinds of cell contents. A portion of these were well structured. Their cytoplasm was slightly shrunken but the vacuole, small vesicles in abundance, and different organelles were visible. In some cells the plasmalemma was loosened from the cell wall and partly broken; cytoplasm was totally destroyed and had a granular appearance, also containing small membrane-bound vesicles and membrane fragments. 80. Ultrastructure of Slowly Frozen Cold-Acclimated Rye Leaf Cells. D. M. R. HARVEY, K. PIHAKASKI, AND U. M. SUORANTA (Depart-
ment of Biology, University of Turku, Turku, Finland). Plant of Secale cereale c.v. Voima were grown for I days at 25°C and were cold acclimated by further growth for 4 weeks at 5°C. LT,,, determined by measuring efflux of ninhydrin-positive substances from leaf pieces, was -8°C; there was 0% survival from temperatures of below - 15°C. Six-centimeter-long leaf pieces were ice nucleated at -3°C and were slowly frozen to - 8 or - 16°C. Specimens from these pieces or from plants maintained at 5°C were prepared for transmission electron microscopy by rapid freezing to - 170°Cfollowed by freeze-substitution-fixation using acetone-OsO,. In sections from plants maintained