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Changes in the resistance of protoplasts and regenerated cells to freezing and osmotic dehydration during protoplast culture of Marchantia polymorpha
ABSTRACTS, 25th ANNUAL lotransplantation, so that NHEK cells may be used as a model system for optimizing cryopreservation parameters for human skin. 26. Changes in the Resistance of Protoplasts and Regenerated Cells to Freezing and Osmotic Dehydration during Protoplast Culture of Marchantia polymorpha. Y. SUGAWARA, AND M.
TAKEUCHI (Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan). Resistance to freezing and osmotic dehydration of protoplasts and regenerated cells was studied in protoplast culture of Marchantia polymorpha in relation to cell wall regeneration and cell division. Protoplasts isolated from callus tissues were cultured on modified Murashige and Skoog’s medium containing 0.23 M mannitol [Z. Pflanzenphysiol. 109, 275(1983)]. Regeneration of the cell wall was initiated immediately in the protoplasts after the beginning of protoplast culture, and more than 90% of the protoplasts formed new, thin cell walls within 24 hr of culture in either light or dark conditions. Division of regenerated cells was observed some time after 48 hr among cells cultured in the light but not in the dark. The protoplasts and regenerated cells were suspended in culture media and frozen to various temperatures at a cooling rate of 0.5-0.8”Cimin. The frozen samples were thawed in water at 40°C. Survival rates at - 40°C of callus cells and freshly isolated protoplasts were less than 10%. The survival rate at -40°C of regenerated cells increased with time of culture, reaching a maximum level (6& 70%) by 18 to 20 hr of culture in the light or dark, and decreased thereafter to the initial level. About 40% of the regenerated cells cultured for 20 hr could survive freezing to - 196°C and grow again normally after thawing. The freezing resistance thus gained was rapidly lost by treatment with cell wall digesting enzyme. A similar trend was observed in the resistance of regenerated cells to osmotic dehydration in balanced salt solution (BSS): The survival rate of protoplasts in 1.O M BSS was about 30%. Whereas, with regenerated cells, no decrease in survival rate was observed even at 2.5 M BSS. Little or no difference between protoplasts and regenerated cells was observed in the degree of volumetric reduction at each concentration of BSS. However, the shapes of regenerated cells observed changing during osmotic contraction in BSS were very different from those of protoplasts. Electron microscopic observations by the freeze-fracture method showed that formation of multilamellar structures under the P face of the plasma membrane is induced in protoplasts by osmotic dehydration in 2.0 M BSS but not in regenerated cells. These data suggest that during the early stage of protoplast culture, the regenerated cell wall and/or some structure of the protoplast, especially in the plasma membrane, play an
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important role in the development of resistance to freezing and osmotic dehydration. 27. A Comparative Study of the Changes in the Morphology of Hyphae during Freezing and Viability upon Thawing for 19 Species of Fungi. G. J.
MORRIS,D. SMITH, AND G. E. COULSON. The changes in morphology of 19 species of fungi during freezing were examined in relation to cooling rate and the presence and absence of glycerol using a cryomicroscope. The viability of fungal hyphae was determined after equivalent rates of cooling to - 196°C in the presence or absence of glycerol. All hyphomycetes, the ascomycete, Sordaria, the zygomycete, Mucor, and the basidiomycete, Schizophyllum, survived freezing and thawing in the absence of glycerol. Cryomicroscopy demonstrated that for these fungi the formation of intracellular ice at rapid rates of cooling was not lethal. Isolates from the Mastigomycotina and some Basidiomycetes required glycerol for survival. The morphological response of Phytophthora, Aschersonia alleyrodis and Volvariella volvacea differed from other isolates, with shrinkage occurring at all rates of cooling. 28. Survival and Stability of Microorganisms during Freeze-Drying K. A. MALIK (Deutsche Sam-
mlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, West Germany) Freeze-drying is a convenient method for the preservation and long-term storage of microorganisms; however, it is not suited to a variety of delicate microorganisms and even after 60 years of intensive development in this field, freeze-drying can still harm several microorganisms. During freeze-drying microorganisms have to undergo many stresses and DNA damage may result which could either cause total death of living cells or a paralysis by which the desired or useful qualities of the living cells could be lost or damaged. Several useful strains which are obtained as a result of gene manipulation, selective screening, mutations, and bear plasmids are mostly affected by freeze-drying and may lose their useful qualities after lyophilization. During freeze-drying gram-positive bacteria usually survive better than gram-negative bacteria. The age of a culture and the cell concentration to be freeze-dried could have a profound effect on the survival of microorganisms. A number of other characteristics may also influence the resistance of several species to freeze-drying as during several years of experimentation it has been observed that strains with spores and cysts are relatively resistant to freeze-drying whereas in particular large-celled gramnegative bacteria, PHB (poly-B-hydroxy butyric acid) filled bulky cells, spirillum-like large cells (due to their abnormal growth in the cell wall), and temporarily de-
TAKEUCHI (Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan). Resistance to freezing and osmotic dehydration of protoplasts and regenerated cells was studied in protoplast culture of Marchantia polymorpha in relation to cell wall regeneration and cell division. Protoplasts isolated from callus tissues were cultured on modified Murashige and Skoog’s medium containing 0.23 M mannitol [Z. Pflanzenphysiol. 109, 275(1983)]. Regeneration of the cell wall was initiated immediately in the protoplasts after the beginning of protoplast culture, and more than 90% of the protoplasts formed new, thin cell walls within 24 hr of culture in either light or dark conditions. Division of regenerated cells was observed some time after 48 hr among cells cultured in the light but not in the dark. The protoplasts and regenerated cells were suspended in culture media and frozen to various temperatures at a cooling rate of 0.5-0.8”Cimin. The frozen samples were thawed in water at 40°C. Survival rates at - 40°C of callus cells and freshly isolated protoplasts were less than 10%. The survival rate at -40°C of regenerated cells increased with time of culture, reaching a maximum level (6& 70%) by 18 to 20 hr of culture in the light or dark, and decreased thereafter to the initial level. About 40% of the regenerated cells cultured for 20 hr could survive freezing to - 196°C and grow again normally after thawing. The freezing resistance thus gained was rapidly lost by treatment with cell wall digesting enzyme. A similar trend was observed in the resistance of regenerated cells to osmotic dehydration in balanced salt solution (BSS): The survival rate of protoplasts in 1.O M BSS was about 30%. Whereas, with regenerated cells, no decrease in survival rate was observed even at 2.5 M BSS. Little or no difference between protoplasts and regenerated cells was observed in the degree of volumetric reduction at each concentration of BSS. However, the shapes of regenerated cells observed changing during osmotic contraction in BSS were very different from those of protoplasts. Electron microscopic observations by the freeze-fracture method showed that formation of multilamellar structures under the P face of the plasma membrane is induced in protoplasts by osmotic dehydration in 2.0 M BSS but not in regenerated cells. These data suggest that during the early stage of protoplast culture, the regenerated cell wall and/or some structure of the protoplast, especially in the plasma membrane, play an
MEETING
517
important role in the development of resistance to freezing and osmotic dehydration. 27. A Comparative Study of the Changes in the Morphology of Hyphae during Freezing and Viability upon Thawing for 19 Species of Fungi. G. J.
MORRIS,D. SMITH, AND G. E. COULSON. The changes in morphology of 19 species of fungi during freezing were examined in relation to cooling rate and the presence and absence of glycerol using a cryomicroscope. The viability of fungal hyphae was determined after equivalent rates of cooling to - 196°C in the presence or absence of glycerol. All hyphomycetes, the ascomycete, Sordaria, the zygomycete, Mucor, and the basidiomycete, Schizophyllum, survived freezing and thawing in the absence of glycerol. Cryomicroscopy demonstrated that for these fungi the formation of intracellular ice at rapid rates of cooling was not lethal. Isolates from the Mastigomycotina and some Basidiomycetes required glycerol for survival. The morphological response of Phytophthora, Aschersonia alleyrodis and Volvariella volvacea differed from other isolates, with shrinkage occurring at all rates of cooling. 28. Survival and Stability of Microorganisms during Freeze-Drying K. A. MALIK (Deutsche Sam-
mlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, West Germany) Freeze-drying is a convenient method for the preservation and long-term storage of microorganisms; however, it is not suited to a variety of delicate microorganisms and even after 60 years of intensive development in this field, freeze-drying can still harm several microorganisms. During freeze-drying microorganisms have to undergo many stresses and DNA damage may result which could either cause total death of living cells or a paralysis by which the desired or useful qualities of the living cells could be lost or damaged. Several useful strains which are obtained as a result of gene manipulation, selective screening, mutations, and bear plasmids are mostly affected by freeze-drying and may lose their useful qualities after lyophilization. During freeze-drying gram-positive bacteria usually survive better than gram-negative bacteria. The age of a culture and the cell concentration to be freeze-dried could have a profound effect on the survival of microorganisms. A number of other characteristics may also influence the resistance of several species to freeze-drying as during several years of experimentation it has been observed that strains with spores and cysts are relatively resistant to freeze-drying whereas in particular large-celled gramnegative bacteria, PHB (poly-B-hydroxy butyric acid) filled bulky cells, spirillum-like large cells (due to their abnormal growth in the cell wall), and temporarily de-