Abstract
A Panicum maximum cell suspension was used to study ultrastructural changes during cryopreservation. Pregrowing the cells in mannitol caused reduction in the vacuolar volume by redistribution of the large central vacuole into a number of smaller vesicles. Invaginations were formed in the plasma membrane of the cells, to accommodate the reduced cell volume. Swelling of organelles occurred during different stages of cryopreservation. The cisternae of the endoplasmic reticulum dilated and formed vesicles. Although some damage was apparent, organelles were still recognizable in cells frozen slowly and freeze-fixed at −10°C. The cells were able to repair such damage within two days in culture, and regained their normal appearance. Cells frozen slowly without any cryoprotection, and cells frozen rapidly by direct immersion into liquid nitrogen after cryoprotection, were lethally damaged by destruction of membranous structures. Osmiophilic granules were found along the plasma membrane of lethally damaged cells, indicating that their formation is a consequence of freeze damage, rather than a mechanism to prevent injury.
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Abbreviations
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- DMSO:
-
dimethyl sulfoxide
References
Dowgert MF and Steponkus PL (1984) Behavior of the plasma membrane of isolated protoplasts during the freeze-thaw cycle. Plant Physiol 75:1139–1151
Farrant J, Walter CA, Lee H and McGann LE (1977) Use of twostep cooling procedures to examine factors influencing cell survival following freezing and thawing. Cryobiology 14:273–286
Gnanapragasam S and Vasil IK (1992) Cryopreservation of immature embryos, embryogenic callus and cell suspension cultures of gramineous species. Pl Sci: (In Press)
Gordon-Kamm WJ and Steponkus PL (1984a) The behavior of plasma membrane following osmotic contraction of isolated protoplasts: Implications in freezing injury. Protoplasma 123:83–94
Gordon-Kamm WJ and Steponkus PL (1984b) The influence of cold acclimation on the behavior of the plasma membrane following osmotic contraction of isolated protoplasts. Protoplasma 123:161–173
Kartha KK (ed) (1985) Cryopreservation of Plant Cells and Organs. CRC Press Inc., Boca Raton, Florida
Mackenzie AP, Kuster TA and Luyet BJ (1975) Freeze-fixation at high subzero temperatures. Cryobiology 12:427–439
Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Pritchard HW, Grout BWW, Reid DS, and Short KC (1982) The effect of growth under water stress on the structure, metabolism and Cryopreservation of cultured sycamore cells. pp. 315–318 in Franks F and SF Mathias (ed): Biophysics of Water. John Wiley, New York.
Reynolds, ES (1963) Use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208–213.
Singh J (1979) Ultrastructural alternations in cells of hardened and non-hardened winter rye during hyperosmotic and extracellular freezing stresses. Protoplasma 98:329–341
Singh J and Miller RW (1985) Biophysical and ultra-structural studies of membrane alterations in plant cells during extracellular freezing: Molecular mechanisms of membrane injury. Pp. 61–73 in Kartha KK (ed): Cryopreservation of Plant Cells and Organs. CRC Press, Inc, Boca Raton, Florida
Spurr AR (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43
Steponkus PL (1985a) Fundamental aspects of cryoinjury as related to cryopreservation of plants cells and organs. Pp. 145–159 in Zaitlin M, Day P and Hollaender A (ed): Biotechnology in Plant Science. Academic Press, New York
Steponkus PL (1985b) Cryobiology of isolated protoplasts: Application to plant cell cryopreservation. Pp. 49–60 in Kartha KK (ed): Cryopreservation of Plant Cells and Organs. CRC Press, Inc., Boca Raton, Florida
Toth R (1982) An introduction to morphometric cytology and its application to botanical research. Am J Bot 69:1694–1706
Withers LA and Davey MR (1978) A fine structural study of the freeze-preservation of plant tissue cultures 1. The frozen state. Protoplasma 94:207–219
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Communicated by F. Constabel
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Gnanapragasam, S., Vasil, I.K. Ultrastructural changes in suspension culture cells of Panicum maximum during cryopreservation. Plant Cell Reports 11, 169–174 (1992). https://doi.org/10.1007/BF00232526
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DOI: https://doi.org/10.1007/BF00232526