Abstract
Cell viability or cell death is an important variable to monitor in many studies of host/pathogen interactions. However for studies that focus on events within the first few hours of the interaction, many of the viability assays currently being used are either too laborious and time consuming or measure the cell's temporary metabolic state rather than irreversible cell death. Evans blue has proven over the years to be a dependable stain for microscopic determination of cell death. We have used this stain to develop a spectrophotometric procedure that allows rapid, reproducible quantification of the stain retained by dead cells. This spectrophotometric procedure was used to compare plant/bacteria interactions involving either soybean/Pseudomonas syringae pv. glycinea or tobacco/P. syringae pv. syringae. Relative increases in cell death during these interactions in suspension cell systems were measured by both the spectrophotometric and microscopic technique and found to be similar. The spectrophotometric procedure was also adapted for leaf disc assays.
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Abbreviations
- HR:
-
hypersensitive response
- SDS:
-
sodium dodecyl sulfate
References
Atkinson MM, Keppler LD, Orlandi EW, Baker CJ & Mischke CF (1990) Involvement of plasma membrane calcium influx in bacterial induction of the K+/H+ and hypersensitive responses in tobacco. Plant Physiol. 92: 215–221
Baker CJ, Atkinson MM & Collmer A (1987) Concurrent loss in Tn5 mutants of Pseudomonas syringae pv. syringae of the ability to induce the hypersensitive response and host plasma membrane K+/H+ exchange in tobacco. Phytopathology 77: 1268–1272
Baker CJ, Mock N, Atkinson MM & Hutcheson S (1990) Inhibition of the hypersensitive response in tobacco by pectate lyase digests of cell wall and of polygalacturonic acid. Physiol. Mol. Plant Pathol. 37: 155–167
Baker CJ, O'Neill NR, Keppler LD & Orlandi EW (1991) Early responses during plant-bacteria interactions in tobacco cell suspensions. Phytopathology 81: 1504–1507
Baker CJ, Mock N, Glazener J & Orlandi EW (1993) Recognition responses in pathogen/non-host and race/cultivar interactions involving soybean (Glycine max) and Pseudomonas syringae pathovars. Physiol. Mol. Plant Pathol. 43: 81–94
Crippen RW & Perrier JL (1974) The use of neutral red and Evans blue for live-dead determinations of marine plankton. Stain Technol. 49: 97–104
Fischer JMC, Peterson CA & Bols NC (1985) A new fluorescent test for cell vitality using Calcofluor White M2R. Stain Technol. 60: 69–79
Gaff DF & Okong 'O-Ogola O (1971) The use of non-permeating pigments for testing the survival of cells. J. Exp. Bot. 22: 756–758
Goodman RN (1968) The hypersensitive reaction in tobacco: a reflection of changes in host cell permeability. Phytopathology 58: 872–873
Keppler LD, Atkinson MM & Baker CJ (1988) Plasma membrane alteration during bacteria-induced hypersensitive reaction in tobacco suspension cells as monitored by intracellular accumulation of fluorescein. Physiol. Mol. Plant Pathol. 32: 209–219
Oprisko MJ, Green RL, Beard JB & Gates CE (1990) Vital staining of root hairs in 12 warm-season perennial grasses. Crop Sci. 30: 947–950
Orlandi EW, Hutcheson SW & Baker CJ (1992) Early physiological responses associated with race-specific recognition in soybean leaf tissue and cell suspensions treated with Pseudomonas syringae pv. glycinea Physiol. Mol. Plant Pathol. 40: 173–180
Palta JP, Levitt J & Stadelmann EJ (1978) Plant viability assay. Cryobiology 15: 249–255
Parker J (1953) Criteria of life: some methods of measuring viability. Amer. Sci. 41: 614–618
Pegg DE (1989) Viability assays for preserved cells, tissues and organs. Cryobiology 26: 212–231
Prosperi E, Croce AC, Bottiroli G & Suprino R (1985) Influence of Daunorubicin on membrane permeability properties: detection by means of intracellular accumulation and efflux of fluorescein. Chem-biol. Interactions 54: 271–280
Smith BA, Reider ML & Fletcher JS (1982) Relationship between vital staining and subculture growth during the senescence of plant tissue cultures. Plant Physiol. 70: 1228–1230
Sukumaran NP & Weiser CJ (1972) Freezing injury in potato leaves. Plant Physiol. 50: 564–567
Taylor JA & West DW (1980) The use of Evan's Blue stain to test the survival of plant cells after exposure to high salt and high osmotic pressure. J. Exp. Bot. 31: 571–576
Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47: 189–194
Zilkah S & Gressel J (1978) The estimation of cell death in suspension cultures evoked by phytotoxic compounds: differences among techniques. Plant Sci. Lett. 12: 305–315
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Jacyn Baker, C., Mock, N.M. An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue. Plant Cell Tiss Organ Cult 39, 7–12 (1994). https://doi.org/10.1007/BF00037585
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DOI: https://doi.org/10.1007/BF00037585