Temperature and Abscisic Acid Can Be Used to Regulate Survival, Growth, and Differentiation of Cultured Guard Cell Protoplasts of Tree Tobacco.

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Guard cell protoplasts isolated from leaves of Nicotiana glauca (Graham) were cultured. Conditions were sought that would maximize survival and maintain cells in their differentiated state. Temperature was an important determinant of survival, growth, and differentiation. As temperatures were increased from 24 to 32[deg]C, survival for 1 week in culture increased from approximately 20% to approximately 80% of cells used to initiate cultures. At all of these temperatures, approximately 90% of surviving cells divided to form callus tissue. "Footprint" areas of cells cultured for 1 week at 32[deg]C increased almost 30-fold. Cells cultured for 1 week at 34 to 40[deg]C also survived in high percentages (approximately 80%), but they retained a morphology similar to that of guard cells and they did not divide. Footprint areas of cells cultured for 1 week at 38[deg]C increased 6-fold. Cells cultured at 36 to 40[deg]C in media containing 0.1 or 1.0 [mu]M abscisic acid survived in high percentages and did not divide. At 38[deg]C their footprint areas did not increase, but cells so cultured increased in diameter when treated with fusicoccin. Morphologies and electrophoretic profiles of total sodium dodecyl sulfate-extractable proteins suggest that cells cultured at 38[deg]C in media containing abscisic acid remain differentiated. L-[alpha]-(2-Aminoethoxyvinyl)-glycine reduced survival to <1% at 26 or 32[deg]C but had no effect at 38[deg]C. At lower temperatures, cell growth and survival appear to be ethylene dependent.

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