Cuard cell protoplasts isolated from leaves of Nicofiana g h c a (Craham) were cultured. Conditions were sought that would maximize survival and maintain cells i n their differentiated state. Temperature was an important determinant of survival, growth, and differentiation. As temperatures were increased from 24 to 32'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°C increased almost 30-fold. Cells cultured for 1 week at 34 t o 40°C also survived i n 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°C increased 6-fold. Cells cultured at 36 t o 4OoC in media containing 0.1 or 1.0 W M abscisic acid survived in high percentages and did not divide. At 38°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°C in media containing abscisic acid remain differentiated. ~-a-(2-Aminoethoxyviny1)-glycine reduced survival t o <1 O/O at 26 or 32°C but had no effect at 38OC. At lower temperatures, cell growth and survival appear to be ethylene dependent.

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