DIVISION OF GUARD-CELL PROTOPLASTS OF NICOTIANA-GLAUCA (GRAHAM) IN LIQUID CULTURES

Guard cells are uniquely differentiated to transduce signals into the metabolic and ion transport processes that result in turgor-driven stomatal movements. We tested the hypothesis that these highly specialized cells are terminally differentiated. Guard cell protoplasts were isolated from abaxial epidermal tissue of leaves of Nicotiana glauca (Graham) and cultured in a medium designed for culturing mesophyll protoplasts of Nicotiana tabacum. Protoplasts were incubated at densities of 2-5 x 10(11) cells m-3 in eight-well microchamber slides under 50-mu-mol m-2 s-1 of photons of continuous fluorescent light at 25-degrees-C. When the medium was modified by the addition of 100 mol m-3 of sucrose and by buffering with 10 mol m-3 of MES buffer at pH 6.1, cell division began within 96 h of the time the culture was initiated. After 9 d of culture, 80% of surviving cells had synthesized new cell walls, had dedifferentiated, and were dividing to form small colonies. Callus tissue was visible after 4-5 weeks. We conclude that guard cells of Nicotiana glauca are not terminally differentiated, and that guard cell protoplasts of this species have the capacity to grow, synthesize cell walls and divide.