MEASURING CYTOPLASMIC CALCIUM LEVEL IN CITRUS PROTOPLASTS USING THE FLUORESCENT-PROBE INDO-1

We measured cytoplasmic free calcium in protoplasts of Citrus sinensis Osb. Protoplasts were isolated from cell-suspension cultures grown as microcalli. The cell surface presented acid polysaccharide filaments, probably responsible for cell adhesion. These filaments were disrupte d by enzymatic digestion liberating the cells and subsequently the pro toplasts. Protoplasts were loaded with the pentapotassium salt of indo -1 by incubation in acidic solutions of this calcium indicator. Loadin g increased with decreasing pH and osmotica. Dye loaded in this manner was distributed only in the cytoplasm. The protoplast autofluorescenc e was very low and did not interfere with measurements. Cytoplasmic fr ee-calcium concentration in Citrus protoplasts, using microspectrofluo rimetry, was estimated to be 154 nmol.L-1. This resting value was incr eased when protoplasts were exposed to high extracellular Ca2+ (1 mmol .L-1) or 6BA (1 mu mol.L-1) suggesting that the pathway in cytokinin s ignal transduction in these cells may involve a Ca2+ uptake facilitati on from extracellular medium.