LOADING OF VICIA-FABA GUARD-CELL PROTOPLASTS WITH INDO-1 TO MEASURE CYTOSOLIC CALCIUM-CONCENTRATION

Calcium selective fluorescence dyes Indo I/AM and Indo 1 were used to load isolated guard cell protoplasts (GCPs) of Vicia faba. Different medium conditions were used with both stains. The loading and/or the cellular conversion of Indo 1/AM to Indo 1 was unsuccessful. Loading was achieved incubating protoplasts with pentapotassium salt of Indo 1 for 90 min at room temperature, in a medium consisting of 0.25 mM Mes-KOH (pH 4.50),0.37 M mannitol, 50 mM KCl. The concentration of cytosolic Ca2+ in GCPs was estimated to be 190-245 nM by fluorescence ratio analysis of Indo 1.