Isolation of protoplast from callus of Populus euphratica and H⁺ fluxes across plasma membrane under NaCl stress

We used callus of Populus euphratica Olive to isolate protoplasts, and H⁺ fluxes across plasma membrane were investigated. The concentration of enzymes for protoplast isolation, e.g. cellulase, pectolyase, macerozyme, hemicellulase, and sorbitol content, incubation time were systemically studied. High yield and viability of protoplast was achieved after 6-8 hours incubation of P. euphratica callus in enzyme solution containing 1.5% (w:v) cellulase R-10, 0.1% (w:v) pectolyase Y-23, 0.2% (w:v) macerozyme R-10, 0.05% (w:v) hemicellulase and 0.75-0.80 mol·L^-1 sorbitol. Non-invasively ion selective microelectrode technique was used to access proton fluxes in the absence and presence of NaCl (20 mmol·L^-1). Salt-induced transient net H⁺ efflux was observed in the plasma membrane of P. euphratica cells. The shift of H+ flux response to NaCl shock and the relevance to salt tolerance were discussed.