Optimization of the technique for maize protoplast isolation and their nativity after electroporation

Abstract

We optimizes the composition and concentration of the enzymes, the time for enzymatic treatment and the volume of the enzyme mixture. We also optimized the concentration osmotic of agent, the centrifugation mode, and filter pore size for protoplasts isolating from epidermal cells of maize roots (Zea mays L.) of the Brown Marker (BM) line. It was found that 150 minutes is the optimal time for 150 mg root tissue maceration. The yield of intact protoplasts was ~ 4.4 ± 0.2 × 105 cells/mL at the following concentrations of enzymes and osmotic stabilizer: cellulase – 17,4, pectolase – 1.2, hemicellulase – 0.07, D-mannitol – 9.3%. The the concentration of protoplasts was to 23 times higher (p < 0.05) in 800 μl, compared with 200 μl of the enzyme mixture with equal concentrations of enzymes and osmotic stabilizer. It was found that filtration of 800 μl protoplast suspension through a filter with a pore size of 15 × 15 microns increases the yield of protoplasts up to 3.3 times, compared with a filter with a pore size of 15×39. Fractional centrifugation without preliminary filtration of the solution and the flotation method did not produce an increase in the yield of protoplasts. The residual number and protoplast wholeness after ~ 20 hours at +3 °C incubation was evaluated. The protoplast number decreased up to 2 times (p < 0.05) after electroporation.