Abstract
Mesophyll protoplasts of Betula platyphylla var. japonica were isolated successfully using a combination of 1% Cellulase Onozuka R-10 and 1% Driselase in 0.6M mannitol. For protoplast isolation, loosening leaves with tweezers after enzymatic treatment was tried. This novel method gave a large yield (5×107/g fresh weight) of highly viable (more than 80%) protoplasts. Effects of plant growth regulators, plating density, levels of nitrogen, inorganic phosphate and pH on cell division activity in the protoplast culture were examined. An optimal plating density was of 5×104 protoplasts/ml. Cell division was promoted with phosphate concentrations of 0.31-0.625mM after one month of culture, whereas its lower and higher levels were rather inhibitory. Active cell division occurred in 1/2 MS medium with 1μM of NAA and 10μM of BAP. The presence of ammonium nitrate in the medium largely influenced the viability of protoplasts. Therefore, protoplasts of B. platyphylla is considered to require a high level of BAP in addition to the presence of ammonium nitrate for efficient cell division.
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