The pieces of plant
tissue were submerged in 10 mL enzyme solution [10 mM MES, pH
5.7, 0.6 M mannitol, 0.1% BSA, 1.5% (w/v) cellulase R10 (Yakult
Honsha, Tokyo, Japan), 0.75% (w/v) macerozyme R10 (Yakult
Honsha), 1 mM CaCl2, 1 mMb-mercaptoethanol, and 0.25 g/ml carbenicillin]. The strips were vacuum infiltrated for 20 min and then
incubated for 4 or 5 h in the dark at 28 C with gentle shaking at
80 rpm. After incubation,