Establishment of Cell Suspension Culture and Isolation of Protoplast of Chinese fir

Callus was induced by embryos from mature seeds of Chinese fir (Cttnninghamia lanceolata Hook.),which had been cultured in MS medium containing 2.0mg/l 2,4-D and 0.2mg/l KT.After successive subculture and selection,cell colonies of the calli which were frible and in rapid growth were obtained.In the period of initial subculture,decrease of sucrose concentration was beneficial to diminishing callus browning.The cell suspension culture,which grew rapidly and consisted mainly of smaller cell aggregates,was established by transferring five-month-old calli to liquid MS medium supplemented with 1.5~2.0mg/l 2.4-D,0.1~0.2mg/l KT and 200 mg/l CH.A great amount of Chinese Fir protoplasts were released from the cell suspension cultures,with protoplast yield (9.3×105/g FW) and viability (above 80%).Enzyme mixture was consisted of 2% cellulase R-10,0.25% pectinase and 13% mannitol.