Callus Proliferation from Protoplasts Isolated from Cell Suspension Cultures of Alnus firma Sieb

Abstract

Protoplasts were isolated from cell suspension cultures induced from a seed of Alnus firma Sieb. in 1/2MS liquid medium containing 1μM 2, 4-D and 0.1μM BAP. The optimal enzyme combination for protoplast isolation was 1% Cellulase Onozuka RS and 0.5% Pectolyase Y-23. Protoplast yield was of 1×10⁷/g fresh weight, and the protoplasts showed high viability of more than 90%. Colony formation from the protoplasts was induced efficiently in 1/2 MS liquid medium containing 0.6M mannitol, 0.09M sucrose and factorial combinations of 0.1-1μM 2, 4-D and 0.1-10μM BAP or 4-PU at the plating density of 5×10⁴/ml. Continuous callus proliferation was achieved by transferring colonies to fresh liquid medium containing 1μM 2, 4-D and 0.1μM BAP without mannitol.