Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Nagata and Takebe's (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana. Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.