Isolation and cell regeneration of protoplasts from sugar pine (Pinus lambertiana)

Protoplasts were isolated at high yields from actively growing callus and cell suspensions of cotyledons and needles of mature trees. The best protoplast growth response was obtained from cell suspensions of cotyledon and needle callus. Lower protoplast yields were obtained directly from young needles of flushing buds on explants from mature shoots (30-year-old trees) growing in vitro. In all cases, the first divisions, promoted by dimethyl sulfoxide, were observed in 10-45% of the protoplasts by 7-10 days. After 25-30 days, colonies of 8-10 cells were established. Browning of protoplast-derived cell cultures was observed within 40-45 days (cotyledons) and 20-25 days (mature tree sources).