Sustained division of protoplast-derived cells from primary leaves of Pinus pinaster, factors affecting growth and change in nuclear DNA content.

Leaf protoplasts were isolated from apical and in vitro-induced axillary buds of Pinus pinaster Ait. seedlings. First divisions were seen after 8-10 days of culture in a 650 mOsm kg H(2)O(-1) medium in which glutamine was the sole nitrogen source. Colony formation was achieved in 6-7 weeks in a modified protoplast culture medium in which a reduction in the concentrations of both calcium and carbon was essential for sustained divisions. To maintain cell suspension growth, it was necessary to subculture every three weeks to a 170 mOsm kg H(2)O(-1) medium. Lowering the C/N ratio did not support better growth. Phenolic compounds were detected in stationary phase cultures. Analysis by HPLC indicated that the cinnamate pathway was involved in their synthesis. After 3 and 7 months of culture, 65 and 74%, respectively, of protoplast-derived cells had a nuclear DNA content comparable to that of leaf protoplasts.