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 ON 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.