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

• Published in: Tree physiology Vol. 1; no. 1; pp. 21 - 30
• Main Authors: David, H, Boucaud, M.T, Gaultier, J.M, David, A
• Format: Journal Article
• Language: English
• Published: Canada 01.06.1986
• Subjects: buds; cell culture; cell division; cell suspension culture; DNA; growth; leaves; nutrient requirements; Pinus pinaster; protoplasts; suspension culture
• ISSN: 0829-318X; 1758-4469
• DOI: 10.1093/treephys/1.1.21

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.