© 1986 Heron Publishing—Victoria, Canada
Sustained division of protoplast-derived cells from primary leaves of Pinus pinaster, factors affecting growth and change
in nuclear DNA content
Helene David (1), Marie-Therese De Boucaud (2), Jean-Maurice Gaultier (3) and Alain David (1)
1. Departement des végétaux ligneux, Université de Bordeaux I, Avenue des Facultés, 33405 Talence
Cedex, France / 2. Laboratoire de Biologie cellulaire, Université de Bordeaux II, 33405 Talence Cedex, France / 3. UER de Mathématique et Informatique, Laboratoire Associé au CNRS 2.26, Université de Bordeaux I, 351, Cours de la Libération,
33405 Talence Cedex, France / Received December 31, 1985
Summary
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 H2O–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 H2O–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.
