© 1991 Heron Publishing—Victoria, Canada
Preparation of protoplasts from callus derived from buds of mature Scots pine and subsequent induction of cell proliferation
Anja Hohtola and Ari-Pekka Kvist
Biocenter and Department of Botany, University of Oulu, SF-90570 Oulu, Finland / Received May 16, 1989
Summary
Callus cultures were established from shoot tip explants excised from buds of mature, 10–40-year-old Scots pine (Pinus sylvestris L.) trees. Protoplast yield and viability were highest when the callus cells were plasmolyzed for 1.5 h in osmoticum (0.35
M mannitol, 0.15 M sorbitol, 1.5 mM CaCl2, 0.7 mM KCl, pH 5.8) before being enzymatically digested in osmoticum containing 0.5% cellulase Onozuka R-10, 0.5% macerase,
0.5% hemicellulase, and 0.5% bovine serum albumin. Bovine serum albumin markedly increased viability of isolated protoplasts.
Protoplasts were purified by flotation on either osmoticum alone or osmoticum containing 9% Nycodenz®.
Cell proliferation was initiated by spreading purified protoplasts on a filter impregnated with a modified Murashige and Skoog
(MS) medium supplemented with 3.5 g casein hydrolysate, 1.3 mM L-glutamine, 0.2 M glucose, 10.7 μM naphthalene acetic acid (NAA), and 4.4 μM benzylaminopurine (BAP). The filter was placed
on a solid, modified MS medium containing pine callus cells as nurse tissue and supplemented with 0.9 μM BAP and 1.0 μM 2,4-dichlorophenoxyacetic
acid (2,4-D). The first microcalli were observed after three weeks, and viability of subcultured cells was about 40% after
six weeks.
