Preparation of protoplasts from callus derived from buds of mature Scots pine and subsequent induction of cell proliferation

Abstract

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.