Seasonal Variation in Callus Proliferation from Explants of Mature Pinus Strobus Trees
In vitro propagation from expiants of mature trees must be achieved before plant tissue culture technique can become a viable method for mass propagation of superior forest trees. One possible route for in vitro mass propagation is differentiation of plantlets from callus cultures. As the first step towards achieving this goal, experiments were done to establish callus cultures from shoots of 15- to 18-year-old Pinus strobus (white pine) trees. The study was started in the spring of 1982 just after the end of dormancy of winter buds. Young shoots were collected early in the morning and were surface sterilized by treatment with 2.625% sodium hypochlorite for 15 min, followed by 70% ethanol for 2 min and 2.625% sodium hypochlorite for an additional 5 min. The surface-sterilized shoots were thoroughly washed with sterilized double-distilled water and cut into approximately 5 mm long segments, which were put on nutrient media. Cultures were incubated at 26 ± 1°C under 450–500 μW/cm2 cool white fluorescent illumination. Day length was 16 hr. Shoot samples were collected at 2-week intervals during May and June, and at monthly intervals during the rest of the year. Best callus proliferation occurred from segments of shoots collected in early spring. A modified Murashige and Skoog medium supplemented with 0.2 mg/1 α-naphthaleneacetic acid (NAA) and 2 mg/1 N6-benzyladenine (BA) was the best for callus proliferation. The callus could be maintained on this medium through several subcultures. The extent of callus proliferation varied with the age of the shoots. Surface contamination was a major problem in shoots collected in late spring or later. Surface contamination could be controlled to a large extent by heat treatment of expiants during the process of surface sterilization.