In vitro culture of Japanese black pine (Pinus thunbergii)

摘要

In vitro-culture from mature embryos of Japanese black pine (Pinus thunbergii) plus trees was developed. For initial adventitious buds induction, Wolter and Skoog (1966) medium with 3 mg/l BAP was the best among screened media. For elongation of shoots, a half strength Lepoivre (LP) medium containing 5 g/l activated charcoal was the best. Rooting percentage was 0-50 percent on the modified Root Induction Medium of Abo El-Nil (RIM). However there were big clonal differences in rooting percentages. Habituated plantlets were potted out to the field and growth performance was checked until 8 years. In vitro plantlet regeneration from 6-7 years old black pine which were selected as resistant clones using pine wood nematode inoculation test was tried. Induced brachyblast buds obtained by topping the trees were the best for explants and the half-strength LP medium containing 2.25 mg/l BAP and half-strength LP medium containing 5 g/1 activated charcoal were used in turn repeatedly for subculture in about 1-2 months interval. After subculture of 2 years, more than 5000 shoots were obtained from one clone. Shoots rooted in RIM containing IBA. Somatic embryogenic cells were induced from immature embryos collected from the end of June to mid July in Tsukuba, Japan. The suitable media for induction, maturation and germination of somatic embryos were different in the constituents. For induction, modified 1/2LP or Smith (1996) medium containing BAP and 2,4-D was used. For maturation , EM-3 medium (original) was added with ABA and PEG. Maltose instead of sucrose was effective for somatic embryo maturation. For germination of the matured somatic embryos, hormone free medium was used with or without activated charcoal powder. Regenerated plantlets were habituated and potted out in the vermiculite.