Plant regeneration from somatic embryos in Pinus thunbergii (Japanese black pine) and Pinus densiflora (Japanese red pine)

Embryogenic tissues were initiated from megagametophyte explants containing pre-cotyledonary embryos. The explants were collected from juvenile cones of two Pinus species, Pinus thunbergii and P. densiflora, in mid and late July. The medium used for initiation was modified DCR medium (inorganic nitrate salts were the same as in DCR but the other components were the same as in MS) containing 1.5g/l glutamine, 10 PM 2,4-D, 5muM BA, 30g/l sucrose and 8g/l agar. The initiation frequencies in Pinus thunbergii and P. densiflora were 8.0 % (31 out of 389 explants) and 2.9 % (3 out of 105 explants), respectively. Embryogenic tissues were subcultured on modified DCR medium containing 1.5g/l glutamine, 2 muM 2,4-D, 1 muM BA, 30g/l sucrose, and 8g/l agar or 4 g/l gelrite. After one year of the initial culture, eleven P. thunbergii and three P. densiflora embryogenic lines were established. Matured somatic embryos were obtained by using Smith embryo development medium containing 50 PM ABA, 75 g/l PEG, 60 g/l sucrose and 5.5 g/l gelrite. Average numbers of mature somatic embryos per dish were 32 and 22 in best cell lines of P. thunbergii and P. densiflora, respectively. The dishes sealed with permeable paper tape benefited for germination of somatic embryos compared with parafilm. Among modified GD medium Smith embryo germination medium, modified DCR medium, and LP medium, the best performance for-germination was carried out by the modified GD medium containing 10 g/l sucrose and 5 g/l gelrite. Germination frequency of selected cell lines of both species was ca. 28 %. Regenerated plantlets have been transferred to soil.