关键词: 黑松, 松材线虫病, 体细胞胚, 聚乙二醇(PEG8000)

Abstract: [Objective]In this study, we investigated the role of medium components in somatic embryo maturation, in order to further improve the quality and quantity of somatic embryos.[Method]The embryogenic cells were incubated for 10 days in the solid proliferation medium. Sterile electronic scales (75% ethanol wipe, ultraviolet sterilize 30 min) were used to weigh 1 g embryonic cells, a 50 mL sterile measuring cylinder (ultraviolet sterilization 30 min) was used to measure 30 mL liquid medium which was poured into a 100 mL conical flask. Then, 1 gram embryonic cells were transferred into a 100 mL conical flask which was transferred into a constant temperature incubator shaker. The culture was incubated 7-8 days in darkness at 25℃, 90 r·min^-1. Subsequently, suspension cells (precipitated volume 3 mL) were taken for the subculture. The subculture was proliferated once a week until all embryogenic cells were evenly dispersed in the culture medium. The suspension cells of 2 mL (fresh mass 200 mg) from the forth culture were sprayed on solid mature media which contained various components of maltose (30,45,60 g·L^-1), abscisic acid (ABA) (0,5,10,20,30,50 mg·L^-1), polyethylene glycol (PEG8000) (0,50,75,100,125,150 g·L^-1), activated carbon (AC) (1 g·L^-1, 2 g·L^-1, 3 g·L^-1), coagulating agent (agar:6, 8, 10, 12 g·L^-1; Phytagel 2, 2.5, 3, 3.5 g·L^-1) and the combination of maltose, ABA and PEG8000.[Result]During the development and maturation of somatic embryos (cell line #1337), the number of somatic embryos obtained from maltose 45 g·L^-1 medium increased significantly; When the ABA concentration ranged from 5 mg·L^-1 to 20 mg·L^-1, the number of somatic embryos increased significantly with ABA concentration, and with abscisic acid (ABA) concentration in 20 mg·L^-1, the maximum quantity of somatic embryos was obtained; The optimal concentration of polyethylene glycol (PEG8000) and activated carbon (AC) for somatic embryogenesis was 125 and 2 g·L^-1, respectively; Agar powder, as coagulant, was added to the mature medium that could not solidify; phytagel was more suitable for solidifying mature medium. phytagel of 3 g·L^-1 was the optimum concentration of somatic embryo maturation. The orthogonal experiment showed that maltose 45 g·L^-1, ABA 10 mg·L^-1, and PEG8000 125 g·L^-1 were the media on which the embryogenic cells (cell line #1337, #1537, #1637) produced the most somatic embryos among the nine maturation medium combinations. Among different combinations of maltose, ABA and PEG8000, the productivity of somatic embryos produced from different clones showed different trends. The optimal combination for somatic embryo development and maturation from embryogenic cell #1337 was maltose 45 g·L^-1, ABA 10 mg·L^-1, PEG8000 125 g·L^-1; from #1537 and #1637, the optimal combination was maltose 30 g·L^-1, ABA 10 mg·L^-1, PEG8000 125 g·L^-1; PEG8000 had the biggest range in the three clones, and thus, PEG8000 had the most important effect on somatic embryos development and maturation of Pinus thunbergii.[Conclusion]In process of somatic embryo maturation (#1337), maltose 45 g·L^-1, ABA 20-30 mg·L^-1, PEG8000 125g·L^-1, AC 2 g·L^-1 and 3 g·L^-1 phytagel promoted somatic embryo maturation. The optimal combination for Japanese black pine somatic embryo maturation is maltose concentration 30 g·L^-1, ABA 10mg·L^-1 and PEG8000 125g·L^-1.

Key words: Pinus thunbergii, pine wilt disease, somatic embryo polyethylene glycol (PEG8000)