Somatic Embryogenesis And Cryopreservation Of Pinus Koraiensis Sieb. Et Zucc.

In this thesis, immature zygotic embryos of Pinus koraiensis Sieb. et Zucc. were used as the initial explants for somatic embryogenesis. The factors affecting somatic embryogenesis, physiological and biochemical characteristics of embryogenic callus as well as the callus cryopreservation were investigated. The results were as follows:1. Immature zygotic embryos of Pinus koraiensis Sieb. et Zucc. sampled at 3 different times were cultured on GLH medium with different concentrations of 2,4-D and 6-BA to induce callus. The results showed that the sampling time (maturity) of immature zygotic embryos, concentration of 2,4-D and 6-BA had a significant effect on induction rates of callus. The result of Duncan's multiple range test confirmed that more efficient induction was achieved from the immature zygotic embryos sampled on July 5 with 2,4-D 2.0mg/L + 6-BA 1.0mg/L. The highest induction rates was 43.33%.2. The growth curve of embryogenic callus during proliferation stage was "S"-shaped and growth became stagnant 20 days after subcultured on fresh GLH medium. The growth rate rose first and then decreased. The maximum growth rate appeared 12 days after subculture. The results illustrated that the appropriate subculture cycle of embryogenic callus during proliferation phase was 2-3 weeks.3. Somatic embryos were induced on GLH medium with different concentrations of sucrose, ABA, PEG.The result showed different concentrations of sucrose, ABA had a significant effect on the induction of somatic embryo, but PEG had no significant impact. GLH + Sucrose 60g/L + ABA 20mg/L was optimal to induce somatic embryo and the frequency was 47.5 somatic embryos per gram callus. The average germination rate of somatic embryos on germination medium (GLH + Sucrose 20g/L) was of 73.3%. The average survival rate of somatic seedlings after transplantation was 64.3%.4. The total soluble sugar and sucrose content of embryogenic callus in embryo-induced phase changed similarly. The content rose in 0-5 days ,dropped in 5-15 days, rosed again in 15-25 days and then kept steady. Soluble protein content of increased in 0-10 days and decreased in 10-20 days, and increased again after 20 days. The change trend of sugar and protein content indicated that the metabolism in embryogenic callus was high and somatic embryos were forming in this stage, at the same time two kind of specific proteins about 63.2KD and 31.7KD appeared. Such specific proteins might functioned as regulator or the structure protein in somatic embryogenesis.5. The callus were treated for cryopreservation with 6 different cryoprotectants after pretreated 0, 12, 24, 36, 48h with 0.4mol/L Sorb separately and thawed by different methods 3days later. The callus survival rate after cryopreservation was higher with 0.4mol/L Sorb as pretreatment for 24h, 5% DMSO + 0.4mol/L Sorb as cryoprotectants, 20℃flowing water as thawing method. The average survival rate of callus after recovery culture was 64.4%. The result laid a solid foundation for mid-long term preservation of Pinus koraiensis Sieb. et Zucc. .