Effects of Subculture Time on Somatic Embryogenesis of Nematode-resistant Pinus thunbergii

The callus of 37^#family used for the experimentwas cultured for 0.5 years,1.5 years and 3.5 years, respectively. During this period, the cell morphology and the maturation stage of somatic embryos were observed. In addition, the proliferation rate, the number of somatic embryos per gram of embryonic callus, the normal germination rate and survival rate of plants were measured. The results revealed that embryonic somatic mass (ESM) gradually became loose and cell stalk was short, thick and deformed in the long-term subculture process. The time of subculture had significant effects not only on the proliferation and differentiation of callus but also somatic embryo germination and survival rate of plants. The proliferation rate of 3 kinds callus was 200.37%, 182.47% and 111.63%, the number of somatic embryos was 77, 31 and 5, the normal germination rate was 84.43%, 51.10% and 11.13%, and the survival rate of plants was 73.00%, 50.00% and 6.70%, respectively, all showing a downward trend. Additionally, there were significant differences in the time of embryo maturation among 3 kinds callus. The callus formed the cotyledon embryo for a minimum of 46 days and a maximum of 74 days. As the subculture time increased, the process of somatic embryo differentiation became slower and slower. It can be seen that the potential of embryogenic callus of nematode-resistant Pinus thunbergii was gradually reduced and the abilities of somatic embryogenesis and plants regeneration were decreased during the subculture. And the short-term cultured embryogenic callus (0.5 years) was an ideal starting material for somatic embryogenesis of nematode-resistant P. thunbergii, and the callus culture time should be less than 1.5 years.