Abstract: [Objective] Exogenous hormones, penetrant, sugar, coagulant and culture model that have impacts on somatic embryogenesis of nematode-resistant Pinus densiflora were studied.[Method] On the basis of investigation and analysis against nematode-resistant P. densiflora germplasm resources database, two clones 22^#-1 and 13^#-1 of nematode-resistant P. densiflora that the induction and proliferation of somatic embryos at a good state were tested. Somatic embryogenesis was determined by comparative tests of different culture methods and different culture media. The optimum medium and method for somatic embryogenesis and germination and plantlet regeneration were screened. SPSS17.0 and other software were used to analyze the somatic embryos.[Result] The number of normal somatic embryos reached 171 embryo·g^-1, higher than other treatments on the LP medium added with 15 mg·L^-1ABA+140 g·L^-1 PEG8000. Regarding the penetrant, the number of normal somatic embryos of nematode-resistant P. densiflora was up to 179 embryo·g^-1, higher than other treatments on 8 g·L^-1 inositol concentration. Embryogenic callus was directly placed on the maturation medium adding inositol. Embryogenic callus was not cultured on the middle medium. Therefore, the culture time could be shortened by 2-3 weeks. Mature cotyledon embryos were formed around 8 weeks. Regarding the carbon source, cultured on LP medium with 60 g·L^-1 maltose, the number of normal somatic embryos reached 235 embryo·g^-1, higher than other treatments; For the coagulant, when plant gel concentration was 3 g·L^-1, solidification of medium displayed a moderate hardness and cotyledon embryo grew well. Supplemented with polyethylene glycol (PEG) and adding coagulant agar concentration from 6 g·L^-1 to 12 g·L^-1, the medium was not solidified. Callus was unable to grow and no cotyledon embryos were formed. Regarding the culture model, after 11-12 weeks the embryogenic callus developed into mature somatic embryos on liquid-solid proliferation-solid maturation. The number of normal somatic embryos of 22^#-1 was up to 239 embryo·g^-1, higher than other treatments. The somatic embryo germination rate was 67.2% and plantlet conversion rate from the germinated somatic embryos was 46.5%. Up to 32.7% of the transplanted plantlets were successfully survived three months in soil.[Conclusion] Embryogenic callus of nematode-resistant P. densiflora was on the LP medium with 15.0 mg·L^-1 ABA +140 g·L^-1 PEG8000+ 8 g·L^-1 inositol+60 g·L^-1 maltose+3 g·L^-1 plant gel. The culture method was liquid-solid proliferation-solid maturation culture. Normal mature somatic embryos were successfully obtained and regenerated plantlets were transplanted to survive. At the same time, the culture duration could be shortened by 2-3 weeks, and the process of somatic embryogenesis of nematode-resistant P. densiflora was speeded up. Regenerated plants survived through transplanting. This study provides a feasible technique for large-scale propagation and mass production of nematode-resistant P. densiflora.

Key words: Pinus densiflora, Bursaphelenchus xylophilus, embryogenic callus, somatic embryogenesis, maturation, germination