Abstract: Using immature Pinus yunnanensis seeds as research materials, select appropriate disinfection methods to observe the development status of zygotic embryos.The effects of explants, genotypes and medium types on embryogenic callus induction were investigated, and embryogenic calli were identified.Results show that the best disinfection method include the following steps: using 75% alcohol to disinfect for 30 s, followed by the rinsing process with sterile water for 2−3 times, soaking in 2% sodium chlorate for 15 min, and rinsing again with sterile water for 4−5 times. Different explant types impose no significant effects on the callus yield, but have significant effects on the formation of embryonic callus. The induction rate of embryonic callus is up to 34.3% when the female gamete is used as the explant, while 14.3% when the zygotic embryo is used as the explant. The average induction rate is between 2.2% and 17.6% when inoculate with 14 different kinds of media. The embryonic callus induction rates is 17.6%, 17.6% and 16.5% when inoculated with MLV+2,4-D 2.0 mg/L+6-BA 1.0 mg/L+KT 1.0 mg/L+ myo-Inositol 0.1 g/L, 1/2LM+2,4-D 2.2 mg/L+6-BA 1.1 mg/L +AgNO₃ 3.4 mg/L+ myo-Inositol 0.1 g/L and DCR +6-BA 0.63 mg/L +KT 0.61 mg/L+NAA 2.0 mg/L+ myo-Inositol 0.2 g/L+ maltose 15 g/L medium respectively, which indicating that the selected 3 types of medium are suitable for embryonic callus induction of female gametophyte of P. yunnanensis. The average induction rate of embryonic callus of 8 immature female gametophytes in P. yunnanensis is 5.1%−19.4%, and genotype 076 and 075 show the highest and lowest induction rate, respectively, suggesting that the genotype have significant influence on the induction of embryonic callus.