| Summary: | Cryopreservation is an important method for the excellent long-term preservation of plant germplasm. This study explores an optimal cryopreservation technology for the embryogenic callus of <i>Fraxinus mandshurica</i> to effectively maintain its genetic stability and morphogenesis potential. The optimal cryopreservation conditions were assessed using the embryogenic callus of <i>F. mandshurica</i> as the material, and the slow cooling method was optimized for its cryopreservation. The results indicated that the preculture of embryogenic callus in 0.4 mol·L<sup>−1</sup> sorbitol solution for 20 h at room temperature, followed by its cryoprotection in 7.5% dimethyl sulfoxide solution at 0 °C for 90 min, constituted the optimal material treatment method. The freezing tube was placed in a −80 °C refrigerator for 2 h and then quickly put into liquid nitrogen for frozen storage. During thawing, the cryopreservation tube was taken out from liquid nitrogen, directly placed in a water bath at 40 °C for 2 min, and used for culturing on the woody plant media + 0.1 mg·L<sup>−1</sup> 6-benzyladenine + 0.15 mg·L<sup>−1</sup> 2, 4-dichlorophenoxyacetic acid. After cryopreservation using the slow cooling method, the highest survival rate of callus cells was 80.82%. The fresh weight reached 1.93 g after 60-day recovery culture. The regeneration rate and the proliferation coefficient of the callus were 100% and 2.79, respectively. The differentiation rate was 56.83%, and the emergence rate was 23.59%. The results provide a scientific basis for the long-term preservation of <i>F. mandshurica</i> germplasm resources.
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