【Objective】To systematically investigate the impact of 6-(2-hydroxy-3-methylbenzylamino) purine (PI-55) on the development of embryogenic callus tissues in European spruce (Picea abies), this study examines both its role during extended periods of callus proliferation and its influence on the subsequent stages of somatic embryo differentiation. By analyzing the morphogenetic responses, cellular proliferation rates, and molecular regulatory mechanisms triggered by PI-55 treatment, this research aims to elucidate the compound’s potential to modulate key developmental processes in coniferous embryogenesis. The findings are anticipated to provide critical insights into optimizing somatic embryogenesis protocols for P. abies, offering a robust scientific foundation for enhancing the efficiency and reproducibility of large-scale plant regeneration techniques in this ecologically and economically important conifer species.【Method】Embryogenic callus tissue derived from mature zygotic embryos of the P. abies genotype 33 was selected as the experimental substrate to investigate the impact of 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) on callus proliferation and somatic embryo development. During the proliferation phase, callus cultures were supplemented with varying concentrations of PI-55 [0 (control), 0.5, 1.0 and 2.0 μmol/L] and maintained under controlled conditions for a duration of 14 days. Subsequent analyses involved comprehensive assessments of physiological and biochemical parameters, including endogenous phytohormone profiles (indole-3-acetic acid [IAA], cytokinins (CTK), zeatin riboside (ZR), and abscisic acid (ABA), antioxidant enzyme activities (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), soluble protein content, and somatic embryo maturation efficiency. These metrics were systematically quantified and compared across treatment groups to elucidate the concentration-dependent effects of PI-55 on callus tissue viability, hormonal balance, oxidative stress tolerance and embryogenic potential.【Result】The application of 0.5-1.0 μmol/L PI-55 has been shown to significantly enhance the differentiation rate of European spruce embryogenic callus. This effect is mediated through PI-55 ability to elevate endogenous hormone levels, antioxidant enzyme activities, and soluble protein content in the embryogenic callus. Notably, comparative analysis revealed that cell lines with weaker somatic differentiation capacity exhibited higher concentrations of cytokinins (CTK) and zeatin riboside (ZR), as well as greater peroxidase (POD) and catalase (CAT) activities compared to those with stronger differentiation potential. These findings suggest that while a certain threshold of endogenous substances is required to promote somatic embryogenesis, supraoptimal levels may conversely inhibit the differentiation process.【Conclusion】These insights provide a robust scientific framework for improving somatic embryogenesis protocols in European spruce. The elucidation of PI-55 regulatory role, in particular, offers valuable opportunities for developing enhanced tissue culture systems capable of large-scale propagation of superior genotypes. Such advancements will significantly contribute to modern breeding initiatives focused on producing improved spruce varieties, thereby meeting both ecological conservation objectives and the growing commercial requirements of the forestry and timber sectors. The potential applications of these findings extend to addressing current challenges in sustainable forest management and wood production.