Abstract: The initial pH of the culture medium is a core microenvironmental factor that reshapes the developmental trajectory of plant in vitro cells.To optimize the somatic embryogenesis and plantlet regeneration system of Liriodendron × sinoamericanum and elucidate its response mechanisms,this study systematically investigated the full-cycle regulatory effects of pH (5.2~6.2)on embryogenic callus proliferation,somatic embryo quality,root system architecture and physiological and biochemical characteristics.The results demonstrated that a slightly acidic environment at pH 5.8 was the optimal steady-state threshold for maintaining high-quality in vitro regeneration of Liriodendron × sinoamericanum.Under this optimal microenvironment,the callus browning rate was only 2.0%,the conversion of normal cotyledonary embryos reached a peak(32 normal embryos out of an average total of 85 per plate),and the lateral root network of somatic seedlings was most ideally developed(averaging 6.8 roots at day 28).Integration of physiological and biochemical traits further revealed the developmental limitation mechanisms deviating from this threshold:overly acidic stress(pH 5.2)triggered a severe burst of reactive oxygen species(ROS)and membrane lipid peroxidation damage(significantly enriched MDA content),which presumably disrupted the stability of the auxin polar transport(PAT)network,leading to a surge in malformation rates such as polycotyledons(94.8%)and hindered main root extension;conversely,a near-neutral environment(pH 6.2)severely restricted chlorophyll synthesis,likely by reducing the bioavailability of trace elements to block porphyrin ring synthesis,ultimately stagnating root expansion due to limited photosynthetic carbon assimilation.In contrast,soluble protein metabolism was significantly upregulated under pH 5.8,suggesting it provides an ideal biochemical microenvironment for the accumulation of functional macromolecules such as late embryogenesis abundant(LEA)proteins.This study clearly indicates that controlling the initial pH within the slightly acidic range around 5.8 is a necessary prerequisite for high-quality somatic embryo conversion and large-scale propagation of this species,providing profound theoretical bases for the microenvironmental regulation of woody plant in vitro regeneration.