【Objective】Woody plants typically have long growth cycles and limited regenerative capacity, which significantly restricts the efficiency of forest tree breeding. In vitro culture techniques can overcome seasonal constraints and accelerate propagation. Calcium, as an essential nutrient and a critical intracellular second messenger, plays a vital role in plant growth and development. However, its specific regulation in woody plant tissue culture remain unclear. Systematic investigation of calcium treatment under the in vitro condition is of great significance for improving tissue culture efficiency and supporting breeding strategies in forestry.【Method】We treated shoots of the woody model speices hybrid poplar ‘84K’ (Populus alba × P. glandulosa) in vitro under Ca²⁺ concentrations of 0, 1.5, 3.0, 6.0, 9.0 and 15.0 mmol/L. The effects of Ca²⁺ on shoot growth and adventitious root induction were systematically analyzed.【Results】Within the 0-9.0 mmol/L range, increasing Ca²⁺ concentrations enhanced shoot growth. The maximum plant height was observed at 6.0 mmol/L Ca²⁺, while leaf expansion was most pronounced at 9 mmol/L, indicating an optimal concentration range of 6.0-9.0 mmol/L. Complete calcium deficiency(0 mmol/L) disrupted apical dominance, resulting in shoot tip necrosis and excessive elongation and thickening of the primary root. In contrast, high Ca²⁺ concentration (15 mmol/L) significantly inhibited overall growth, with reduced plant height and smaller leaves. Ca²⁺ promoted stem elongation mainly by increasing internode length rather than node number. While the response intensity varied across different basal salts, the overall trend was consistent. Different concentration of Ca²⁺ treatments had no significant effect on the number of adventitious roots, but calcium deficiency led to pronounced elongation and thickening of adventitious roots, suggesting a specific root morphogenetic response to Ca²⁺ dificiency.【Conclusion】Ca²⁺ exerts concentration-dependent and tissue-specific effects on poplar in vitro. Moderate Ca²⁺ levels help maintain apical dominance and promote shoot elongation, while the Ca²⁺ deficiency triggers architectural remodeling in roots. These findings provide preliminary insights into the differential regulation of shoot and root development by Ca²⁺ in woody plants and lay a theoretical foundation for improving tissue culture systems and calcium-mediated molecular breeding in forestry.