Abstract: INTRODUCTION: Hydrangea macrophylla is essential in landscaping, ecology, and medical care, with significant development prospects. Chikushi-no-kaze is an ideal low-maintenance variety for microlandscaping and potted H. macrophylla, which are widely favored by consumers. However, the extremely low setting rate of H. macrophylla and poor seed development under natural conditions render traditional reproduction methods inadequate for meeting the demands of large-scale annual production in the market. The breeding of H. macrophylla plantlets through tissue culture technology is currently the most efficient method for producing high-quality plantlets. RATIONALE: Regeneration efficiency in plant tissue culture is a key factor in achieving factory seedling production. Therefore, this study investigated the regeneration efficiency of isolated leaves from tissue culture plantlets of Chikushi-no-kaze under optimal culture conditions at each key stage, considering different leaf positions, dark culture durations, and other factors. The aim was to establish an efficient regeneration technology system that provides technical guidance for large-scale plantlet production and serves as a reference for establishing a genetic transformation system for H. macrophylla in the future. RESULTS: The 3^rd to 5^th leaves (middle mature leaves) of Chikushi-no-kaze tissue culture plantlets were identified as the optimal sampling leaves. A dark culture duration of 10–14 days was conducive to callus formation. The most suitable medium for the induction and regeneration of adventitious buds was MS+3.0 mg·L^–1 CPPU+0.1 mg·L^–1 2,4-D, with induction and regeneration rates of 97.78% and 93.33%, respectively. The optimal medium for adventitious bud proliferation was MS+2.0 mg·L^–1 6-BA+0.1 mg·L^–1 IBA, yielding a proliferation coefficient of 8.33. For elongation growth, the optimal medium was MS+1.0 mg·L^–1 6-BA+0.1 mg·L^–1 IBA, resulting in an average stem length of 4.10 cm. The optimal medium for rooting culture was MS+0.3 mg·L^–1 IBA, achieving a rooting rate of 87.20%. CONCLUSION: This study initially established a technical system for in vitro leaf regeneration of the large leaf H. macrophylla ‘Chikushi-no-kaze’, which effectively solved the problem of low efficiency of adventitious bud regeneration of H. macrophylla and helped to achieve efficient reproduction and recycling. This study lays the foundation for the large-scale production and genetic improvement of H. macrophylla.

Key words: callus, Hydrangea macrophylla, leaf regeneration, rapid propagation of plantlets, tissue culture