INTRODUCTION: Blackberry (Rubus fruticosus L.) is an important economic crop whose fruits are not only rich in fiber, vitamins, and phenolic metabolites but also possess significant health benefits. Traditional blackberry propagation methods, including seed propagation, layering, cutting, and suckering, have various limitations. For instance, seed propagation has a long cycle, low germination rate, and variable offspring traits. In contrast, tissue culture-based rapid propagation technology offers distinct advantages such as season-independent operation, high multiplication coefficient, and preservation of maternal superior traits, making it an effective solution for blackberry seedling propagation. Therefore, it is necessary to establish an efficient blackberry rapid propagation system, which would lay the foundation for large-scale production of high-quality virus-free seedlings and germplasm innovation. 

RATIONALE: The establishment of an in vitro regeneration system for blackberry stem segments is based on the theory of plant cell totipotency and hormonal regulation mechanisms, under which the meristematic cells of stem segments can regenerate into complete plants under suitable culture conditions. To establish a stable regeneration system for blackberry 'APF-190T', we investigated the effects of sterilization conditions, medium types, and the types and concentrations of plant growth regulators on primary culture, proliferation culture, and rooting culture. Additionally, the influence of different substrates on the growth of tissue-cultured seedlings was further analyzed. 

RESULTS: Comparative experiments on disinfection durations revealed that a 7-minute treatment yielded optimal results for blackberry explants, demonstrating the lowest contamination rate of 20.00%, a relatively low browning rate of 6.67%, and the highest survival rate of 73.33%. In MS medium, these conditions produced the best stem segment growth with a maximum bud induction rate of 63.33%, characterized by abundant germinated buds reaching an average length of 1.41 cm. For primary culture, the combination of 1.0 mg·L⁻¹ 6-BA + 0.2 mg·L⁻¹ NAA proved most effective, achieving 100% bud induction rate and producing robust seedlings with dark green leaves. Subsequent proliferation culture demonstrated superior results using a combination of 0.8 mg·L⁻¹ 6-BA and 0.1 mg·L⁻¹ NAA, achieving the highest adventitious bud proliferation coefficient of 12.51 and the tallest average bud height of 3.83 cm, along with vigorous growth. Rooting efficiency peaked in 1/2 MS medium supplemented with 1.0 mg·L⁻¹ NAA, attaining a 97.78% rooting rate with an average of 5.11 thick roots per plant, featuring well-developed lateral roots and abundant fine roots. Finally, transplantation success was maximized using a 2:1 peat-vermiculite substrate, achieving 95.56% survival rate with robust plant growth, expanded leaves, and continuous root development. 

CONCLUSION: For in vitro propagation of blackberry stem explants, the optimal sterilization protocol was achieved using 75% ethanol for 30 seconds followed by 2% sodium hypochlorite for 7 minutes.In primary culture, the most suitable medium was MS + 1.0 mg·L⁻¹ 6-BA + 0.2 mg·L⁻¹ NAA.For shoot proliferation, the best results were obtained with MS + 0.8 mg·L⁻¹ 6-BA + 0.1 mg·L⁻¹ NAA, promoting optimal shoot multiplication. In the rooting stage, the highest rooting efficiency was observed in 1/2MS + 1.0 mg·L⁻¹ NAA. For ex vitro acclimatization, a peat (2:1) mixture proved most effective for seedling survival and growth under controlled greenhouse conditions, and this technique can be potentially applied for commercialization of the plant.