Establishment of an <italic>In Vitro</italic> Regeneration System for Stem Segments of Blackberry APF-190T

INTRODUCTION: Blackberry (Rubus fruticosus) is an important economic crop whose fruits are not only rich in fiber, vitamins, and phenolic metabolites, but also have significant health benefits. Traditional blackberry propagation methods, including seed propagation, layering, cutting, and suckering, have various limitations. For example, seed propagation has a long cycle, a low germination rate, and variable offspring traits. In contrast, tissue culture-based rapid propagation technology offers distinct advantages, such as season-independent operation, a high multiplication coefficient, and preservation of superior maternal traits, making it an effective solution for blackberry plantlet 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 plantlets 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 regulatory 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 plantlets was further analyzed. RESULTS: Comparative experiments on disinfection durations revealed that a 7-minute treatment yielded optimal results for blackberry explants, with 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 media, 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 was the most effective, achieving a 100% bud induction rate and producing robust seedlings with dark green leaves. Subsequent proliferation culture with a combination of 0.8 mg∙L–1 6-BA and 0.1 mg∙L–1 NAA resulted in the highest adventitious bud proliferation coefficient of 12.51 and the tallest average bud height of 3.83 cm, along with vigorous growth. The rooting efficiency peaked in 1/2MS 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 when a 2:1 (v/v) peat-vermiculite substrate was used, resulting in a 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 s followed by 2% sodium hypochlorite for 7 min. For 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. During the rooting stage, the highest rooting efficiency was observed in the 1/2MS+1.0 mg∙L–1 NAA group. For ex vitro acclimatization, a peat mixture proved most effective for seedling survival and growth under controlled greenhouse conditions, and this technique can potentially be applied for commercialization of the plant. Establishment of an in vitro regeneration system for stem segments of blackberry APF-190T. A series of studies was carried out on sterile seedling establishment, multiplication culture, rooting culture and other aspects of the blackberry variety APF-190T.