Comparative Transcriptomic Analysis of Two Apple Cultivars in Response to Dual Cytokinin Applied In Vitro

The application of dual cytokinins can significantly enhance shoot multiplication rates in specific apple cultivars compared to standard protocols using a single cytokinin. This study presents a comprehensive analysis of shoot multiplication parameters and the underlying transcriptomic response of two distinct apple scion cultivars, cvs. Húsvéti rozmaring and McIntosh, to the simultaneous application of two cytokinins (BA and KIN). Morphological parameters were recorded, followed by comparative RNA-seq analysis and RT-qPCR validation. Our results demonstrate that the BA+KIN treatment induces a unique transcriptomic signature in both cultivars, which cannot be explained by a simple dose–response effect. In cv. McIntosh, 76% of the DEGs were uniquely regulated by the combination, while in cv. Húsvéti rozmaring, although the overlap with single treatments was higher, 17% of the DEGs (representing 1218 genes) were still exclusively activated by the BA+KIN treatment. The fact that the combined treatment recruits specific gene sets and metabolic pathways that remain silent under single BA or KIN applications—regardless of the cultivar—strongly supports a synergistic or non-additive hormonal interaction rather than a response to increased total cytokinin concentration. The dual treatment revealed 3209 DEGs in the inter-cultivar comparison, reflecting distinct strategies: cv. Húsvéti rozmaring achieved high efficiency growth by down-regulating internal hormones, whereas cv. McIntosh exploited intense auxin signaling and hormonal plasticity to maximize bud release. These results prove that distinct molecular pathways can lead to peak performance depending on the apple cultivar.