Subculture age-dependent de novo organogenesis in Agapanthus praecox callus is governed by integrated auxin and ROS signaling

Callus-mediated regeneration is critical for large-scale propagation of Agapanthus praecox , yet the regulatory mechanisms underlying subculture age-dependent callus regenerative potential remain elusive. In dicotyledonous plants, auxin and reactive oxygen species (ROS) work synergistically to control de novo organogenesis. By contrast, this regulatory mechanism in monocotyledonous callus under long-term subculture has not yet been elucidated. Here, we integrated transcriptomic sequencing, targeted metabolomics (plant hormones, sugars), untargeted LC-MS metabolomics, and ROS/antioxidant enzyme activity assays to systematically investigate the regenerative potential and intrinsic regulatory networks of A. praecox calli across four subculture stages (3, 6, 9, and 12 months; 3 M, 6 M, 9 M and 12 M). Phenotypically, 9 M calli exhibited the highest organogenesis efficiency, whereas 12 M calli displayed enhanced proliferation but drastically diminished regenerative capacity, a finding that confirmed subculture age as the decisive factor governing regenerative competence. Transcriptomic analysis identified a 582-gene module tightly correlated with the superior regenerative capacity of 9 M calli. Integrated transcriptome-metabolome analysis further revealed three core regulatory pathways: plant hormone signaling, sugar metabolism, and oxidative stress response. Targeted metabolite analyses verified that indole-3-acetic acid (IAA), cis-zeatin riboside (cZR), and sucrose are critical regulators of regeneration efficiency. Notably, the high regenerative efficiency of 9 M calli was accompanied by elevated IAA levels, increased H₂O₂ accumulation, and enhanced SOD activity. Dynamic ROS accumulation at 6 M and 9 M facilitated regenerative reprogramming, while disrupted ROS homeostasis in 12 M calli contributed to regenerative decline; the expression of antioxidant genes was strongly correlated with cellular ROS levels. Overall, this study identifies a synergistic regulatory module of auxin and ROS that governs de novo organogenesis in long-term subcultured callus of the perennial monocotyledonous plant. These findings clarified the subculture age-dependent regulatory mechanisms of callus regeneration and laid a theoretical foundation for optimizing A. praecox callus subculture protocols, shortening de novo organogenesis induction cycles, and enhancing induction efficiency.