Variable NAA concentrations regulate efficient somatic embryogenesis and plant regeneration mediated by FELBs and RTBs in Allium fistulosum.

Somatic embryogenesis (SE) underpins plant regeneration, genetic transformation and virus-free seedling propagation, yet efficient SE systems using root explants remain lacking in Allium fistulosum. Here, we establish and characterize two novel high-efficiency SE pathways mediated by frog egg-like bodies (FELBs) and rhizoid tubers (RTBs) in A. fistulosum, and clarify the regulatory effects of NAA, 2,4-D and TDZ on morphogenesis and plant regeneration. Screening of multiple explants demonstrated that only root segments and stem tips could form translucent mucilaginous callus, while single 2,4-D treatment failed to induce callus at all tested concentrations. Under dark conditions, 10 mg/L NAA maximized callus induction (94.233%) and FELB formation (87.833%) in root explants with significant dose-dependent effects. Phytocytohistological analysis confirmed that FELBs/RTBs develop via three unique stages (pro-embryo, globular embryo, heart/torpedo transitional embryo), distinct from the five canonical stages of typical somatic embryos. For RTB pathway, 5.0 mg/L NAA induced the maximum rhizoid number (54.867 per explant), and subsequent treatment with 20.0 mg/L TDZ under high light achieved optimal RTB production (51.333 per explant). Stage-specific BAP application yielded 100% FELB germination and over 90% RTB plantlet regeneration, with both regenerated seedlings exhibiting > 90% acclimatization survival rates. This study first elucidates the ontogeny of FELB and RTB in monocot A. fistulosum. This is the first study characterizing FELB/RTB developmental stages and ontogeny in a monocot, showing striking homology to eudicotyledons, confirming that FELB- and RTB-mediated somatic embryogenesis can also be achieved in monocots. These optimized systems provide robust technical support for high-frequency regeneration, genetic modification and virus-free seedling production of Allium crops.