Welsh onion (Allium fistulosum L.), a globally significant vegetable, flavoring agent, and phytomedicine resource, has remained unavailable with established transient expression platforms for functional genomic investigations. To address this critical methodological limitation, we present systematically optimized protocols for both Agrobacterium-mediated hairy root transformation and protoplast transient expression systems, achieving significant advances in transformation efficiency for this species. Through systematic optimization of key parameters, including Agrobacterium rhizogenes (A. rhizogenes) strain selection (with Ar.Qual demonstrating superior performance), explant type efficacy, bacterial suspension optical density (OD₆₀₀ = 0.3), and acetosyringone induction concentration (100 μM), we established a highly efficient stem disc infection methodology, achieving 88.75% hairy root induction efficiency. Subsequent optimization of protoplast isolation protocols identified the optimal enzymatic digestion conditions: 6-h dark digestion of young leaves using 1.0% (w/v) Cellulase R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M mannitol, yielding 3.3 × 10⁶ viable protoplasts g⁻¹ FW with 90% viability. System functionality validation through PEG-mediated transient transformation demonstrated successful green fluorescent protein (GFP) reporter gene expression, confirmed by fluorescence microscopy. As the first documented transient expression platforms for Welsh onion, these protocols enable essential molecular investigations, including in planta promoter activity profiling, subcellular protein localization, and CRISPR-based genome-editing validation. This methodological breakthrough overcomes previous technical constraints in Welsh onion molecular biology, providing critical tools for accelerated gene functional characterization in this agriculturally important species.

Plants exhibit diverse pathways to regulate the timing of flowering. Some plant species require a vegetative phase before being able to perceive cold stimuli for the acceleration of flowering through vernalization. This research confirms the correlation between the vernalization process and seedling age in Welsh onions. Findings from two vernalization experiments conducted at different time intervals demonstrate that seedlings must reach a vegetative phase of at least 8 weeks to consistently respond to vernalization. Notably, 8–week–old seedlings subjected to 6 weeks of vernalization displayed the shortest time to bolting, with an average duration of 138.1 days. Transcriptome analysis led to the identification of genes homologous to those in Arabidopsis thaliana that regulate flowering. Specifically, AfisC7G05578 (CO), AfisC2G05881 (AP1), AfisC1G07745 (FT), AfisC1G06473 (RAP2.7), and AfisC2G01843 (VIM1) were identified and suggested to have potential significance in age–dependent vernalization in Welsh onions. This study not only presents a rapid vernalization method for Welsh onions but also provides a molecular foundation for understanding the interplay between seedling age and vernalization.