Establishment of Transient Transformation Systems in Welsh Onion (Allium fistulosum L.): Hairy Root Induction and Protoplast Transformation

Welsh onion (<i>Allium fistulosum</i> 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 <i>Agrobacterium</i>-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 <i>Agrobacterium rhizogenes</i> (<i>A. rhizogenes</i>) 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% (<i>w</i>/<i>v</i>) Cellulase R-10, 0.7% (<i>w</i>/<i>v</i>) 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.