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.

Carpel number (CN) is an important trait affecting the fruit size and shape of melon, which plays a crucial role in determining the overall appearance and market value. A unique non-synonymous single nucleotide polymorphism (SNP) in CmCLAVATA3 (CmCLV3) is responsible for the variation of CN in C. melo ssp. agrestis (hereafter agrestis), but it has been unclear in C. melo ssp. melo (hereafter melo). In this study, one major locus controlling the polymorphism of 5-CN (multi-CN) and 3-CN (normal-CN) in melo was identified using bulked segregant analysis (BSA-seq). This locus was then fine-mapped to an interval of 1.8 Mb on chromosome 12 using a segregating population containing 1451 progeny. CmCLV3 is still present in the candidate region. A new allele of CmCLV3, which contains five other nucleotide polymorphisms, including a non-synonymous SNP in coding sequence (CDS), except the SNP reported in agrestis, was identified in melo. A cis-trans test confirmed that the candidate gene, CmCLV3, contributes to the variation of CNs in melo. The qRT-PCR results indicate that there is no significant difference in the expression level of CmCLV3 in the apical stem between the multi-CN plants and the normal-CN plants. Overall, this study provides a genetic resource for melon fruit development research and molecular breeding. Additionally, it suggests that melo has undergone similar genetic selection but evolved into an independent allele.