Wang Dan, Liu Yin, Zhang Yao, Huang Xiumei, Wang Jiaxuan, Wang Yi, Liu Yue, Yan Chao, Lv Bingsheng, Jia Yue
College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China.
College of Horticulture, Jilin Agricultural University, Changchun 130118, China.
Plants (Basel). 2025 Aug 26;14(17):2664. doi: 10.3390/plants14172664.
Welsh onion ( 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 -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 () strain selection (with Ar.Qual demonstrating superior performance), explant type efficacy, bacterial suspension optical density (OD = 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% (/) Cellulase R-10, 0.7% (/) Macerozyme R-10, and 0.4 M mannitol, yielding 3.3 × 10 viable protoplasts g 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.
大葱（L.）是一种具有全球重要意义的蔬菜、调味剂和植物药资源，但在用于功能基因组研究的既定瞬时表达平台上一直无法获得。为了解决这一关键的方法学限制，我们提出了针对介导的毛状根转化和原生质体瞬时表达系统的系统优化方案，在该物种的转化效率方面取得了显著进展。通过对关键参数的系统优化，包括（）菌株选择（Ar.Qual表现出卓越性能）、外植体类型效果、细菌悬浮液光密度（OD = 0.3）和乙酰丁香酮诱导浓度（100 μM），我们建立了一种高效的茎盘感染方法，毛状根诱导效率达到88.75%。随后对原生质体分离方案的优化确定了最佳酶解条件：使用1.0%（/）纤维素酶R - 10、0.7%（/）离析酶R - 10和0.4 M甘露醇对幼叶进行6小时暗消化，每克鲜重产生3.3×10个有活力的原生质体，活力为90%。通过聚乙二醇介导的瞬时转化进行系统功能验证，证实了绿色荧光蛋白（GFP）报告基因的成功表达，荧光显微镜观察确认。作为首次记录的大葱瞬时表达平台，这些方案能够进行重要的分子研究，包括植物体内启动子活性分析、亚细胞蛋白质定位以及基于CRISPR的基因组编辑验证。这一方法学突破克服了大葱分子生物学先前的技术限制，为加速这一农业重要物种的基因功能表征提供了关键工具。
