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

Wang, Dan; Liu, Yin; Zhang, Yao; Huang, Xiumei; Wang, Jiaxuan; Wang, Yi; Liu, Yue; Yan, Chao; Lv, Bingsheng; Jia, Yue

doi:10.3390/plants14172664

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

by

Dan Wang

^1,†,

Yin Liu

^2,†

,

Yao Zhang

¹,

Xiumei Huang

¹,

Jiaxuan Wang

¹,

Yi Wang

¹,

Yue Liu

¹,

Chao Yan

¹,

Bingsheng Lv

^1,*

and

Yue Jia

^1,*

¹

College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China

²

College of Horticulture, Jilin Agricultural University, Changchun 130118, China

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Plants 2025, 14(17), 2664; https://doi.org/10.3390/plants14172664

Submission received: 8 July 2025 / Revised: 4 August 2025 / Accepted: 25 August 2025 / Published: 26 August 2025

(This article belongs to the Special Issue Applications of Plant Biotechnology: In Vitro Propagation and Plant Transformations)

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Abstract

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.

Keywords: Welsh onion; transient transformation; hairy root induction; protoplast transformation

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MDPI and ACS Style

Wang, D.; Liu, Y.; Zhang, Y.; Huang, X.; Wang, J.; Wang, Y.; Liu, Y.; Yan, C.; Lv, B.; Jia, Y. Establishment of Transient Transformation Systems in Welsh Onion (Allium fistulosum L.): Hairy Root Induction and Protoplast Transformation. Plants 2025, 14, 2664. https://doi.org/10.3390/plants14172664

AMA Style

Wang D, Liu Y, Zhang Y, Huang X, Wang J, Wang Y, Liu Y, Yan C, Lv B, Jia Y. Establishment of Transient Transformation Systems in Welsh Onion (Allium fistulosum L.): Hairy Root Induction and Protoplast Transformation. Plants. 2025; 14(17):2664. https://doi.org/10.3390/plants14172664

Chicago/Turabian Style

Wang, Dan, Yin Liu, Yao Zhang, Xiumei Huang, Jiaxuan Wang, Yi Wang, Yue Liu, Chao Yan, Bingsheng Lv, and Yue Jia. 2025. "Establishment of Transient Transformation Systems in Welsh Onion (Allium fistulosum L.): Hairy Root Induction and Protoplast Transformation" Plants 14, no. 17: 2664. https://doi.org/10.3390/plants14172664

APA Style

Wang, D., Liu, Y., Zhang, Y., Huang, X., Wang, J., Wang, Y., Liu, Y., Yan, C., Lv, B., & Jia, Y. (2025). Establishment of Transient Transformation Systems in Welsh Onion (Allium fistulosum L.): Hairy Root Induction and Protoplast Transformation. Plants, 14(17), 2664. https://doi.org/10.3390/plants14172664

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

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