Establishment of Hairy Root Genetic Transformation System in Xanthoceras sorbifolium Mediated by Agrobacterium rhizogenes

doi:10.13560/j.cnki.biotech.bull.1985.2025-1162

Abstract

Abstract:

Objective Xanthoceras sorbifolium is a woody oil crop with both ecological restoration and economic value, and characteristic of Northwest China. Establishing an efficient hairy root genetic transformation system for X. sorbifolium may provide technical support and research foundation for gene function validation and the targeted breeding of stable, high-yielding superior varieties through molecular breeding techniques. Method Using the hypocotyls of four superior X. sorbifolium seedling genotypes and five Agrobacterium rhizogenes strains as experimental materials, the cut-soak-bud method was employed to induce transgenic hairy roots to > 2 cm in length. Phenotypic observation, PCR and sequencing techniques were utilized to evaluate the expression and editing efficiency of the gene in this technical system. Result Using the RUBY gene as the reporter gene, the selected most suitable strain of A. rhizogenes was K599, with the plant induction rate of 81.67% and the positive rate of over 51.64% for the transgenic hairy roots. The successful integration of the RUBY gene into the genome of X. sorbifolium hairy roots were detected by PCR amplification and RT-qPCR quantitative methods. Using the GUS-GFP gene as the reporter gene, the transgenic hairy root was stained to be blue with GUS staining solution and observed with obvious green fluorescence under the microscope, further confirming that this technical system can be used for gene expression and functional localization research. In addition, the CRISPR/Cas9 gene editing vector for the XsPDS3 gene was constructed, and its target editing efficiency was detected to be 20% through sequencing, demonstrating the potential of this system in gene editing. Conclusion This study established a simple and efficient genetic transformation system for the hairy roots of Wenguan fruit, providing a key technical platform for gene function verification of Wenguan fruit. It is expected to establish a genetic transformation system for Wenguan fruit through root regeneration for genetic improvement.

Key words: Xanthoceras sorbifolium, hairy roots, transformation system, gene function identification

ZHOU Tao, LI Ya-qiang, HE Hui-juan, HU Hai-fang, CHEN Tong-seng, PAN Yue, WANG Tian-bin. Establishment of Hairy Root Genetic Transformation System in Xanthoceras sorbifolium Mediated by Agrobacterium rhizogenes[J]. Biotechnology Bulletin, 2026, 42(7): 97-104.

Figures/Tables 7

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Usage
RUBY-PF RUBY-PR	AGACACTCCGCCTCCATCC	RUBY基因片段扩增
RUBY-PF RUBY-PR	TTCTACATCACCTGCCTGCTT	RUBY基因片段扩增
RUBY-RT-PF RUBY-RT-PR	ACGCATGAAGGTGAAGAAACA	RUBY基因表达检测
RUBY-RT-PF RUBY-RT-PR	CCATCCGCAGTGGGTGAG	RUBY基因表达检测
XsPDS3-T1	TTTCGAGGGAGTGAATCCATGGG	靶点序列
F2-tgRNA1-T1 R2-tgRNA1-T1	tgcagTTTCGAGGGAGTGAATCCAT	基因编辑载体构建
F2-tgRNA1-T1 R2-tgRNA1-T1	aaacATGGATTCACTCCCTCGAAAC	基因编辑载体构建
T1-PF T1-PR	GTCGGAGATGATGGTGATGAAT	靶点片段扩增
T1-PF T1-PR	AGTCCAGGCAAACAACCTGATA	靶点片段扩增

Fig. 1 Influence of Agrobacterium strain types on the transformation efficiency of hairy roots in X. sorbifoliumDifferent letters indicate significant difference (P<0.05). The same below

Fig. 2 Process of hairy root transformation technology for X. sorbifolium

Fig. 3 Phenotype and PCR detection of red transgenic roots in X. sorbifoliumA: Transforming the RUBY gene to produce a red transgenic hairy root phenotype. B: PCR amplification and electrophoresis identification of RUBY gene

Fig. 4 Transgenic expression detection of red transgenic roots of X. sorbifoliumA: Detection of RT-qPCR expression level. B: Detection of GUS staining. C: Detection of GFP fluorescence signal

Fig. 5 Optimization of induction efficiency of transgenic hairy rootsA: Positive rate of transgenic hairy roots at different seedling germination times. B: Induction efficiency of transgenic hairy root at different seedling germination times. C: Positive rate of transgenic hairy roots across different varieties. D: Induction efficiency of transgenic hairy root across different varieties

Fig. 6 CRISPR-Cas9 gene editing of X. sorbifolium hairy rootsA: The gene structure and selected targets of XsPDS3, with blue characters for sgRNA and red characters for PAM sequences. B: WT is wild-type sequencing result. C: Sequencing results of M1 as the target gene mutation type 1. D: M2 is the sequencing result of target sequence mutation type 2

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