Enhancing Agrobacterium-Mediated Hairy Root Transformation Efficiency in Peanut Through the Application of GRF, GIF and WOX Genes

1. Introduction

Peanut (Arachis hypogaea L.) is an important oil crop cultivated in more than 100 countries [1]. Their seeds are rich in lipids, proteins, folate, tocopherols, phytosterols, and polyphenols [2,3]. With rapid advances in biotechnology, genetic engineering has become a powerful approach for crop improvement [4,5]. However, peanut transformation still suffers from low efficiency and long regeneration cycles, limiting functional gene studies and molecular breeding [6]. Agrobacterium rhizogenes-induced hairy roots are fast-growing roots generated after infection and have been widely used for gene function studies, secondary metabolite production, and trait improvement [7]. In peanut, the system is still constrained by low induction efficiency, insufficient root biomass, and large variability among explants.

Growth-Regulating Factors (GRFs) are plant-specific transcription factors that promote growth and organ development. GRF-Interacting Factors (GIFs) act as transcriptional co-regulators and are essential for cell proliferation, organogenesis, and regeneration [8,9]. Overexpressing AtGRF1 or AtGRF2 in Arabidopsis increases leaf size, and overexpression of the pear gene PbGRF18 in tomato can promote fruit development and sugar accumulation [10,11]. Notably, GRF-GIF fusion (chimera) constructs can enhance transformation and regeneration efficiency and shorten regeneration cycles in crops such as maize, wheat, and soybean [12,13,14]. In peanut, however, the application of GRF/GIF has rarely been reported, highlighting the need to evaluate GRF-GIF tools and develop a more efficient and practical transformation platform.

WOX proteins are key transcription factors that maintain meristem activity and regulate organogenesis by controlling cell fate and re-differentiation [15]. In Arabidopsis, the WOX family (WUS and WOX1-WOX14) participates in development of multiple organs, including roots, stems, leaves, flowers, and fruits [16]. WOX11 directly activates LBD16 to initiate root primordia and thereby promotes lateral and adventitious root formation [17]. In woody species, BpWOX11 (Betula platyphylla) increases adventitious rooting of cuttings, and JrWOX5 (walnut) promotes root formation and influences plant architecture [18,19]. Despite these advances, the use of WOX genes to enhance induction performance in peanut hairy-root systems remains limited, and a WOX-based tool that improves rooting efficiency would be valuable.

In this study, we identified and cloned multiple peanut GRF genes, GRF-GIF fusion genes and WOX genes, and constructed high-expression vectors for peanut hairy-root transformation. With the exception of 2S-PL-GUS and GRF-2A-T-GUS, which showed lower transformation efficiency than the empty-vector control, most constructs improved transformation efficiency, with GRF-2A(396)-GIF-GUS achieving the highest rate (85.14 ± 2.94%; Table 1). Compared with the empty-vector control, GRF, GIF, and WOX treatments also promoted hairy-root growth, increasing the number of roots per explant by 1.3–2.4-fold and producing thicker, more highly branched roots. These tools can strengthen peanut hairy-root platforms for rapid gene function validation, trait improvement, secondary metabolite production, and related applications.

2. Results

2.1. Phylogenetic Selection of Peanut GRF/GIF/WOX Candidates and Construction of Expression Vectors

To identify peanut developmental regulators with potential utility in transformation enhancement, we performed phylogenetic analyses of GRF, GIF and WOX proteins from peanut and representative model/crop species. In the GRF clade, several peanut proteins grouped closely with established regulators such as AtGRF5 and TaGRF4, supporting functional conservation and motivating the selection of GRF-V829EQ, GRF-2A7ZAY and GRF-FF6C67 for downstream testing (Figure 1). Similarly, the peanut candidate GIF-HK1F5C clustered with the reference OsGIF, indicating it represents a likely functional GIF ortholog (Figure 1). For the WOX family, WOX-PLVV0P and an additional candidate, WOX-ZS5XSZ grouped with the reference AtWOX5, suggesting conserved roles associated with meristematic or regenerative competence (Figure 1).

Based on these phylogenetic relationships, we generated a compact vector set to evaluate single and combinatorial regulator modules in peanut hairy-root assays. All constructs shared a common architecture including a 35S::HygR selection cassette and a pAhUBQ4::GUS reporter cassette for rapid quantification of transformation output (Figure 2). Regulator expression cassettes were driven by pAhUBQ4, including single-gene constructs (GRF-V829EQ, GRF-2A7ZAY, WOX-PLVV0P) as well as combinatorial modules, notably GRF-GIF co-expression constructs (GRF-2A7ZAY–GIF-HK1F5C and GRF-FF6C67–GIF-HK1F5C) and a miR396-modified GRF-GIF module to mitigate miR396-associated repression (Figure 2). Together, these designs established a standardized genetic toolkit for comparing developmental regulator modules under the same promoter and reporter framework (Figure 1 and Figure 2).

2.2. Induction of Hairy Roots by GRF, GIF and WOX Genes

In our preliminary work, we established a peanut hairy-root transformation system. Using 30-day-old rootless seedlings as explants, Agrobacterium rhizogenes K599 carrying a GUS reporter and developmental regulator constructs was used to infect peanut stem segments. Driven by the peanut endogenous promoter AhUBQ4, transgenic hairy roots were efficiently induced (Figure 3) [20].

Developmental regulators such as GRF, GIF and WOX—and their combinations—can improve transgenic efficiency and shorten regeneration cycles in multiple species [12,21,22]. To evaluate their effects in peanut, we quantified transformation performance and performed GUS staining in transgenic hairy roots. Except for 2S-PL-GUS and GRF-2A-T-GUS, which showed lower transformation efficiency than the empty-vector control, all other constructs improved transformation efficiency. Among them, GRF-2A(396)-GIF-GUS showed the highest transformation efficiency (85.14 ± 2.94%; Table 1). Relative to the empty-vector control, GRF, GIF and WOX constructs also promoted root growth, increasing the number of hairy roots per explant by 1.3–2.4-fold and producing thicker, more highly branched roots (Table 1).

Explants from the same infection batch were transferred to MS medium and grown for 2 weeks before GUS staining. The staining results further supported that GRF, GIF and WOX constructs promote peanut hairy-root growth and branching (Figure 4).

3. Discussion

Peanut is an allotetraploid legume formed through hybridization and domestication of the wild diploid ancestors Arachis duranensis (AA genome) and Arachis ipaensis (BB genome) [23,24]. As a major oilseed crop, peanut seeds contain abundant storage proteins [25]. Establishing an efficient genetic transformation system is critical for genetic engineering-based germplasm innovation. However, current peanut transformation methods often suffer from chimerism, low transformation efficiency, and poor regeneration efficiency, which constrain functional genomics and molecular breeding [26]. Agrobacterium rhizogenes-mediated hairy-root transformation integrates T-DNA from the Ri plasmid into the plant genome, inducing transgenic hairy roots [27]. Because it bypasses complex tissue culture and whole-plant regeneration, this approach enables rapid generation of composite plants with relatively short cycles and good genetic stability [28]. It is therefore well suited for root-specific gene function studies, metabolite production, and heterologous protein expression [29].

Overexpression of developmental regulators has broad utility for improving plant regeneration and transformation. GRF-GIF fusion expression can substantially enhance transformation efficiency, increasing rates by 3.0–4.7-fold in wheat, rye, tomato, and citrus [12,30]. WOX genes also influence root development and differentiation: for example, WOX11 overexpression in rice increases adventitious root formation, whereas WOX11 mutants show root defects [31]. Plants expressing TaWOX9 in Arabidopsis develop longer roots than controls, indicating a role in promoting root growth [32]. Consistent with these reports, our peanut GRF, GRF-GIF and WOX constructs improved hairy-root induction and growth, increasing roots per explant by 1.3–2.4-fold and generating thicker, more highly branched roots. GUS staining confirmed stable transgene expression, supporting the use of these constructs as practical tools for peanut functional validation and metabolic engineering.

Although GRF-2A(396)-GIF-GUS produced transgenic hairy roots with high transformation efficiency and genetic stability, regenerating whole plants from hairy roots remains challenging. To date, stable transgenic plants regenerated from hairy roots have been reported for only a limited number of species, including sweet potato, radish, apple, and several medicinal plants [22,33,34]. Previous studies show that developmental regulators such as WOX, GRF and GIF play important roles in organ regeneration. For example, the Wus2-ipt combination can promote callus formation and directly induce regeneration buds at wound sites without exogenous hormones; using early-bolting genotypes, transgenic seeds can be obtained within 6–7 months, helping overcome radish transformation bottlenecks [22]. In sugar beet, AtGRF5 promotes shoot formation and improves transformation of recalcitrant varieties, and GRF5/GRF6/GRF9 can induce callus formation and differentiation in rapeseed [35]. GRF-GIF chimeras in Arabidopsis, wheat, rice, and maize enhance regeneration, potentially through activation of transcription via interactions with SWI2/SNF2 complexes [12,36]. Because GRF and GIF proteins are highly conserved, GRF-GIF chimeras may be broadly applicable to species with low regeneration efficiency [9]. Our results demonstrate that these developmental regulators can improve peanut hairy-root transformation and growth; future work should test their utility in peanut regeneration from hairy roots.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

We followed previously published protocols with modifications. Seeds from four red-seeded peanut varieties were surface-rinsed and soaked overnight. After removing the seed coat under a laminar-flow hood, seeds were surface-sterilized in 75% ethanol for 1 min and then in 2.5% sodium hypochlorite (available chlorine) for 15 min. Seeds were rinsed five times with sterile water. Half of each cotyledon was removed with a scalpel, and cotyledons with intact embryos were placed on MS medium for germination. Seedlings were grown in a tissue-culture room under a 28 °C day/25 °C night cycle with 16 h light/8 h dark (Figure 3A,B).

4.2. Identification and Cloning of GRF, GIF and WOX Genes in Peanut

Candidate members of the GRF, GIF and WOX gene families were identified from the Arachis hypogaea cv. Tifrunner genome (https://data.legumeinfo.org/Arachis/hypogaea/genomes/Tifrunner.gnm2.J5K5/) [37]. Protein sequences of reported GRF/GIF/WOX regulators from Arabidopsis, wheat, and rice were used as queries for homology-based searches against the peanut proteome. Putative candidates were further validated by confirming the presence of conserved domains using standard domain databases (NCBI CDD).

To infer evolutionary relationships and support candidate selection, representative GRF/GIF/WOX proteins from peanut and reference species were aligned and used to construct phylogenetic trees (Figure 1). Based on phylogenetic placement relative to known regulators (AtGRF5, TaGRF4, OsGIF, AtWOX5), several peanut candidates were prioritized for cloning and functional evaluation, including GRF-V289EQ, GRF-2A7ZAY, GRF-FF6C67, GIF-HK1F5C, WOX-PLVV0P and WOX-ZS5XSZ (Figure 1 and Figure 2).

Total RNA was extracted from young peanut leaves (Tifrunner) and reverse-transcribed into cDNA. Full-length coding sequences (CDSs) were amplified using gene-specific primers (Table S1), purified, cloned into an intermediate vector, and verified by Sanger sequencing prior to binary vector assembly.

4.3. Vector Construction

Binary vectors were constructed using a backbone reported previously [20]. As illustrated in Figure 1B, all constructs were designed within the T-DNA region and included: (i) a hygromycin resistance cassette (Hyg) driven by the CaMV35S promoter for selection, and (ii) a GUS reporter cassette driven by the AhUBQ4 promoter (pAhUBQ4) to facilitate rapid scoring of transformed hairy roots.

For functional testing of developmental regulators, individual CDSs and combinatorial modules were expressed under pAhUBQ4 (Figure 2), including GRF-V829EQ, GRF-2A7ZAY, WOX-PLVV0P, and a dual-WOX construct (WOX-ZS5XSZ-WOX-PLVV0P) connected by a linker. To enable co-expression of GRF and GIF, GRF-GIF combinatorial constructs were generated by linking GRF-2A7ZAY (or GRF-FF6C67) with GIF-HK1F5C using a linker sequence (Figure 2). In addition, a miR396-related modified GRF module (as indicated by “Mutant miR396” in Figure 2) was generated to reduce miR396-mediated repression while maintaining the encoded protein sequence, and then assembled with GIF-HK1F5C for co-expression.

All plasmids were verified by restriction digestion and sequencing, and then introduced into Agrobacterium rhizogenesstrain K599 using standard procedures. Transformed strains were selected on appropriate antibiotics before infection assays.

4.4. Hairy Root Transformation

Stem segments (3–5 cm) from in vitro–grown seedlings were wounded with a scalpel and infected with Agrobacterium rhizogenes. For infection, A. rhizogenes was resuspended in infection medium (1/2 MS liquid medium supplemented with 100 μM acetosyringone) and adjusted to OD600 = 0.6; the suspension was incubated at 28 °C for 0.5–2 h. Explants were soaked in the infection suspension and shaken at 150 rpm for 30 min (Figure 3C). After infection, explants were washed 2–3 times with sterile water, blotted dry, and placed on 1/2 MS solid medium over filter paper for co-cultivation at 23 °C for 3 days (Figure 3D). Explants were then transferred to 1/2 MS solid medium containing timentin (300 mg/L) to suppress Agrobacterium and induce hairy roots (Figure 3E). Cultures were maintained at 28 °C under 16h light/8h dark, and the medium was refreshed every 2 weeks (Figure 3F).

4.5. GUS Histochemical Staining

Infected hairy roots were incubated in GUS staining solution (GUS staining kit, COOLABER, SL7160) at 37 °C for 24 h. Samples were then rinsed 2–3 times with sterile water and photographed for documentation (Figure 4).

4.6. Data Statistics and Analysis

Data were analyzed in Excel. The transformation (positive root) rate (%) was calculated as: (number of explants producing positive transgenic hairy roots / number of explants producing hairy roots) × 100%.

5. Conclusions

This study demonstrates that GRF, GIF and WOX developmental regulators can enhance peanut hairy-root transformation and promote root growth. Compared with the empty-vector control, these treatments increased hairy-root induction and growth, raising the number of roots per explant by 1.3–2.4-fold and producing thicker, more highly branched roots. These tools provide a practical platform for peanut genetic engineering, including rapid gene function validation, CRISPR construct evaluation, and metabolite production.