Search Results (108)

Cowpea (Vigna unguiculata (L.) Walp.) is a globally cultivated leguminous crop, but an efficient and stable genetic transformation system for cowpea is lacking. Thus, in this study, using different cowpea accessions, the main factors (genotype, explant, Agrobacterium strain for infection, and vector) affecting genetic transformation efficiency were systematically screened. Among the 43 cowpea accessions, two accessions (JD-0212 and A132) whose in vitro regeneration frequencies were high (propagation coefficient > 0.8 and adventitious bud induction index > 0.7) were identified. A system with a high infection rate for the two cowpea accessions was subsequently constructed, including cotyledonary nodes with cotyledons as the optimal explants, the Agrobacterium rhizogenes strain K599 and the rbcs-RUBY vector. Next, the system was optimized for its transformation conditions, such as infection duration, vacuum infiltration parameters and cocultivation time. The maximum transformation efficiency of genotype JD-0212 reached 82.79% under the optimal transformation conditions: 60 min of infection combined with 30 s of vacuum infiltration (−0.08 MPa), followed by four days of cocultivation. Furthermore, the transformation efficiency was validated in 86 cowpea accessions using two distinct vectors (rbcs-RUBY and bs-EGFP), indicating an average transformation efficiency of 42.09% (ranging between 4.04% and 82.79%). An efficient hairy root genetic transformation system for cowpea was established in this study. Full article

Cultivated lettuce (Lactuca sativa L.) is a major leafy crop and an emerging model for functional genomics within the Asteraceae family, supported by high-quality reference genomes and efficient transformation systems. Although CRISPR/Cas technology offers powerful opportunities for crop improvement, editing efficiency depends on optimized construct architecture and reliable guide RNA (gRNA) validation. However, a rapid platform for evaluating CRISPR reagents in lettuce is still lacking. Here, we developed an efficient hairyroot-based system to accelerate CRISPR/Cas genome editing optimization in L. sativa. Four Agrobacterium rhizogenes strains were compared for hairy root induction in two cultivars, ‘Saladin’ and ‘Osiride’, identifying strain ATCC15834 as the most effective based on transformation frequency and root production. Using this platform, we evaluated multiple CRISPR construct configurations, including alternative promoters for nuclease and gRNA expression. A plant-derived promoter combined with At-pU6-26 variant significantly improved editing efficiency. As a proof of concept, we targeted LsHB2, the putative ortholog of Arabidopsis thaliana ATHB2, a key regulator of the shade avoidance response using SpCas9, SaCas9, and LbCas12a nucleases. The system enabled rapid genotyping and quantitative indel profiling. Overall, this workflow provides a robust framework for efficient guide selection and construct optimization in lettuce genome editing. Full article

The Lateral Organ Boundaries Domain (LBD) gene family encodes plant-specific transcription factors that play pivotal roles in growth, development, and stress responses. However, a comprehensive genome-wide analysis of the LBD family in watermelon (Citrullus lanatus) has not been conducted until now. In this study, we identified 39 ClLBD genes using the latest watermelon reference genome and systematically analyzed the function of ClLBD2 in root development. These ClLBDs are unevenly distributed across 10 chromosomes except Chr4. Evolutionary analysis grouped the gene family members into six subgroups: Class I (a–e) and Class II. Physicochemical properties and gene structure analysis showed that the ClLBD protein members are tightly conserved. In the promoter regions of ClLBD genes, we identified abundant cis-acting regulatory elements related to abiotic stress and hormone responses. Through RNA-seq analysis from a cucurbit database, we found that several ClLBD genes showed high relative expression in roots, with ClLBD2 being the most highly expressed. Since its subfamily includes AtLBD25, a known root development-related gene, we hypothesized that ClLBD2 might be involved in root development. To validate this, ClLBD2-edited roots were generated using the CRISPR-Cas9 system and Agrobacterium rhizogenes-mediated transformation. Compared to the wild type, the ClLBD2 edited roots exhibited significant reduction in taproot length and lateral root numbers, indicating that ClLBD2 may regulate root development. This study provides the first comprehensive analysis of the LBD gene family in watermelon, offering valuable insights for evolutionary and further functional studies of ClLBD genes. Full article

Powdery mildew poses a significant threat to watermelon production. The development of disease-resistant varieties through gene editing represents a major focus in current breeding research. In this study, we identified an MLO family gene in watermelon, denoted by ClMLO5b, which is phylogenetically closely related to cucumber CsaMLO8 and melon CmMLO5. Homology modeling revealed high conservation of the three-dimensional protein structures among these orthologs. Expression analysis demonstrated that ClMLO5b is significantly up-regulated upon powdery mildew infection, and the protein localizes to the plasma membrane. To validate its function, we first employed an Agrobacterium rhizogenes-mediated hairy root transformation system to rapidly verify the editing efficiency of two CRISPR/Cas9 targets designed for ClMLO5b. Subsequently, stable transgenic watermelon plants were generated via Agrobacterium tumefaciens-mediated transformation, and a mutant line with homozygous substitutions at target site 2 was obtained. Disease resistance assays showed that, compared to wild-type plants, the Clmlo5b exhibited strongly inhibited mycelial growth, significantly reduced disease severity, and a substantial decrease in spore production after inoculation with powdery mildew. Our findings confirm that ClMLO5b is a key susceptibility gene in watermelon and provide both a promising genetic target and valuable breeding material for developing powdery mildew-resistant watermelon varieties. Full article

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is a prized Chinese medicinal plant renowned for its medicinal and economic importance. In our previous study, a key auxin-related gene ThGH3.1 (encoding amide synthetase) was identified by quantitative transcriptome sequencing. To explore ThGH3.1 function in root development, we generated ThGH3.1-overexpressing and RNA interference (RNAi) transgenic hairy root lines via Agrobacterium rhizogenes (A. rhizogenes)-mediated genetic transformation. The results showed that overexpression of ThGH3.1 significantly inhibited the total length and the lateral root number of hairy roots, accompanied by significantly increased levels of methyl indole-3-acetate (MeIAA) and indole-3-acetyl-aspartate (IAA-Asp). In contrast, ThGH3.1 knockdown displayed an opposite trend. To further confirm the function of ThGH3.1, the overexpression vector was heterologously transformed into wild-type Arabidopsis. After single-copy and homozygous line screening, three overexpressing lines (named G19, G29, and G32) were obtained. The primary root length of transgenic Arabidopsis was significantly shortened, with a significant increase in indole-3-acetonitrile (IAN) levels. Further pot experiments revealed that transgenic Arabidopsis grew more slowly, had significantly smaller leaf areas, and lower plant height. The indole-3-acetic acid (IAA) treatment suggested that ThGH3.1 responded to IAA. Collectively, these findings highlight the crucial roles of ThGH3.1 in regulating root development, which will deepen our understanding of the molecular mechanisms underlying root development in T. hemsleyanum. Full article

Sweet potato (Ipomoea batatas) is a globally important crop and one of a growing number of plants recognized as naturally transgenic, harboring Agrobacterium-derived T-DNA genes whose functions remain largely uncharacterized. In this proof-of-concept study, we applied CRISPR/Cas9 technology to generate targeted knockouts of the Ib-rolB/C and Ib-rolD-like genes located within the sweet potato cellular T-DNA2 (IbT-DNA2) region. Mutations were introduced into sweet potato callus cultures using an optimized genome editing protocol, with most edits consisting of single-nucleotide insertions. Knockout of Ib-rolB/C did not affect callus growth but significantly reduced levels of chlorogenic acid derivatives. Validation in planta using transient expression in I. batatas leaves confirmed the suppressive effect of Ib-rolB/C disruption on polyphenol content. In contrast, Ib-rolD-like knockout lines showed reduced biomass accumulation and downregulation of cell cycle–related genes, but did not display significant changes in metabolite content in either callus cultures or leaf tissues. These findings suggest that Ib-rolB/C and Ib-rolD-like may differentially contribute to growth and secondary metabolism in sweet potato. Full article

Japanese apricot (Prunus mume Sieb. et Zucc.) is a dicotyledonous plant from the Rosaceae family that originated in China. Functional genomic studies in Japanese apricot are essential to elucidate the molecular mechanisms underlying key agronomic traits and to accelerate crop improvement. However, the lack of an efficient genetic transformation system has hindered gene function analysis and impeded molecular breeding efforts. Agrobacterium rhizogenes-mediated transformation has emerged as a robust tool for functional gene validation and studying root-specific processes across diverse plant species, due to its simple protocol and rapid turnaround time. Notably, Agrobacterium-mediated transformation remains notoriously recalcitrant in Rosaceae species, particularly in Japanese apricot. Through screening of ten Japanese apricot varieties, we identified ‘Muguamei’ (MGM) as the optimal cultivar for tissue culture. Using its genotype, we established an Agrobacterium rhizogenes-mediated transformation system for Japanese apricot via an in vitro approach. The binary vector incorporated the RUBY reporter for visual selection and eYGFPuv for fluorescent validation of transformation events. Furthermore, CRISPR/Cas9-mediated knockout of PmPDS in ‘Muguamei’ calli generated albino phenotypes, confirming successful genome editing. Through optimization of antibiotics, the study achieved an 80% explant survival rate using Woody Plant Medium (WPM) supplemented with 6-BA (0.5 mg/L) and TDZ (0.05 mg/L). For in vitro micropropagation, we found that ‘Muguamei’ exhibited optimal shoot growth in the presence of 6-BA (0.06 mg/L) and TDZ (0.1 mg/L), and up to 8 bud proliferation lines could be reached under 4.0 mg/L 6-BA. During the rooting of micro shoots, ½MS medium performed better and reached the optimum root length (35.70 ± 4.56 mm) and number (6.00 ± 1.00) under IAA (0.5 mg/L) and IBA (0.4 mg/L). Leaf explants were cultured on WPM supplemented with TDZ (4.0 mg/L) and NAA (0.2 mg/L). 50 mg/L kanamycin concentrations were the suitable screening concentration. Full article

Agrobacterium rhizogenes (A. rhizogenes)-mediated transformation of hairy roots is a favored and flexible method for root gene functional analysis. However, the selection of transformants can be complex and time-consuming. Here, we describe our simplified method for the A. rhizogenes-mediated hairy root induction in young peanut shoots using an expression vector with RUBY for direct visual selection of transformants. Analyses verified that this method provides a high-efficiency gene transformation technique for peanut, with transformant frequencies between 46.2 and 73.7%. To test the utility of this method in gene functional analyses, it was used to overexpress AhLRX6 in hairy roots and we present our preliminary results indicating the production of thicker cells walls in root tips relative to the WT. Full article

Background/Objectives: Licorice (Glycyrrhiza glabra L.) is a highly important medicinal plant that is widely used in China owing to its active ingredients. Its main active components are flavonoids, including liquiritigenin, liquiritin and licochalcone A. The hairy roots (HRs) induced by Agrobacterium rhizogenes are a commonly used chassis in synthetic biology to enhance the production of active compounds in medicinal plants. Methods: A biosynthesis system to acquire the active ingredients of G. glabra was established using an HR culture system. It employed a transcriptome analysis to identify the change in gene expression following treatment with methyl jasmonate (MeJA). Results: After 28 days of suspension culture, the biomass of HRs increased by approximately 34.5-fold and reached 1.83 g/100 mL flask. Treatment with MeJA significantly increased the contents of liquiritigenin, liquiritin, and glabridin in the HRs. The transcriptome data indicated that MeJA activated the flavonoid biosynthetic pathway genes in the HRs, which was largely consistent with the qRT-PCR results. Furthermore, the overexpression of the GgCHS6 gene substantially increased the content of flavonoids in HRs. Conclusions: Collectively, this study established an HR system to biosynthesize the active ingredients of G. glabra using metabolic engineering and genetic engineering techniques and provides several valuable candidate genes for further functional study. Full article

The soybean cyst nematode (Heterodera glycines, SCN) is the leading pathogen causing economic losses in soybean production worldwide. Using resistant cultivars is the most sustainable control method, yet the molecular basis of this resistance remains unclear. Heinong 531 (HN531), a high-yield soybean variety rich in seed oil, shows broad resistance to multiple SCN races. In this research, we studied HN531’s resistance to SCN races 3 and 5 through phenotypic assessment and comparative transcriptomics. Although initial infection rates were similar between resistant HN531 and the susceptible Dongsheng 1 (DS1), HN531 limited later nematode development inside roots, with fewer progressing to the J2 stage and maturing females. RNA-seq at 5 days post-infection revealed 1459 differentially expressed genes (DEGs) in HN531, mainly involved in secondary metabolite pathways, especially phenylpropanoid biosynthesis. We pinpointed a β-glucosidase gene (Glyma.12G053800, BGLU) upregulated after SCN infection and naturally more expressed in HN531 roots than DS1. Functional tests using Agrobacterium rhizogenes-mediated hairy root transformation showed that overexpressing Glyma.12G053800 in the susceptible DS1 significantly decreased SCN development and adult female counts by around 65%, without affecting initial infection. These findings suggest Glyma.12G053800 contributes to SCN resistance via phenylpropanoid-driven secondary metabolism, offering new insights into nematode resistance pathways and a valuable genetic resource for breeding broad-spectrum resistant soybean varieties. Full article

Woody plants, comprising forest and fruit tree species, provide essential ecological and economic benefits to society. Their genetic improvement is challenging due to long generation intervals and high heterozygosity. Genetic transformation, which combines targeted DNA delivery with plant regeneration from transformed cells, offers a powerful alternative to accelerating their domestication and improvement. Agrobacterium tumefaciens, Rhizobium rhizogenes, and particle bombardment have been widely used for DNA delivery into a wide variety of explants, including leaves, stems, hypocotyls, roots, and embryos, with regeneration occurring via direct organogenesis, callus-mediated organogenesis, somatic embryogenesis, or hairy root formation. Despite successes, conventional approaches are hampered by low efficiency, genotype dependency, and a reliance on challenging tissue culture. This review provides a critical analysis of the current landscape in woody plant transformation, moving beyond a simple summary of techniques to evaluate the co-evolution of established platforms with disruptive technologies. Key advances among these include the use of developmental regulators to engineer regeneration, the rise in in planta systems to bypass tissue culture, and the imperative for DNA-free genome editing to meet regulatory and public expectations. By examining species-specific breakthroughs in key genera, including Populus, Malus, Citrus, and Pinus, this review highlights a paradigm shift from empirical optimization towards rational, predictable engineering of woody plants for a sustainable future. Full article

Soil alkalinity severely restricts the cultivation of Lupinus angustifolius, a valuable legume. Wild soybean (Glycine soja) is a leguminous plant with extremely strong alkaline resistance (pH 8.5). Transferring the alkali-tolerant genes from wild soybeans into lupinus can effectively enhance the alkali tolerance. In this study, we combined transcriptome profiling and genetic transformation to elucidate the molecular basis of alkaline stress response in lupinus. RNA-seq analysis of root tips under acid (HCl, pH 4.0) and alkali (NaHCO₃, pH 8.5) stress revealed 104,353 annotated unigenes, with differential expression patterns highlighting enrichment in cellular component, binding, and catalytic activity categories. KEGG pathway analysis indicated that early responses involved ribosome-related pathways, while later stages activated plant hormone signaling and MAPK pathways. Notably, no homeodomain-leucine zipper (HD-Zip) family genes were identified in the lupinus genome. Therefore, we transferred GsHZ4, an alkali-resistant HD-Zip transcription factor from wild soybean into lupinus hairy roots via Agrobacterium rhizogenes-mediated transformation. Overexpression of GsHZ4 significantly enhanced antioxidant enzyme activities (CAT, POD, and SOD) and reduced malondialdehyde content under NaHCO₃ stress. Furthermore, the promoter of GsHZ4 expression was strongly induced by indole-3-acetic acid (IAA). Key alkali-responsive genes (LaKIN, LaMYB34, LaDnaJ1, LaDnaJ20, LaNAC22, and LaNAC35) were upregulated in transgenic lines, suggesting that GsHZ4 integrates into the endogenous stress-regulation network. Our findings demonstrate that heterologous expression of GsHZ4 can enhance alkaline tolerance of lupinus, providing a novel strategy for breeding stress-resistant varieties and expanding lupinus cultivation in saline–alkali soils. Full article

Fusarium wilt, caused by Fusarium oxysporum f. sp. phaseoli (Fop), is a major constraint to global common bean (Phaseolus vulgaris L.) production. Thiamine (vitamin B1), an essential coenzyme in plant metabolism, has recently emerged as a potential regulatory factor in plant defense. Here, we performed a comprehensive genome-wide analysis of thiamine biosynthesis-related genes in common bean and elucidated their roles in resistance to Fusarium wilt. Five key thiamine biosynthetic genes were identified and characterized, showing conserved functional domains and evolutionary conservation across species. Expression profiling revealed tissue-specific patterns, with PvTHI1-1 and PvTHIC being highly expressed in reproductive and photosynthetic organs, with their relative expression levels 0.28–0.57 higher than other members in the same tissue, while PvTPK maintained a basal expression level in the roots. Upon Fop infection, resistant genotypes exhibited significantly higher expression of thiamine biosynthetic genes and greater accumulation of endogenous thiamine and its derivatives than susceptible ones. Functional analysis using Agrobacterium rhizogenes-mediated transformation demonstrated that overexpression of PvTPK enhanced thiamine metabolism and conferred resistance in susceptible genotypes. Similarly, exogenous application of thiamine upregulated biosynthetic genes and improved disease resistance. Together, these results reveal that thiamine biosynthesis is intricately linked to Fusarium wilt resistance and that both genetic and biochemical manipulation of thiamine pathways can enhance disease tolerance. This study provides new insights into thiamine-mediated plant immunity and establishes a foundation for its application in the control of Fusarium wilt in common bean. Full article

We investigated the effect of Agrobacterium rhizogenes-mediated transformation mof rolB on adventitious root development and endogenous hormones in ‘duli’ (Pyrus betulaefolia) via transcriptomic analysis of wild-type (WT) and rolB-transformed plants. The formation of root primordia occurred earlier in transgenic ‘duli’ than in the WT plants. At seven days, 57% of the transgenic seedlings had formed root primordia, whereas root primordia first appeared at seven days in WT ‘duli’. The rooting rate of transgenic ‘duli’ and WT plants was 90% and 77.14%, respectively. rolB significantly promoted the formation of secondary roots. Within 20 days, auxin (IAA), gibberellic acid (GA₃), and zeatin riboside (ZR) were higher and abscisic acid (ABA) was lower in transgenic ‘duli’ than in WT plants. Gene Ontology analysis revealed high enrichment in signaling pathways and ADP binding, and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that several differentially expressed genes were enriched in flavonoid and carotenoid-related pathways and plant hormone signal transduction. rolB induced changes in the expression patterns of several genes involved in hormone biosynthesis, metabolism, and signal transduction pathways in ‘duli’. Weighted gene co-expression network analysis identified the DEGs associated with endogenous hormone levels and indicated that the central genes of modules most strongly correlated with ABA, ZR, IAA, and GA₃ regulate protein synthesis, signaling, and root tissue meristem activity. Protein–protein interaction analysis yielded a co-expression network of physiological and transcriptomic data during rooting and identified key genes at the network core. These findings provide valuable insights into the regulatory mechanisms of rolB and its influence on root development in ‘duli’. Full article

Dracocephalum kotschyi Boiss., an endangered and endemic medicinal plant of Iran belonging to the Lamiaceae family, is a valuable source of methoxylated flavonoids with proven anticancer activity. In this study, hairy roots were effectively induced from two-week-old hypocotyl explants using Agrobacterium rhizogenes strain ATCC 15834. Biomass optimization showed that maximum seedling growth occurred in ¼-strength MS medium, while the highest hairy root induction (76.55%) was achieved in ½-strength MS medium supplemented with 1 mM L-arginine. Root induction varied significantly with explant type and age, with the lowest response (14.66%) observed in five-week-old leaf explants. To increase rosmarinic acid (RA) accumulation, transgenic hairy roots were treated with yeast extract (200 mg L⁻¹) as a biotic elicitor and titanium dioxide (TiO₂) nanoparticles (200 and 400 mg L⁻¹) as abiotic elicitors for 24 and 48 h. HPLC analysis revealed that treatment with yeast extract (200 mg L⁻¹ for 48 h) resulted in the highest accumulation of rosmarinic acid (5.65 mg g⁻¹ DW), corresponding to a 26% increase over the control, accompanied by a substantial enhancement of antioxidant activity (63.4%). Yeast extract application also elevated total protein content and glutathione peroxidase (GPX) activity, while markedly suppressing ascorbate peroxidase (APX) and polyphenol oxidase (PPO) activities. In contrast, TiO₂ nanoparticles, particularly at a concentration of 400 mg L⁻¹ for 48 h, augmented APX and PPO activities, indicating the induction of oxidative stress. These findings demonstrate that yeast extract and low concentrations of TiO₂ nanoparticles can serve as effective elicitors to enhance rosmarinic acid accumulation in D. kotschyi hairy root cultures, providing valuable insights for applications in plant biotechnology. Full article