Search Results (320)

Passion fruit (Passiflora edulis Sims), belonging to the Passifloraceae family, is an economically important plant in tropical and subtropical regions. The advances in functional genomics research of passion fruit have been significantly hindered by its recalcitrance to regeneration and stable transformation. This study establishes the first efficient Agrobacterium rhizogenes-mediated hairy root transformation system for passion fruit. Utilizing the eGFP marker gene, transformation efficiencies of 11.3% were initially achieved with strains K599, MSU440, and C58C1, with K599 proving most effective. Key transformation parameters were systematically optimized to achieve the following: OD₆₀₀ = 0.6, infection duration 30 min, acetosyringone concentration 100 μM, and a dark co-cultivation period of 2 days. The system’s utility was further enhanced by incorporating the red visual marker RUBY, enabling direct, instrument-free identification of transgenic roots via betaxanthin accumulation. Finally, this system was applied for functional analysis using PeMYB123, which may be involved in proanthocyanidin accumulation. Overexpression of PeMYB123 produced a higher content of proanthocyanidin in hairy roots. Additionally, the PeANR gene involved in the proanthocyanidin pathway was strongly activated in the transgenic hairy roots. This rapid and efficient visually simplified hairy root transformation system provides a powerful tool for functional gene studies in passion fruit. Full article

A nanoparticle formulation was generated from distiller dried grains with solubles (DDGS), and its effect on the production of anthraquinones (AQs) was evaluated on Rubia tinctorum hairy roots. The DDGS material was washed with water and ethyl acetate to remove mainly the soluble organic/inorganic molecules and reduce the fat content, respectively, followed by an alkaline treatment to remove the polysaccharides. The resulting alkaline solutions were then lyophilized and redispersed in deionized water to generate a monodispersed nanoparticulate formulation (DDGS-NP) with a hydrodynamic diameter and zeta potential of 227 ± 42 nm and −53 ± 7 mV, respectively. The formulation demonstrated good colloidal stability over time, and sterilized DDGS-NPs maintained comparable physicochemical properties. The nanoparticles were enriched in protein fractions, unsaturated fatty acids, and orthophosphate anion components from DDGS, as determined by solid-state Nuclear Magnetic Resonance (NMR), X-ray photoelectron spectroscopy (XPS), organic elemental analysis (OEA), and inductively coupled plasma optical emission spectrometry (ICP-OES) techniques. The DDGS-NPs were tested at different concentrations on Rubia tinctorum hairy roots, in comparison to or in combination with methyl jasmonate (MeJ), for their capacity to induce the production of AQs. All DDGS-NP concentrations increased the production of specific AQs to 7.7 (100 mg L⁻¹), 7.8 (200 mg L⁻¹), and 9.3 µmol/gFW (500 mg L⁻¹), with an extracellular AQ accumulation of 18 µM for the highest DDGS-NP concentration, in comparison with the control hairy roots (~2 µM AQ). The plant growth was not affected at any of the tested nanoparticle concentrations. Interestingly, the combination of DDGS-NPs and MeJ resulted in the highest extracellular AQ accumulation in R. tinctorum root cultures. Full article

Flavonoid synthases (FNSs) are key enzymes catalyzing the conversion of flavanones to flavonoids, yet their functions in citrus remain functionally uncharacterized. In this study, we identified three FNSII genes in the citrus genome. Phylogenetic analysis revealed that citrus FNSII genes share the closest evolutionary distance with apple FNSII genes. Chromosomal localization demonstrated that the three FNSII genes are distributed across two out of nine chromosomes. Gene structure analysis indicated that the majority of motifs within these three FNSII genes are highly conserved. We cloned a gene called CitFNSII-1 from citrus. Transient overexpression of CitFNSII-1 in citrus leaves significantly increased flavonoid content, while simultaneous virus-induced silencing of CitFNSII-1 led to synchronously and significantly reduced gene expression levels and flavonoid content in citrus seedlings. Through the Agrobacterium rhizogenes-mediated genetic transformation system, overexpression of CitFNSII-1 was found to markedly enhance flavonoid accumulation in hairy roots, whereas knockout of CitFNSII-1 resulted in a significant decrease in flavonoid content in hairy roots. Further experiments verified an interaction between CitFNSII-1 and the Chalcone isomerase-1 (CHI-1) protein. The results demonstrated that the flavonoid accumulation patterns of CHI-1 and CitFNSII-1 are highly similar. In conclusion, this study advances the understanding of the flavonoid biosynthesis pathway in citrus and provides a theoretical foundation for molecular breeding strategies in citrus. Full article

Hairy root cultures induced by Agrobacterium rhizogenes (Rhizobium rhizogenes) provide a sustainable approach to meet the growing demand for economically valuable plant-derived compounds in the face of depleting natural resources. These cultures exhibit rapid, hormone-independent growth and genetic stability, making them viable for producing bioactive compounds, plant-specialized metabolites, and recombinant proteins. However, challenges remain in optimizing large-scale production, improving bioreactor efficiency, and enhancing metabolite synthesis across different plant species. This review addresses these challenges by exploring the mechanisms behind the induction of hairy root cultures, their applications in genetic and metabolic engineering, and their potential in environmental remediation. The review further highlights recent advances in biotechnology and illustrates how the hairy root system can sustainably meet industrial, pharmaceutical, and agricultural needs. In addition, by pointing out essential research areas such as optimizing culture conditions, increasing metabolite yields, and scaling up production, this work strengthens the significance of hairy root cultures in meeting the demand for high-value products while ensuring sustainable resource utilization. In particular, the integration of hairy root systems with advanced genomic tools such as transcriptomics and CRISPR technology holds immense potential for accelerating pathway-specific metabolic engineering, enhancing biosynthetic flux, and expanding their applications in sustainable agriculture and pharmaceutical innovation. This convergence is expected to drive substantial economic value by optimizing the production of high-value bioactive compounds, improving crop resilience, and facilitating precision medicine. Future work involving systems and synthetic biology will be instrumental in unlocking novel functions and ensuring broader deployment of hairy root cultures across industrial biotechnological platforms. Full article

The rhizome of Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial properties. It is utilized to treat nyctalopia and problems related to the gastrointestinal tract. However, its yield is limited because of its endangered status, long growth period, and restricted reproductive ability. Ancillary approaches have not been established to ensure sustainable resource utilization by applying efficient plant regeneration technologies and producing bioactive metabolites via genome editing. This study reports the effects of explants, hormones, and culture conditions on embryogenic callus induction, plant regeneration, adventitious and hairy root cultivation, and essential oil production. Embryogenic calli were successfully induced in MS and 2.0 mg/L 2,4-D and 1.0 mg/L NAA and 1/2MS medium supplemented with 4.0 mg/L 6-BA and 0.4 mg/L NAA, which were optimal for callus differentiation. Maximum proliferation (12-fold) of cluster buds was observed with a select combination of hormones [NAA (0.2 mg/L) and 6-BA (2.0 mg/L)]. “Efficient plant regeneration and bioactive metabolite production” can provide technical support for the protection and sustainable utilization of A. lancea germplasm resources in terms of resource preservation and new variety breeding, natural product production, and industrial breeding of medicinal plants. Full article

Soybean, an important leguminous crop valued for its high protein and oil content, obtains most of its nitrogen through symbiotic fixation processes. The symbiosis between soybeans and rhizobium can provide sufficient nitrogen for soybean growth. However, the signaling pathways underlying the establishment of the symbiosis are not so clear, especially the rhizobial type III effector-induced host response. In this study, we found that the single mutant HH103 nopAA::kan significantly affected the nodule number in soybeans. To further demonstrate the NopAA-triggered response in soybeans. Initial quantitative real-time PCR (qRT-PCR) tests showed that NopAA affects the expression of the soybean gene GmERF5, which was significantly upregulated upon inoculation with HH103 nopAA::kan, acting as a positive regulator of nodulation. The direct interaction between NopAA and GmERF5 was confirmed through yeast-two hybrid analysis. Furthermore, overexpression of GmERF5 in hairy roots indicated that GmERF5 may underlie the nodule phenotype of soybeans in response to NopAA. These findings provide new insights into the mechanisms by which soybean genes respond to rhizobial type III effectors to regulate symbiosis. Full article

Auxin is the most important phytohormone, regulating root growth and development in plants. ARFs function as central regulators in auxin signaling in model plants, and the functions of ARF activators have been widely investigated, while the functions of ARF repressors remain elusive. In this study, we investigated the family of GmARFs in soybean through a genome-wide expression pattern and functional study of roots highly expressing ARFs. In total, we identified 59 GmARF members in the soybean genome. GmARFs harbor canonical B3 DNA-binding (B3), ARF (Aux rep), and PB1 functional protein domains. We identified two potential repressor genes, GmARF9b (Glyma.03G070500) and GmARF2a (Glyma.05G200800), which are specifically or highly expressed in the roots. Histochemical staining suggested that both genes are primarily expressed in the stele, root tips, and lateral root primordia. Subcellular localization analysis showed they were mainly localized in the nucleus. Overexpression of GmARF9b and GmARF2a significantly inhibited root growth using a transgenic hairy root assay. Root section analysis further suggested that GmARF9b and GmARF2a negatively regulated cortical cell layers and the longitudinal cell length of roots, thereby modulating root growth. Overall, this study has preliminarily identified two GmARFs that inhibit root growth in soybean. This discovery has deepened our understanding of the functions of ARFs in root development and has provided guidance for breeding crop roots with improved nutrient use efficiency. Full article

Lignification of juice sacs is a primary contributor to reductions in fruit quality, with impacts on taste and economic value of pomelo (Citrus grandis). To date, information on the regulation of fruit lignification remains fragmentary. In this study, we first analyzed the relationship between lignification and nutrient status of pomelo juice sacs, which revealed a significant positive correlation between nitrate (NO₃⁻) concentration and lignin concentration, with over 60% of lignin accumulation explained by NO₃⁻ levels in three models of machine learning-based regression. Results from field trails in 11 pomelo orchards, as well as in pear fruits and soybean roots exposed to low or high NO₃⁻ supplies, further demonstrated that nitrate plays an important role in lignification. Transcriptomic analysis further showed that pomelo laccases (CgLACs) were more intensively up-regulated upon addition of NO₃⁻ than any of the genes encoding one of the other 12 enzymes involved in lignin biosynthesis. Among the nine identified CgLACs, CgLAC3 was the most significantly up-regulated CgLAC in high nitrate treated plants. Over-expressing CgLAC3 increased lignin concentrations in both pomelo albedo and soybean hairy roots. Taken together, we conclude that nitrate modulates fruit lignification in pomelo through regulation of CgLAC3 expression, which suggests that NO₃⁻-N fertilization may affect fruit lignification, and thereby can be managed to improve fruit quality. Full article

Stephania tetrandra S. MOORE, a medicinal plant, is recognized for tetrandrine production, which is extensively accepted for its therapeutic benefits. However, the slow growth of S. tetrandra limits tetrandrine’s availability, which can be addressed by in vitro hairy root cultivation using Rhizobium rhizogenes and optimization of medium components. The present study attempted the three-step optimization of three components of woody plant medium (ammonium nitrate, calcium nitrate, and sucrose), including two-level factorial design, path of steepest ascent (PSA), and central composite design (CCD) to obtain high hairy root biomass and tetrandrine production. The CCD-based optimization for enhanced hairy root biomass resulted in a hairy root biomass of 9.75 g dw/L at optimal concentrations of ammonium nitrate (NH₄NO₃), calcium nitrate (Ca(NO₃)₂), and sucrose of 631.96 mg/L, 651 mg/L, and 41.35 g/L, respectively. The optimal concentration of 550.31 mg/L, 862.88 mg/L, and 25.89 g/L for NH₄NO₃, Ca(NO₃)₂, and sucrose, respectively, obtained after CCD analysis for enhanced tetrandrine production led to the maximum tetrandrine of 70.48 mg/L. Medium optimization resulted in a 1.47-fold increase in hairy root biomass and a 1.37-fold increase in tetrandrine production under individually optimized conditions. The present study findings confirmed the important role of process optimization for enhanced product yield. Full article

Loropetalum chinense is a significant small tree and ornamental shrub known for its colorful foliage and is widely used in landscaping in tropical and subtropical regions. This study aimed to establish an efficient, tissue culture-independent genetic transformation system for L. chinense. Cuttings from two varieties, ‘Xiangnong Xiangyun’ and ‘Hei Zhenzhu’, were infected with different strains of Agrobacterium rhizogenes. The results showed that the K599 strain significantly induced hairy roots in both varieties, with ‘Xiangnong Xiangyun’ demonstrating a higher survival rate (60%), rooting rate (51.66%), and hairy root induction efficiency (45%) compared to ‘Hei Zhenzhu’. Based on these findings, ‘Xiangnong Xiangyun’ and the K599 strain were selected for further optimization through an orthogonal L9 (3³) experiment, which focused on optimizing the infection solution composition, bacterial concentration, and infection duration, Finally, the genetic transformation system established at the beginning of the experiment was validated on ‘Xiangnong Xiangyun’ plants using the pre-screening LcDREB-43 gene of our group. Among these factors, infection duration was identified as the most influential for improving transformation efficiency. The optimal conditions were determined as an infection solution containing MES solution, a bacterial concentration of OD₆₀₀ = 0.8, and a 15 min infection duration. Under these optimized conditions, the survival rate, rooting rate, induction efficiency, and transformation efficiency reached 86.67%, 70%, 61.67%, and 43.33%, respectively. Furthermore, the transgenic plants with LcDREB-43 overexpression and pCAMBIA1305-GFP were obtained through the established transformation system, the authenticity of the system was proved, and the production application was carried out through phenotypic observation, molecular identification, and auxiliary verification of physiological indicators. Full article

Ballota acetabulosa (L.) Benth. (syn. Pseudodictamnus acetabulosus (L.) Salmaki and Siadati), f. Lamiaceae, the Greek horehound, is a compact evergreen small shrub native to Greece, with hairy grey-green leaves, that bears small pink-purple flowers with green conical calyxes along its erect stems in late spring. The species stands out for its high resistance in xerothermic conditions and therefore it is advisable to promote its use in xeriscaping. The aim of this study was to develop an efficient protocol for in vitro propagation of B. acetabulosa for introduction into the horticultural and pharmaceutical industries. Shoot tip and single node explants derived from in vitro seedlings were cultured on MS medium with various cytokinin types and concentrations. Explants responded at almost 100% to produce high number of shoots on a medium with 1.0 mg L⁻¹ zeatin or 6-benzyladenine. However, there was intense hyperhydricity in the cultures, which was addressed in further experiments by increasing agar concentration from 8 to 12 g L⁻¹, preserving high multiplication indices (92% response, 10.2 shoots per explant). Microcuttings with 2–3 visible nodes, either from the apical part, including the apical meristem, or from the basal part of microshoots, as well as microshoot clusters, rooted 100% on full- or half-strength MS medium, respectively, regardless of the addition of indole-3-butyric acid (ΙΒA, 0.5–4.0 mg L⁻¹) in the rooting medium. However, middle level concentrations of IBA increased the number and length of roots produced, while the higher its concentration, the more and longer axillary shoots developed in the microcuttings during the rooting period. The acclimatization of all plantlets was completely successful (100%) in ex vitro conditions on peat/perlite substrate (1:1, v/v). Thus, efficient methods of producing propagation material to promote Ballota acetabulosa as a horticultural and medicinal plant were developed. In particular, rooting of microshoot clusters or microcuttings without the shoot tip, in the presence of 1.0 mg L⁻¹ IBA, leads to a plant of suitable shape for the floricultural market, without the need for further manipulation (pruning) in the nursery. Full article

Plumbagin is an important naphthoquinone with potent anticancer properties besides multitudinous uses in healthcare. It is produced in a limited number of species and families but mostly in the roots of Plumbaginaceae family members. The biosynthetic pathway and the genes that regulate plumbagin synthesis are not completely known, but details of these are being revealed. Several species, including Plumbago, Drosera, and others, are being uprooted for the extraction of plumbagin by pharmaceutical industries, leading to the destruction of natural habitats. The pharmaceutical industry is therefore facing an acute shortage of plant material. This necessitates enhancing the accumulation of plumbagin using suspensions and hairy roots to meet market demands. Many factors, such as the aggregate size of the inoculum, stability of the culture, and the sequential effects of elicitors, immobilization, and permeabilization, have been demonstrated to act synergistically and markedly augment plumbagin accumulation. Hairy root cultures can be used for the large-scale production, growth, and plumbagin accumulation, and the exploration of their efficacy is now imperative. The secretion of compounds into the spent medium and their in situ adsorption via resin has remarkable potential, but this has not been thoroughly exploited. Improvements in the quality of biomass, selection of cell lines, and production of plumbagin in bioreactors have thus far been sporadic, and these parameters need to be further exploited. In this review, we report the advances made relating to the importance of stable cell line selection for the accumulation of compounds in long-term cultures, hairy root cultures for the accumulation of plumbagin, and its semicontinuous production via total cell recycling in different types of bioreactors. Such advances might pave the way for industrial exploitation. The steps in the biosynthetic pathway that are currently understood might also aid us in isolating the relevant genes in order to examine the effects of their overexpression or heterologous downregulation or to edit the genome using CRISPR-Cas9 technology in order to enhance the accumulation of plumbagin. Its potential as an anticancer molecule and its mode of action have been amply demonstrated, but plumbagin has not been exploited in clinics due to its insolubility in water and its highly lipophilic nature. Plumbagin-loaded nanoemulsions, plumbagin–silver, or albumin nanoparticle formulations can overcome these problems relating to its solubility and are currently being tried to improve its bioavailability and antiproliferative activities, as discussed in the current paper. Full article

This study reports the first successful establishment of Perovskia atriplicifolia hairy root cultures using Rhizobium rhizogenes and evaluates their potential for bioactive phenolic acid production, particularly rosmarinic acid (RA). Hairy roots were induced using two R. rhizogenes strains, A4 and ATCC 15834; transformation was confirmed by PCR analysis targeting the rol and aux genes. The A4 strain exhibited higher transformation efficiency (41.3%) than ATCC 15834 (30.2%). Eight transgenic root clones (C1–C8) were established and confirmed as transformed. The clones exhibited significant variation in biomass accumulation and phenolic acid production. RA production was most strongly correlated with PAL, RAS, and CYP98A14 expression. Hierarchical clustering clustered the clones into three groups based on growth, metabolite content, and gene expression. Lines C1 and C2 exhibiting the highest RA, total polyphenol content, and the highest productivity were selected for further experiments. McCown Woody Plant (WP) and Schenk and Hildebrandt (SH) media demonstrated the greatest biomass accumulation, with growth indexes exceeding 13. Conversely, Gamborg (B5) medium enhanced RA content, achieving 38.3 and 40.8 mg/g dry weight (DW) for clones C1 and C2, respectively, representing a fourfold increase compared to the least favorable Murashige and Skoog (MS) medium. These findings establish P. atriplicifolia hairy roots as efficient systems for RA biosynthesis and can provide a basis for metabolic engineering and scale-up production of phenolic acids in medicinal plants. Full article

For the first time, adventitious and hairy root cultures of Salvia apiana (white sage) have been established and analyzed for the content of secondary metabolites. Non-transformed roots derived from sterile seedlings were maintained on a full-strength IBA-supplemented SH medium. Adventitious roots yielded up to 44.5 mg/g and 18.7 mg/g DW rosmarinic acid when grown in shake flasks and immersion-column bioreactors, respectively. Transformed root cultures were established from S. apiana microshoots, infected with A4 and LBA9402 strains of Rhizobium rhizogenes. The obtained hairy root cultures (three and two clonal lines established using A4 and LBA9402 strains, respectively) were maintained in the PGR-free, full-strength SH medium. The most productive root line, established using A4 strain, accumulated rosmarinic acid at 38.1 and 39.6 mg/g DW when grown in shake flasks and spray bioreactors, respectively. Neither adventitious nor transformed roots of S. apiana produced diterpenoids, identified in roots of the field-grown plants, and instead proved to be a selective source of rosmarinic acid. Full article

In vitro cultivation of arbuscular mycorrhizal fungi (AMF) is challenging due to their biotrophic symbiosis. The principal aim of this study was to demonstrate the effect of establishing in vitro dual cultures of arbuscular mycorrhizal fungi (AMF) inoculated on Swietenia macrophylla (mahogany) roots on plant growth. Furthermore, it was sought to demonstrate that plant colonization by Glomeromycota can be achieved with a replicable protocol. This study established monoxenic cultures of carrot (Daucus carota) Ri T-DNA ROC inoculated with Glomus sp. on two-compartment plates. At 75 days, hyphal growth reached 223.93 mm in the root compartment and 103.71 mm in the hyphal compartment. Spores produced in vitro measured 26.14 ± 1.70 µm, smaller than ex vitro spores (101.2 ± 4.22 µm). Rhodotorula mucilaginosa was isolated from cultures and appeared to stimulate hyphal growth and root–fungal contact. From these cultures, a dual culture of mahogany inoculated with Glomus sp. was established. No significant differences were observed between inoculated and non-inoculated plants in stem length, root length, root number, or leaf number at 30 days. Spore production ranged from 10,166 to 27,696 per plate, averaging 14,795 ± 3301, with hyphal lengths of 3655.46 ± 308.75 mm. Hyphal development included running and branching patterns, with solitary and clustered spores. Spore diameter averaged 27.68 ± 3.85 µm. Arbuscular colonization reached 41.49% at 30 days and 52.13% at 75 days, exceeding rates reported for other culture systems. Monoxenic cultures are a reliable, aseptic source of high-quality inoculum, supporting biofertilizer production and biotechnological applications. These methods provide valuable tools for studies involving AMF, such as those demonstrated with mahogany. Full article