Search Results (35)

This study investigates the transformation of Acmella oleracea with the Agrobacterium rhizogenes rolC gene and evaluates its impact on phytochemical composition and biological activity. A total of 480 plant nodes were subjected to Agrobacterium−mediated transformation, leading to the regeneration of 35 putative transgenic plants. Molecular analysis confirmed the presence of the rolC transgene in 17 clones, of which four (C123, C127, C129, and C132) exhibited rolC mRNA expression. Phytochemical profiling of hydroalcoholic extracts of aerial parts (AP) and roots (R) revealed significant differences (p ≤ 0.05) between transgenic and non-transgenic plants (CTR). Compared to non−transgenic plants, transgenic AP exhibited lower total phenolic content but retained or increased flavonoid concentrations, particularly flavan−3−ols, whereas R extracts consistently showed reduced secondary metabolite levels. LC−DAD−ESI−MS analysis identified a diverse metabolite profile, with AP being notably rich in flavonoids (48.65%) and alkylamides (32.43%), including spilanthol. Functional assessments across antioxidant and anti−inflammatory assays demonstrated that R extracts exhibited stronger bioactivity compared to AP extracts, as indicated by lower IC₅₀ values (0.004–2.18 mg/mL for R vs. 0.007–7.24 mg/mL for AP). However, iron−chelating capacity was higher in AP extracts, correlating with flavonoid concentration. Hierarchical clustering confirmed that transgenic lines C123 and C127 most closely resembled the control, while C129 and C132 displayed distinct metabolic profiles. These findings highlight rolC’s role in modulating secondary metabolite synthesis, influencing both the phytochemical composition and functional properties of A. oleracea extracts. 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

Dittrichia viscosa is a ruderal plant species growing along roadsides and well adapting to extreme environmental conditions. D. viscosa plant tissues, especially leaves, are known to be a rich source of bioactive metabolites which have antioxidant, cytotoxic, antiproliferative and anticancer properties. Hairy root cultures are a suitable biotechnological system for investigating plant metabolic pathways and producing specialized metabolites in in vitro conditions. In this study, D. viscosa hairy root transformed lines induced by Agrobacterium rhizogenes ATCC15834 were obtained using leaf explants, and the integration of rolB and rolC genes in the genomes of transformed hairy roots were confirmed by PCR analysis. Three hairy root D. viscosa lines (DvHrT1, DvHrT4 and DvHrT5) having different phenotypic features were characterized in terms of total phenolics, flavonoids and antioxidant activity. Correlated with antioxidant activity, phenolic and flavonoid content of DvHrT1 was significantly higher than control roots and the other DvHrT lines. Our results suggest that D. viscosa hairy roots can be a valuable tool for producing various bioactive compounds having antioxidant activity and are to be further investigated to produce other specific molecules that could find application in agricultural or pharmaceutical fields. Full article

Kalanchoë gastonis-bonnieri Raym.-Hamet & Perrier is a plant used for medicinal purposes in the treatment of several ailments. The aim of this study was to analyze the chemical profile of extracts from K. gastonis-bonnieri embryogenic calli, generated from genetically transformed roots by Agrobacterium rhizogenes. Putative transformants were verified by PCR. Hydroalcoholic extracts were obtained and the chemical profile was analyzed by LC-ESI-MS/MS. Root formation was obtained from 80% of infected seedlings. Fifteen root lines were isolated, and two lines showed prominent longitudinal growth and profuse branching in the B5 semi-solid medium. In all lines, the formation of nodules and later embryogenic callus was observed. Putative transgenic root lines were cultivated in free-plant growth regulators B5 medium. In the two selected lines, the PCR amplification of rolA, rolB, rolC, rolD, and aux1 genes was detected. The extract of embryogenic calli showed 60 chemical compounds tentatively identified, such as ferulic acid, quinic acid, neobaisoflavone, and malic acid, among others, and the chemical profile was different in comparison to wild-type extracts. This is the first study reporting the analysis of the chemical profile of hairy root extracts derived from Kalanchoë gastonis-bonnieri. This work displays the great potential for obtaining chemical compounds of pharmacological importance from hairy roots and facilitates the identification of new useful drugs against human chronic-degenerative diseases. Full article

The presence of polycyclic aromatic hydrocarbons (PAHs) in soil poses a significant global environmental concern, particularly in coastal wetlands. Mangrove ecosystems exhibit enormous potential in environmental purification; however, the underlying mechanisms involved in the degradation of pollutants (e.g., PAHs) remain ambiguous. In the present investigation, a soil pot experiment was conducted with the addition of pyrene to evaluate the effect of radial oxygen loss (ROL) from roots on PAH degradation using three mangrove seedlings (Rhizophora stylosa, Aegiceras corniculatum, and Avicennia marina). The results showed that mangrove plantation can significantly promote the efficiency of pyrene removal. As for the three mangrove species studied, the greatest removal rate (90.75%) was observed in the soils associated with A. marina, followed by A. corniculatum (83.83%) and R. stylosa (77.15%). The higher PAH removal efficiency of A. marina can be partially attributed to its distinctive root anatomical structure, characterized by a thin exodermis and high porosity, which facilitates ROL from the roots. The results from qPCR further demonstrate that ROL is beneficial for promoting the abundance of PAH-ring hydroxylating dioxygenase gene, leading to a higher removal efficiency. Additionally, Rhizobiales, Defferrisomatales, and Ardenticatenales may also play important roles in the process of pyrene degradation. In summary, this study provides evidence for elucidating the mechanism of PAH removal from the perspective of ROL, thereby contributing valuable insights for species selection during mangrove restoration and remediation. Full article

Centella asiatica is a medicinal plant with a rich tradition of use for its therapeutic properties. Among its bioactive compounds are centellosides, a group of triterpenoid secondary metabolites whose potent pharmacological activities have attracted significant attention. Metabolic engineering has emerged as a powerful biotechnological tool to enhance the production of target compounds. In this study, we explored the effects of overexpressing the squalene synthase (SQS) gene and transcription factor TSAR2 on various aspects of C. asiatica hairy root lines: the expression level of centelloside biosynthetic genes, morphological traits, as well as squalene, phytosterol, and centelloside content. Three distinct categories of transformed lines were obtained: LS, harboring At-SQS; LT, overexpressing TSAR2; and LST, simultaneously carrying both transgenes. These lines displayed noticeable alterations in morphological traits, including changes in branching rate and biomass production. Furthermore, we observed that the expression of T-DNA genes, particularly aux2 and rolC genes, significantly modulated the expression of pivotal genes involved in centelloside biosynthesis. Notably, the LS lines boasted an elevated centelloside content but concurrently displayed reduced phytosterol content, a finding that underscores the intriguing antagonistic relationship between phytosterol and triterpene pathways. Additionally, the inverse correlation between the centelloside content and morphological growth values observed in LS lines was countered by the action of TSAR2 in the LST and LT lines. This difference could be attributed to the simultaneous increase in the phytosterol content in the TSAR2-expressing lines, as these compounds are closely linked to root development. Overall, these discoveries offer valuable information for the biotechnological application of C. asiatica hairy roots and their potential to increase centelloside production. Full article

This study delves into the novel utilization of Aristolochia manshuriensis cultured cells for extracellular silver nanoparticles (AgNPs) synthesis without the need for additional substances. The presence of elemental silver has been verified using energy-dispersive X-ray spectroscopy, while distinct surface plasmon resonance peaks were revealed by UV-Vis spectra. Transmission and scanning electron microscopy indicated that the AgNPs, ranging in size from 10 to 40 nm, exhibited a spherical morphology. Fourier-transform infrared analysis validated the abilty of A. manshuriensis extract components to serve as both reducing and capping agents for metal ions. In the context of cytotoxicity on embryonic fibroblast (NIH 3T3) and mouse neuroblastoma (N2A) cells, AgNPs demonstrated varying effects. Specifically, nanoparticles derived from callus cultures exhibited an IC₅₀ of 2.8 µg/mL, effectively inhibiting N2A growth, whereas AgNPs sourced from hairy roots only achieved this only at concentrations of 50 µg/mL and above. Notably, all studied AgNPs’ treatment-induced cytotoxicity in fibroblast cells, yielding IC₅₀ values ranging from 7.2 to 36.3 µg/mL. Furthermore, the findings unveiled the efficacy of the synthesized AgNPs against pathogenic microorganisms impacting both plants and animals, including Agrobacterium rhizogenes, A. tumefaciens, Bacillus subtilis, and Escherichia coli. These findings underscore the effectiveness of biotechnological methodologies in offering advanced and enhanced green nanotechnology alternatives for generating nanoparticles with applications in combating cancer and infectious disorders. Full article

Aristolochia manshuriensis is a relic liana, which is widely used in traditional Chinese herbal medicine and is endemic to the Manchurian floristic region. Since this plant is rare and slow-growing, alternative sources of its valuable compounds could be explored. Herein, we established hairy root cultures of A. manshuriensis transformed with Agrobacterium rhizogenes root oncogenic loci (rol)B and rolC genes. The accumulation of nitrogenous secondary metabolites significantly improved in transgenic cell cultures. Specifically, the production of magnoflorine reached up to 5.72 mg/g of dry weight, which is 5.8 times higher than the control calli and 1.7 times higher than in wild-growing liana. Simultaneously, the amounts of aristolochic acids I and II, responsible for the toxicity of Aristolochia species, decreased by more than 10 fold. Consequently, the hairy root extracts demonstrated pronounced cytotoxicity against human glioblastoma cells (U-87 MG), cervical cancer cells (HeLa CCL-2), and colon carcinoma (RKO) cells. However, they did not exhibit significant activity against triple-negative breast cancer cells (MDA-MB-231). Our findings suggest that hairy root cultures of A. manshuriensis could be considered for the rational production of valuable A. manshuriensis compounds by the modification of secondary metabolism. Full article

Various strategies have been used to increase the efficiency of secondary metabolite production in Salvia plants. This report is the first to examine the spontaneous development of Salvia bulleyana shoots transformed by Agrobacterium rhizogenes on hairy roots and the influence of light conditions on the phytochemical profile of this shoot culture. The transformed shoots were cultivated on solid MS medium with 0.1 mg/L of IAA (indole-3-acetic acid) and 1 mg/L of m-Top (meta-topolin), and their transgenic characteristic was confirmed by PCR-based detection of the rolB and rolC genes in the target plant genome. This study assessed the phytochemical, morphological, and physiological responses of the shoot culture under stimulation by light-emitting diodes (LEDs) with different wavelengths (white, WL; blue, B; red, RL; and red/blue, ML) and under fluorescent lamps (FL, control). Eleven polyphenols identified as phenolic acids and their derivatives were detected via ultrahigh-performance liquid chromatography with diode-array detection coupled to electrospray ionization tandem mass spectrometry (UPLC-DAD/ESI-MS) in the plant material, and their content was determined using high-performance liquid chromatography (HPLC). Rosmarinic acid was the predominant compound in the analyzed extracts. The mixed red and blue LEDs gave the highest levels of polyphenol and rosmarinic acid accumulation (respectively, 24.3 mg/g of DW and 20.0 mg/g of DW), reaching two times greater concentrations of polyphenols and three times greater rosmarinic acid levels compared to the aerial parts of two-year-old intact plants. Similar to WL, ML also stimulated regeneration ability and biomass accumulation effectively. However, the highest total photosynthetic pigment production (1.13 mg/g of DW for total chlorophyll and 0.231 mg/g of DW for carotenoids) was found in the shoots cultivated under RL followed by BL, while the culture exposed to BL was characterized as having the highest antioxidant enzyme activities. Full article

A variety of plant species found in nature contain agrobacterial T-DNAs in their genomes which they transmit in a series of sexual generations. Such T-DNAs are called cellular T-DNAs (cT-DNAs). cT-DNAs have been discovered in dozens of plant genera, and are suggested to be used in phylogenetic studies, since they are well-defined and unrelated to other plant sequences. Their integration into a particular chromosomal site indicates a founder event and a clear start of a new clade. cT-DNA inserts do not disseminate in the genome after insertion. They can be large and old enough to generate a range of variants, thereby allowing the construction of detailed trees. Unusual cT-DNAs (containing the rolB/C-like gene) were found in our previous study in the genome data of two Vaccinium L. species. Here, we present a deeper study of these sequences in Vaccinium L. Molecular-genetic and bioinformatics methods were applied for sequencing, assembly, and analysis of the rolB/C-like gene. The rolB/C-like gene was discovered in 26 new Vaccinium species and Agapetes serpens (Wight) Sleumer. Most samples were found to contain full-size genes. It allowed us to develop approaches for the phasing of cT-DNA alleles and reconstruct a Vaccinium phylogenetic relationship. Intra- and interspecific polymorphism found in cT-DNA makes it possible to use it for phylogenetic and phylogeographic studies of the Vaccinium genus. Full article

Difenoconazole (DFZ) is a broad-spectrum triazole fungicide that is widely utilized in agriculture. Although DFZ has been demonstrated to induce reproductive toxicity in aquatic species, its toxic effects on the mammalian reproductive system have yet to be fully elucidated. In vivo, male mice were administered 0, 20 or 40 mg/kg/d of DFZ via oral gavage for 35 days. Consequently, DFZ significantly decreased testicular organ coefficient, sperm count and testosterone levels, augmented sperm malformation rates, and elicited histopathological alterations in testes. TUNEL assay showed increased apoptosis in testis. Western blotting results suggested abnormally high expression of the sperm meiosis-associated proteins STRA8 and SCP3. The concentrations of retinoic acid (RA), retinaldehyde (RE), and retinol (ROL) were increased in the testicular tissues of DFZ-treated groups. The mRNA expression level of genes implicated in RA synthesis significantly increased while genes involved in RA catabolism significantly decreased. In vitro, DFZ reduced cell viability and increased RA, RE, and ROL levels in GC-2 cells. Transcriptome analysis revealed a significant enrichment of numerous terms associated with the RA pathway and apoptosis. The qPCR experiment verified the transcriptome results. In conclusion, our results indicate that DFZ exposure can disrupt RA signaling pathway homeostasis, and induce testicular injury in mice testes. Full article

Secondary metabolites have been shown to possess a range of biological functions. Flavonoids, due to their ability to scavenge ROS, are famous antioxidants. The plants of Artemisia species are rich sources of flavonoids; however, the amount of these metabolites is less. In the current study, the flavonoid content was detected and then enhanced by genetically modifying the Artemisia carvifolia Buch with Agrobacterium tumefaciens strain GV3101 carrying rol A gene. The transformation of rol A gene was confirmed with PCR and the gene copy number was confirmed by Southern blot analysis. The HPLC analysis revealed the presence of catechin (3.19 ug/mg DW) and geutisic acid (2.22 ug/mg DW) in transformed plants, unlike wild-type plants. In transformed plants, all detected flavonoids (vanillic acid, rutin, catechine, gallic acid, syringic acid, caffeic acid, coumaric acid, geutisic acid, ferulic acid, and cinnamic acid) were increased up to several folds. Real-time qPCR revealed the higher expression levels of the genes for flavonoid biosynthesis enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) in plants transformed with rol A genes, as the expression levels were increased up to 9–20-fold and 2–6-fold, respectively. The rol A transgenic lines T3 and T5 carrying two copies of rol A gene, particularly showed higher expression of both PAL and CHS gene, with the highest expression in T3 line. The transgenic lines demonstrated an average increase of 1.4-fold in the total phenolic content and 1–2-fold in the total flavonoid content as compared to wild-type plants. Total antioxidant capacity and total reducing power were increased up to an average of 1–2-fold and 1.5–2-fold respectively, along with increased free radical scavenging ability. Furthermore, the rol A gene transgenics were found to have much greater cytotoxic capacity than the A. carvifolia wild-type plant against the MCF7, HeLA, and HePG2 cancer cell lines. Current findings show that the rol A gene effectively increases the flavonoid content of A. carvifolia Buch, boosting the plant’s capacity as an antioxidant and an anticancer. This is the first-ever report, demonstrating the genetic transformation of Artemisia carvifolia Buch with rol A gene. Full article

During Agrobacterium rhizogenes–plant interaction, the rolB gene is transferred into the plant genome and is stably inherited in the plant’s offspring. Among the numerous effects of rolB on plant metabolism, including the activation of secondary metabolism, its effect on plant defense systems has not been sufficiently studied. In this work, we performed a proteomic analysis of rolB-expressing Arabidopsis thaliana plants with particular focus on defense proteins. We found a total of 77 overexpressed proteins and 64 underexpressed proteins in rolB-transformed plants using two-dimensional gel electrophoresis and MALDI mass spectrometry. In the rolB-transformed plants, we found a reduced amount of scaffold proteins RACK1A, RACK1B, and RACK1C, which are known as receptors for activated C-kinase 1. The proteomic analysis showed that rolB could suppress the plant immune system by suppressing the RNA-binding proteins GRP7, CP29B, and CP31B, which action are similar to the action of type-III bacterial effectors. At the same time, rolB plants induce the massive biosynthesis of protective proteins VSP1 and VSP2, as well as pathogenesis-related protein PR-4, which are markers of the activated jasmonate pathway. The increased contents of glutathione-S-transferases F6, F2, F10, U19, and DHAR1 and the osmotin-like defense protein OSM34 were found. The defense-associated protein PCaP1, which is required for oligogalacturonide-induced priming and immunity, was upregulated. Moreover, rolB-transformed plants showed the activation of all components of the PYK10 defense complex that is involved in the metabolism of glucosinolates. We hypothesized that various defense systems activated by rolB protect the host plant from competing phytopathogens and created an effective ecological niche for A. rhizogenes. A RolB → RACK1A signaling module was proposed that might exert most of the rolB-mediated effects on plant physiology. Our proteomics data are available via ProteomeXchange with identifier PXD037959. Full article

Ipomoea batatas is a vital root crop and a source of caffeoylquinic acid derivatives (CQAs) with potential health-promoting benefits. As a naturally transgenic plant, I. batatas contains cellular T-DNA (cT-DNA) sequence homologs of the Agrobacterium rhizogenes open reading frame (ORF)14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n of unknown function. This study aimed to evaluate the effect of abiotic stresses (temperature, ultraviolet, and light) and chemical elicitors (methyl jasmonate, salicylic acid, and sodium nitroprusside) on the biosynthesis of CQAs and cT-DNA gene expression in I. batatas cell culture as a model system. Among all the applied treatments, ultraviolet irradiation, methyl jasmonate, and salicylic acid caused the maximal accumulation of secondary compounds. We also discovered that I. batatas cT-DNA genes were not expressed in cell culture, and the studied conditions weakly affected their transcriptional levels. However, the Ib-rolB/C gene expressed under the strong 35S CaMV promoter increased the CQAs content by 1.5–1.9-fold. Overall, our results show that cT-DNA-encoded transgenes are not involved in stress- and chemical elicitor-induced CQAs accumulation in cell cultures of I. batatas. Nevertheless, overaccumulation of RolB/RolC transcripts potentiates the secondary metabolism of sweet potatoes through a currently unknown mechanism. Our study provides new insights into the molecular mechanisms linked with CQAs biosynthesis in cell culture of naturally transgenic food crops, i.e., sweet potato. Full article

People have used carnivorous plants of the genus Sarracenia in folk medicine for centuries due to the biochemical composition of Sarracenia plants, which are rich in numerous bioactive compounds with anti-inflammatory, antioxidant, antiviral and antibacterial properties. The subject of this study was the genetic transformation of Sarracenia purpurea L. with Agrobacterium rhizogenes strains 15834, 9402 and A4 using two different methods: bacterial injection or co-culture of the bacteria with plant explants. This study confirmed the possibility of hairy root induction in S. purpurea using A. rhizogenes strain 15834 and the injection method. Seven lines of transformed plants, exhibiting the integration of the rolB gene, were obtained. The hairy roots formed showed morphological differences in comparison to the roots of unmodified plants. A mathematical model was used to optimize the conditions for the extraction of bioactive compounds. Extracts isolated under optimal conditions from the transformed plants showed biochemical changes, i.e., an increase in the accumulation of total polyphenols (line 7#1 in hairy roots: 71.048 mg GAE g⁻¹ DW; in leaves: 9.662 mg GAE g⁻¹ DW) and triterpenes (line 7#1 in hairy roots: 1.248 mg BA g⁻¹ DW; in leaves: 0.463 mg BA g⁻¹ DW) in comparison to untransformed plants (polyphenols in roots: 7.957 mg GAE g⁻¹ DW and in leaves: 5.091 mg GAE g⁻¹ DW; triterpenes in roots: 0.298 mg BA g⁻¹ DW and in leaves: 0.296 mg BA g⁻¹ DW), especially when induced roots were analyzed. HPLC analysis showed an increase in the level of betulinic acid in some transformed Sarracenia lines. Betulinic acid is a pentacyclic triterpenoid compound with high pharmacological significance. Full article