Organic extracts from sustainable hybrid poplar hairy root cultures as potential natural antimicrobial and antibiofilm agents.

In order to meet growing consumer demands in terms of naturalness, the pharmaceutical, food, and cosmetic industries are looking for active molecules of plant origin. In this context, hairy roots are considered a promising biotechnological system for the sustainable production of compounds of interest. Poplars (genus Populus, family Salicaceae) are trees of ecological interest in temperate alluvial forests and are also cultivated for their industrial timber. Poplar trees also produce specialized metabolites with a wide range of bioactive properties. The present study aimed to assess the hybrid poplar hairy root extracts for antimicrobial and antibiofilm activities against four main life-threatening strains of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Ethyl acetate extracts from two hairy root lines (HP15-3 and HP A4-12) showed significant antibacterial properties as confirmed by disc diffusion assay. Antibiofilm activities were found to be dose dependent with significant biofilm inhibition (75-95%) recorded at 1000 µg.mL-1 in all the bacterial strains tested. Dose-dependent enhancement in the release of exopolysaccharides was observed in response to treatment with extracts, possibly because of stress and bacterial cell death. Fluorescence microscopy confirmed loss of cell viability of treated bacterial cells concomitant with increased production of reactive oxygen species compared to the untreated control. Overall, this study demonstrates for the first time a high potential of poplar hairy root extracts as a natural and safe platform to produce antimicrobial agents in pharmaceutical, food, industrial water management, or cosmetic industries.

An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.

Genetic transformation is a critical tool for gene editing and genetic improvement of plants. Although many model plants and crops can be genetically manipulated, genetic transformation systems for fruit trees are either lacking or perform poorly. We used Rhizobium rhizogenes to transfer the target gene into the hairy roots of Malus domestica and Actinidia chinensis. Transgenic roots were generated within 3 weeks, with a transgenic efficiency of 78.8%. Root to shoot conversion of transgenic hairy roots was achieved within 11 weeks, with a regeneration efficiency of 3.3%. Finally, the regulatory genes involved in stem cell activity were used to improve shoot regeneration efficiency. MdWOX5 exhibited the most significant effects, as it led to an improved regeneration efficiency of 20.6% and a reduced regeneration time of 9 weeks. Phenotypes of the overexpression of RUBY system mediated red roots and overexpression of MdRGF5 mediated longer root hairs were observed within 3 weeks, suggesting that the method can be used to quickly screen genes that influence root phenotype scores through root performance, such as root colour, root hair, and lateral root. Obtaining whole plants of the RUBY system and MdRGF5 overexpression lines highlights the convenience of this technology for studying gene functions in whole plants. Overall, we developed an optimized method to improve the transformation efficiency and stability of transformants in fruit trees.

Characterization of soybean chitinase genes induced by rhizobacteria involved in the defense against Fusarium oxysporum.

Rhizobacteria are capable of inducing defense responses via the expression of pathogenesis-related proteins (PR-proteins) such as chitinases, and many studies have validated the functions of plant chitinases in defense responses. Soybean (Glycine max) is an economically important crop worldwide, but the functional validation of soybean chitinase in defense responses remains limited. In this study, genome-wide characterization of soybean chitinases was conducted, and the defense contribution of three chitinases (GmChi01, GmChi02, or GmChi16) was validated in Arabidopsis transgenic lines against the soil-borne pathogen Fusarium oxysporum. Compared to the Arabidopsis Col-0 and empty vector controls, the transgenic lines with GmChi02 or GmChi16 exhibited fewer chlorosis symptoms and wilting. While GmChi02 and GmChi16 enhanced defense to F. oxysporum, GmChi02 was the only one significantly induced by Burkholderia ambifaria. The observation indicated that plant chitinases may be induced by different rhizobacteria for defense responses. The survey of 37 soybean chitinase gene expressions in response to six rhizobacteria observed diverse inducibility, where only 10 genes were significantly upregulated by at least one rhizobacterium and 9 genes did not respond to any of the rhizobacteria. Motif analysis on soybean promoters further identified not only consensus but also rhizobacterium-specific transcription factor-binding sites for the inducible chitinase genes. Collectively, these results confirmed the involvement of GmChi02 and GmChi16 in defense enhancement and highlighted the diverse inducibility of 37 soybean chitinases encountering F. oxysporum and six rhizobacteria.

Nanoparticle elicitation: A promising strategy to modulate the production of bioactive compounds in hairy roots.

Hairy root culture is one of the promising biotechnological tools to obtain the stable and sustainable production of specialized metabolites from plants under controlled environment conditions. Various strategies have been adopted to enhance the accumulation of bioactive compounds in hairy roots yet their utilization at the commercial scale is restricted to only a few products. Recently, nanotechnology has been emerged as an active technique that has revolutionized the many sectors in an advantageous way. Elicitation using nanoparticles has been recognized as an effective strategy for enhancing the bioactive compounds of interest in plants. Nanoparticles elicit the activity of defense-related compounds through activation of the specific transcription factors involved in specialized metabolites production. This review discusses the recent progress in using nanoparticles to enhance specialized metabolite biosynthesis using hairy root culture system and the significant achievements in this area of research. Biotic and abiotic elicitors to improve the production of bioactive compounds in hairy roots, different types of nanoparticles as eliciting agents, their properties as dependent on shape, most widely used nanoparticles in plant hairy root systems are described in detail. Further challenges involved in application of nanoparticles, their toxicity in plant cells and risks associated to human health are also envisaged. No doubt, nanoparticle elicitation is a remarkable approach to obtain phytochemicals from hairy roots to be utilized in various sectors including food, medicines, cosmetics or agriculture but it is quite essential to understand the inter-relationships between the nanoparticles and the plant systems in terms of specifics such as type, dosage and time of exposure as well as other important parameters.

Insights into enhancing Centella asiatica organ cell biofactories via hairy root protein profiling.

Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica.

Auxin-dependent regulation of growth via rolB-induced modulation of the ROS metabolism in the long-term cultivated pRiA4-transformed Rubiacordifolia L. calli.

Gene transfer from Agrobacterium to plants is the best studied example of horizontal gene transfer (HGT) between prokaryotes and eukaryotes. The rol genes of A. rhizogenes (Rhizobium rhizogenes) provide uncontrolled root growth, or "hairy root" syndrome, the main diagnostic feature. In the present study, we investigated the stable pRiA4-transformed callus culture of Rubia cordifolia L. While untransformed callus cultures need PGRs (plant growth regulators) as an obligatory supplement, pRiA4 calli is able to achieve long-term PGR-free cultivation. For the first time, we described the pRiA4-transformed callus cultures' PGR-dependent ROS status, growth, and specialized metabolism. As we have shown, expression of the rolA and rolB but not the rolC genes is contradictory in a PGR-dependent manner. Moreover, a PGR-free pRiA4 transformed cell line is characterised as more anthraquinone (AQ) productive than an untransformed cell culture. These findings pertain to actual plant biotechnology: it could be the solution to troubles in choosing the best PGR combination for the cultivation of some rare, medicinal, and woody plants; wild-type Ri-plants and tissue cultures may become freed from legal controls on genetically modified organisms in the future. We propose possible PGR-dependent relationships between rolA and rolB as well as ROS signalling targets. The present study highlighted the high importance of the rolA gene in the regulation of combined rol gene effects and the large knowledge gap in rolA action.

Alternative Rooting Methods for Medicinal Cannabis Cultivation in Denmark-Preliminary Results.

Cannabis sativa L. is typically propagated through micropropagation or vegetative cuttings, but the use of root-inducing hormones, such as indole-3-butyric acid (IBA), is not allowed for growing medicinal cannabis in Denmark. This study examined alternative rooting treatments, including Rhizobium rhizogenes inoculation, water-only as well as IBA treatments, in eight cannabis cultivars. PCR on root tissue suggested that 19% of R. rhizogenes-inoculated cuttings were transformed. These were derived from "Herijuana", "Wild Thailand", "Motherlode Kush", and "Bruce Banner", indicating a variation in cultivar susceptibility toward R. rhizogenes. A 100% rooting success was achieved regardless of cultivar and treatment, suggesting that alternative rooting agents are not required for efficient vegetative propagation. However, rooted cuttings differed in shoot morphology with improved shoot growth in cuttings treated with R. rhizogenes (195 ± 7 mm) or water (185 ± 7 mm) while inhibited shoot growth under IBA treatment (123 ± 6 mm). This could have advantageous economic implications should cuttings not treated with hormone reach maturity faster than those exposed to the hormone, thereby contributing to completing a full growing cycle more effectively. IBA exposure increased root length, root dry weight, and root/shoot dry weight ratio compared to cuttings treated with R. rhizogenes or water but simultaneously inhibited shoot growth compared to these.

Hairy Root Cultures as a Source of Phenolic Antioxidants: Simple Phenolics, Phenolic Acids, Phenylethanoids, and Hydroxycinnamates.

Plant-derived antioxidants are intrinsic components of human diet and factors implicated in tolerance mechanisms against environmental stresses in both plants and humans. They are being used as food preservatives and additives or ingredients of cosmetics. For nearly forty years, Rhizobium rhizogenes-transformed roots (hairy roots) have been studied in respect to their usability as producers of plant specialized metabolites of different, primarily medical applications. Moreover, the hairy root cultures have proven their value as a tool in crop plant improvement and in plant secondary metabolism investigations. Though cultivated plants remain a major source of plant polyphenolics of economic importance, the decline in biodiversity caused by climate changes and overexploitation of natural resources may increase the interest in hairy roots as a productive and renewable source of biologically active compounds. The present review examines hairy roots as efficient producers of simple phenolics, phenylethanoids, and hydroxycinnamates of plant origin and summarizes efforts to maximize the product yield. Attempts to use Rhizobium rhizogenes-mediated genetic transformation for inducing enhanced production of the plant phenolics/polyphenolics in crop plants are also mentioned.

Nicotiana hairy roots for recombinant protein expression, where to start? A systematic review.

BACKGROUND: Hairy roots are a plant-tissue culture raised by Rhizobium rhizogenes infection (formerly known as Agrobacterium rhizogenes). Nowadays, these roots have been gaining more space in biotechnology due to their benefits for the recombinant expression of valuables proteins; it includes simplified downstream processing, protein rhizosecretion, and scalability in bioreactors. However, due to methodological inconsistency among reports, the tissue platform is still a promising technology. METHODS AND RESULTS: In the current paper, we propose the first step to overcome this issue through a systematic review of studies that employ Nicotiana hairy roots for recombinant expression. We conducted a qualitative synthesis of 36 out of 387 publications initially selected. Following the PRISMA procedure, all papers were assessed for exclusion and inclusion criteria. Multiple points of root culture were explored, including transformation methods, root growth curve, external additives, and scale-up with bioreactors to determine which approaches performed best and what is still required to achieve a robust protocol. CONCLUSION: The information presented here may help researchers who want to work with hairy roots in their laboratories trace a successful path to appraisal the literature status.

Peat-based hairy root transformation using Rhizobium rhizogenes as a rapid and efficient tool for easily exploring potential genes related to root-knot nematode parasitism and host response.

BACKGROUND: Root-knot nematodes (RKNs) pose a worldwide threat to agriculture of many crops including cucumber. Genetic transformation (GT) has emerged as a powerful tool for exploration of plant-RKN interactions and genetic improvement of RKN resistance. However, it is usually difficult to achieve a highly efficient and stable GT protocol for most crops due to the complexity of this process. RESULTS: Here we firstly applied the hairy root transformation system in exploring root-RKN interactions in cucumber plants and developed a rapid and efficient tool transformation using Rhizobium rhizogenes strain K599. A solid-medium-based hypocotyl-cutting infection (SHI) method, a rockwool-based hypocotyl-cutting infection (RHI) method, and a peat-based cotyledon-node injection (PCI) method was evaluated for their ability to induce transgenic roots in cucumber plants. The PCI method generally outperformed the SHI and RHI methods for stimulating more transgenic roots and evaluating the phenotype of roots during nematode parasitism. Using the PCI method, we generated the CRISPR/Cas9-mediated malate synthase (MS) gene (involved in biotic stress responses) knockout plant and the LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16, a potential host susceptibility gene for RKN) promoter-driven GUS expressing plant. Knockout of MS in hairy roots resulted in effective resistance against RKNs, while nematode infection induced a strong expression of LBD16-driven GUS in root galls. This is the first report of a direct link between these genes and RKN performance in cucumber. CONCLUSION: Taken together, the present study demonstrates that the PCI method allows fast, easy and efficient in vivo studies of potential genes related to root-knot nematode parasitism and host response.

Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots.

In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated.

Hairy Root Cultures as a Source of Polyphenolic Antioxidants: Flavonoids, Stilbenoids and Hydrolyzable Tannins.

Due to their chemical properties and biological activity, antioxidants of plant origin have gained interest as valuable components of the human diet, potential food preservatives and additives, ingredients of cosmetics and factors implicated in tolerance mechanisms against environmental stress. Plant polyphenols are the most prominent and extensively studied, albeit not only group of, secondary plant (specialized) metabolites manifesting antioxidative activity. Because of their potential economic importance, the productive and renewable sources of the compounds are desirable. Over thirty years of research on hairy root cultures, as both producers of secondary plant metabolites and experimental systems to investigate plant biosynthetic pathways, brought about several spectacular achievements. The present review focuses on the Rhizobium rhizogenes-transformed roots that either may be efficient sources of plant-derived antioxidants or were used to elucidate some regulatory mechanisms responsible for the enhanced accumulation of antioxidants in plant tissues.

Improving galegine production in transformed hairy roots of Galega officinalis L. via elicitation.

Galega officinalis L. is an herbaceous legume used to treat symptoms associated with hyperglycemia or diabetes mellitus because of its dominant alkaloid, galegine. In this study, we induced hairy roots in this plant using Rhizobium rhizogenes strain A4, and investigated the effect of type, concentration, and duration of elicitor application on galegine content and some phytochemical characteristics in the hairy roots. Hence, the best growing hairy root line in terms of growth rate was selected and subcultured for treatment with elicitors. Then, at the end of the log phase of growth, chitosan (100, 200, and 400 mg/L), salicylic acid (100, 200, and 300 mM), and ultrasound (1, 2, and 4 min) were applied to hairy roots culture medium. High-performance liquid chromatography (HPLC) showed that the content of galegine was significantly increased after elicitation compared with the control. Thus, the highest content of galegine (14.55 mg/g FW) was obtained 2 days after elicitation when ultrasonic waves were applied to the hairy root culture medium for 4 min. Also, elicitation resulted in a significant increase in the content of total phenol, flavonoid, H2O2 and MDA compared with the control. So that the highest total flavonoid content was obtained in hairy roots that were treated with ultrasonic waves for 4 min and harvested 2 days after elicitation; while, application of 400 mg/L chitosan for 4 days resulted in the highest total phenol (16.84 mg/g FW).

Rhizobium rhizogenes infection in threatened Indian orchid Dendrobium ovatum mobilises 'Moscatilin' to enhance plant defensins.

The present study illustrates the transformation ability of two wild-type bacterial strains of Rhizobium rhizogenes (MTCC 532 and MTCC 2364) on the embryogenic callus and callus-derived plantlets of a threatened Indian orchid, Dendrobium ovatum. Co-culture of the bacterium with the explants gave marginal hairy root phenotype that failed to multiply in the culture medium. Some primary and secondary metabolites were subdued in infected explants. Moscatilin, the stilbenoid active principle in D. ovatum, was found below the detection limit. The presence of two metabolites viz., Laudanosine, a benzyltetrahydroisoquinoline alkaloid and Lyciumin B, a cyclic peptide, were detected exclusively in the infected explants. The subjugated amino acids and phenolics in the infected plantlets were routed to produce phytoanticipins, and phenanthrenes, strengthening the defence mechanism in infected tissues. This research implies that the plant's defence mechanism activation could have prevented the extensive hairy root formation in the explants, even though nodulations and phenotype transitions were witnessed. Moscatilin has a structural resemblance with Resveratrol, a phytoalexin that combats bacterial and fungal pathogens. The study favours the possibility of Moscatlin being a precursor for phenanthrene compounds, thereby serving as a 'phytoanticipin' during the infection phase. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03180-9.

Generation of Stable Catharanthus roseus Hairy Root Lines with Agrobacterium rhizogenes.

Agrobacterium rhizogenes is the bacterial agent that causes hairy root disease in dicots and is purposefully engineered for the development of transgenic hairy root cultures. Due to their genetic and metabolic stability, hairy root cultures offer advantages as a tissue culture system for investigating the function of transgenes and as a production platform for specialized metabolites or proteins. The process for generating hairy root cultures involves first infecting the explant with A. rhizogenes, excising and eliminating A. rhizogenes from the emerging hairy roots, selecting for transgenic hairy roots on plates containing the selective agent, confirming genomic integration of transgenes by PCR, and finally adapting the hairy roots in liquid media. Here we provide a detailed protocol for developing and maintaining transgenic hairy root cultures of our medicinal plant of interest, Catharanthus roseus.

Response of physiological parameters in Dionaea muscipula J. Ellis teratomas transformed with rolB oncogene.

BACKGROUND: Plant transformation with rol oncogenes derived from wild strains of Rhizobium rhizogenes is a popular biotechnology tool. Transformation effects depend on the type of rol gene, expression level, and the number of gene copies incorporated into the plant's genomic DNA. Although rol oncogenes are known as inducers of plant secondary metabolism, little is known about the physiological response of plants subjected to transformation. RESULTS: In this study, the physiological consequences of rolB oncogene incorporation into the DNA of Dionaea muscipula J. Ellis was evaluated at the level of primary and secondary metabolism. Examination of the teratoma (transformed shoots) cultures of two different clones (K and L) showed two different strategies for dealing with the presence of the rolB gene. Clone K showed an increased ratio of free fatty acids to lipids, superoxide dismutase activity, synthesis of the oxidised form of glutathione, and total pool of glutathione and carotenoids, in comparison to non-transformed plants (control). Clone L was characterised by increased accumulation of malondialdehyde, proline, activity of superoxide dismutase and catalase, total pool of glutathione, ratio of reduced form of glutathione to oxidised form, and accumulation of selected phenolic acids. Moreover, clone L had an enhanced ratio of total triglycerides to lipids and accumulated saccharose, fructose, glucose, and tyrosine. CONCLUSIONS: This study showed that plant transformation with the rolB oncogene derived from R. rhizogenes induces a pleiotropic effect in plant tissue after transformation. Examination of D. muscipula plant in the context of transformation with wild strains of R. rhizogenes can be a new source of knowledge about primary and secondary metabolites in transgenic organisms.

Preliminary Phytochemical Analysis and Evaluation of the Biological Activity of Leonotis nepetifolia (L.) R. Br Transformed Roots Extracts Obtained through Rhizobium rhizogenes-Mediated Transformation.

According to the present knowledge, this is the first report on establishing transformed root cultures of Leonotis nepetifolia after Rhizobium rhizogenes-mediated transformation. The preliminary phytochemical analysis showed differences in the content of phenols and flavonoids in transformed and nontransformed roots. The dominant compounds in the analyzed extracts were (+)-catechin (5464 and 6808 µg/g DW), p-coumaric acid (2549 and 4907 µg/g DW), m-coumaric acid (1508 and 2048 µg/g DW) and rosmarinic acid (1844 and 2643 µg/g DW) for nontransformed (LNNR) and transformed (LNTR4) roots, respectively. Initial biological studies carried out on LNNR, and LNTR4 extracts showed a cytotoxic effect on the A549 lung, HCC1937 breast and leukemia NALM-6 cell lines, antioxidants, as well as repair and protection against DNA damage induced by H2O2 in HUVEC cells. Due to the stronger effect of the LNTR4 root extract, which can be a relatively efficient and cheap source of bioactive secondary metabolites, further biological analyses are needed to discover in detail their potentially valuable biological properties.

Opine biosynthesis in naturally transgenic plants: Genes and products.

The plant pathogen Agrobacterium transfers DNA into plant cells by a specific transfer mechanism. Expression of this transferred DNA or T-DNA leads to crown gall tumors or abnormal, hairy roots and the synthesis of specific compounds, called opines. Opines are produced from common plant metabolites like sugars, amino acids and α-keto acids, which are combined into different low molecular weight structures by T-DNA-encoded opine synthase enzymes. Opines can be converted back by Agrobacterium into the original metabolites and used for agrobacterial growth. Recently it has been discovered that about 7% of Angiosperms carry T-DNA-like sequences. These result from ancient Agrobacterium transformation events, followed by spontaneous regeneration of transformed cells into natural genetically transformed organisms (nGMOs). Nearly all nGMOs identified up to date carry opine synthesis genes, several of these are intact and potentially encode opine synthesis. So far, only tobacco and cuscuta have been demonstrated to contain opines. Whereas opines from crown gall and hairy root tissues have been studied for over 60 years, those from the nGMOs remain to be explored.

Transformed tissue of Dionaea muscipula J. Ellis as a source of biologically active phenolic compounds with bactericidal properties.

The Venus flytrap (Dionaea muscipula J. Ellis) is a carnivorous plant able to synthesize large amounts of phenolic compounds, such as phenylpropanoids, flavonoids, phenolic acids, and 1,4-naphtoquinones. In this study, the first genetic transformation of D. muscipula tissues is presented. Two wild-type Rhizobium rhizogenes strains (LBA 9402 and ATCC 15834) were suitable vector organisms in the transformation process. Transformation led to the formation of teratoma (transformed shoot) cultures with the bacterial rolB gene incorporated into the plant genome in a single copy. Using high-pressure liquid chromatography, we demonstrated that transgenic plants were characterized by an increased quantity of phenolic compounds, including 1,4-naphtoquinone derivative, plumbagin (up to 106.63 mg × g-1 DW), and phenolic acids (including salicylic, caffeic, and ellagic acid), in comparison to non-transformed plants. Moreover, Rhizobium-mediated transformation highly increased the bactericidal properties of teratoma-derived extracts. The antibacterial properties of transformed plants were increased up to 33% against Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli and up to 7% against Pseudomonas aeruginosa. For the first time, we prove the possibility of D. muscipula transformation. Moreover, we propose that transformation may be a valuable tool for enhancing secondary metabolite production in D. muscipula tissue and to increase bactericidal properties against human antibiotic-resistant bacteria. KEY POINTS: • Rhizobium-mediated transformation created Dionaea muscipula teratomas. • Transformed plants had highly increased synthesis of phenolic compounds. • The MBC value was connected with plumbagin and phenolic acid concentrations.