Plant Cell Cultures Research Articles

Activation of Cryptic Secondary Metabolite Biosynthesis in Tobacco BY-2 Suspension Cells by Epigenetic Modifiers.

Cultured plant cells often biosynthesize secondary metabolites to a lesser extent relative to the mother plants. This phenomenon is associated with epigenetic alterations of the biosynthetic gene(s). Here we investigated the effectiveness of epigenetic modifiers, such as inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT), to activate cryptic secondary metabolite biosynthesis in tobacco (Nicotiana tabacum) BY-2 cells. The BY-2 suspension cells cultured with an HDAC inhibitor, suberoyl bis-hydroxamic acid, exhibited strong biosynthesis of four compounds that were originally present at trace concentrations. The induced compounds were identified as caffeoylputrescine (1), 4-O-β-D-glucopyranosylferulic acid (2), 5-O-caffeoylquinic acid (3), and feruloylputrescine (4). Biosynthetic activation of compounds 1-4 was reproduced by two other HDAC inhibitors. Treatment of the cells with a DNMT inhibitor (zebularine) also activated the biosynthesis of compounds 1-4, but had a limited effectiveness relative to the HDAC inhibitors, indicating that histone acetylation levels are involved more than DNA methylation levels in the epigenetic regulation of the biosynthesis of compounds 1-4 in the BY-2 cells. Following our previous demonstration using cultured cells of a monocotyledonous plant, this study demonstrates the utility of epigenetic modifiers to activate cryptic secondary metabolite biosynthesis in cultured cells of a dicotyledonous plant.

Production of phenolic compounds, flavonoids, and lupeol by Lycium schweinfurthii suspension cultures in shake flasks and a rocking-motion bioreactor

Lycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.

Targeted regulation of 5-aminolevulinic acid enhances flavonoids, anthocyanins and proanthocyanidins accumulation in Vitis davidii callus

BackgroundSpine grape (Vitis davidii) is a promising source of high-quality anthocyanins, with vast potential for application in food, pharmaceutical, and cosmetic industries. However, their availability is limited by resource constraints. Plant cell culture has emerged as a valuable approach for anthocyanin production and serves as an ideal model to investigate the regulation of anthocyanin biosynthesis. Elicitors are employed to achieve targeted enhancement of anthocyanin biosynthesis. The present study investigated the impact of 5-aminolevulinic acid (ALA) as an elicitor on the accumulation of anthocyanins and flavonoids during spine grape callus growth. Specifically, we examined the effects of ALA on anthocyanin and its component accumulation in callus, and biosynthetic anthocyanin gene expression.ResultsALA at 25 µg/L increased the biomass of spine grape callus. ALA induction enhanced the levels of flavonoids, anthocyanins and proanthocyanidins in callus, with maximum values reaching 911.11 mg/100 g DW, 604.60 mg/100 g DW, and 5357.00 mg/100 g DW, respectively, after callus culture for 45 days. Notably, those levels were 1.47-, 1.93- and 1.83-fold higher than controls. ALA induction modulated the flavonoid profile, and among 97 differential flavonoid metabolites differing from controls, 77 were upregulated and 20 were downregulated. Six kinds of anthocyanins, namely cyanidin (8), delphinidin (6), peonidin (5), malvidin (4), petunidin (3) and pelargonidin (3), were detected in callus, with peonidin most abundant. Compared with controls, anthocyanin components were increased in ALA-treated callus. The key genes PAL1, PAL2, PAL4, CHI, CHS3, F3’H, F3H, FLS, DFR, UFGT, MYBA1, LDOX, OMT3, GT1 and ACT involved in anthocyanin biosynthesis were upregulated following ALA treatment, resulting in anthocyanin accumulation.ConclusionThis study revealed a novel mode of ALA-mediated promotion of plant anthocyanin biosynthesis and accumulation at the cellular level, and a strategy for enhancing anthocyanin content in spine grape callus. The findings advance commercial-scale production of anthocyanins via spine grape callus culture. we also explored the accumulation patterns of flavonoids and anthocyanins under ALA treatment. Augmentation of anthocyanins coincided with elevated expression levels of most genes involved in anthocyanin biosynthesis within spine grape callus following ALA treatment.

Morphological, cytogenetic, and phytochemical estimation of Lychnis flos-cuculi L. cell cultures: can cell biomass be a source of bioactive compounds?

The ragged robin (Lychnis flos-cuculi L.) from the Caryophyllaceae family contains flavonoids, phenolic acids, flavonoids, triterpenoid saponins, and ecdysteroids, making it potential for biological and medicinal application. Plant cell culture technologies could be beneficial for obtaining low-cost biomass sources with valuable phytochemicals sustainably. The current study reports the induction and proliferation of ragged robin cell culture and evaluation of various plant growth regulators (PGRs) impact on the secondary metabolites profile using UPLC-HRMS/MS. Callus was initiated from different explants on the Murashige and Skoog medium with various combinations and concentrations of PGRs. It was found that prolonged proliferation on MS medium enriched with a combination of 1.0 mg l− 1 2,4-Dichlorophenoxyacetic acid and 0.1 mg l− 1 N6-furfuryl adenine (Kinetin) yields the maximum biomass with 504.05% growth index. The results of flow cytometric analysis suggested no genetic alteration between differentiated and dedifferentiated cultures. Furthermore, the UPLC-HRMS/MS result distinguished 101 significant signals that were manually identified as ecdysteroids (16 metabolites), flavonoids (11 metabolites), phenolic acids (10 metabolites), triterpene saponins and sapogenins (53 metabolites), and some primary metabolites. Our study demonstrates that the cell culture of L. flos-cuculi displays significant differences in its phytochemical makeup to plants cultivated in vitro. Moreover, the type of growth regulators used for callus cultures was identified to cause substantial shifts in the identified metabolites.Graphical

Exhausted plant cell culture media as potential biostimulants to enhance plant growth and nitrogen use efficiency in tomatoes under optimal and reduced nitrogen supply

AbstractBackgroundInefficient nitrogen (N) use leads to economic losses and environmental harm. Improving N use efficiency (NUE) is crucial. The use of plant biostimulants (PBs), integrated with good agronomic practices, offers a sustainable solution.AimsThe study explores the potential use of exhausted cell culture media from plant cell cultures (Nicotiana sp. and Gardenia sp.) as PBs, assessing their role in enhancing plant physiological status and NUE. It also compares their effectiveness against commercial PBs.MethodsTwo experiments (Experiments 1 and 2) were conducted using cherry tomato plants. In the first one, the two media were applied at different concentrations as a foliar spray to define the optimal dose. This outcome was used to set up Experiment 2, in which both media were compared to commercial PBs under optimal and reduced N supply. Measurements included plant growth, chlorophyll (Chl) content, and NUE indices. Moreover, Experiment 2 examined gene expression related to N assimilation, transport, and response to oxidative stress.ResultsBoth experiments demonstrated a significant increase in leaf area (≈11%) and aboveground biomass (≈13%) using exhausted media. Furthermore, Experiment 2 demonstrated enhancements in Chl content (≈10%) and plant N accumulation (≈20%). These findings indicate that exhausted media has a comparable efficacy to commercial PBs.ConclusionsThe study underscores the practical viability of exhausted cell culture media as effective PBs for tomato growth in both N conditions. This approach aligns with sustainability objectives by repurposing by‐products to enhance plant resilience against abiotic stress and potentially improve the efficiency of N fertilizers.

Exosome-like Nanoparticles, High in Trans-δ-Viniferin Derivatives, Produced from Grape Cell Cultures: Preparation, Characterization, and Anticancer Properties.

Background: Recent interest in plant-derived exosome-like nanoparticles (ENs) has surged due to their therapeutic potential, which includes antioxidant, anti-inflammatory, and anticancer activities. These properties are attributed to their cargo of bioactive metabolites and other endogenous molecules. However, the properties of ENs isolated from plant cell cultures remain less explored. Methods: In this investigation, grape callus-derived ENs (GCENs) were isolated using differential ultracentrifugation techniques. Structural analysis through electron microscopy, nanoparticle tracking analysis, and western blotting confirmed that GCENs qualify as exosome-like nanovesicles. Results: These GCENs contained significant amounts of microRNAs and proteins characteristic of plant-derived ENs, as well as trans-δ-viniferin, a notable stilbenoid known for its health-promoting properties. Functional assays revealed that the GCENs reduced the viability of the triple-negative breast cancer cell line MDA-MB-231 in a dose-dependent manner. Moreover, the GCENs exhibited negligible effects on the viability of normal human embryonic kidney (HEK) 293 cells, indicating selective cytotoxicity. Notably, treatment with these GCENs led to cell cycle arrest in the G1 phase and triggered apoptosis in the MDA-MB-231 cell line. Conclusions: Overall, this study underscores the potential of grape callus-derived nanovectors as natural carriers of stilbenoids and proposes their application as a novel and effective approach in the management of cancer.

Potato and dairy industry side streams as feedstock for fungal and plant cell cultures

Utilization of industrial side streams as nutrient source for cellular agriculture is a promising option to improve the sustainability of the production processes. The aim of the study was to evaluate usability of two liquid food industry side streams, potato cell fluid and acid whey, as nutrient source for production of food ingredients with plant cell cultures (arctic bramble (Rubus arcticus L.) and tobacco BY-2 (Nicotiana tabacum L.)) and filamentous fungi (Paecilomyces variotii, Rhizopus oligosporus and Trichoderma reesei). The side streams were found to contain various sugars and other potential nutrients suitable for the studied organisms. The side streams were used as a sole nutrient source (fungi) or as a replacement of selected nutrients in the growth media (fungi and plant cells) in flask scale cultivations. Acid whey was successfully used as a growth medium for filamentous fungi, but it inhibited plant cell growth presumably due to high organic acid content (14 g/l). Potato side stream was found suitable as a fungal and plant cell growth media supplement, and it was used together with glucose for filamentous fungi, or as a partial replacer of macronutrients for plant cells, in bioreactor cultivations yielding high fungal (up to 36 g/l dry weight) and good plant cell (9.5 g/l dry weight) biomass production. Analyses of the produced biomasses revealed good nutritional value in terms of amino acid (17–27 % of dry matter) and dietary fiber (23–30 % of dry matter) contents, giving good premises for utilization in human nutrition.

Role of biosynthesized silver nanoparticles in mitigating hyperhydricity in micropropagated watermelon (Citrullus lanatus Thunb.) and assessment of genetic fidelity using RAPD and SCoT markers

Watermelon (Citrullus lanatus) is one of the five most consumed fresh fruits and is a commercially important horticulture crop. The consumer demand for seedless watermelons has considerably increased due to their superior taste and ease of consumption. Seedless watermelon plants are triploids hence plant tissue culture technology which is the cultivation of plant cells, tissues, or organs on specially formulated nutrient media can facilitate the rapid multiplication of seedless watermelon varieties. However, hyperhydricity has been reported as a frequent problem for in vitro growth and development of watermelon. Hence, the present study highlights the application of silver nanoparticles biosynthesized from Parthenium hysterophorus (PHAgNPs) for reversal of hyperhydricity and enhancing in vitro shoot production in watermelon. The biosynthesized PHAgNPs was characterized using UV–vis spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis and Transmission electron microscopy. Cotyledonary nodes used as explant and cultured in three types of cytokinins (BAP, TDZ and KIN) became hyperhydric, losing their ability to regenerate at high cytokinin concentrations (> 1.5 mg/L) irrespective of the cytokinin type. The optimum concentration of BAP (1.0 mg/L) fortified with PHAgNPs (15.0 mg/L) exhibited an improved response of 91 % and induced a maximum of 32.33 shoots with a maximum shoot length of 5.66 cm after 6 weeks. The incorporation of PHAgNPs reduced the percentage of hyperhydricity from 29 % to 1.33 % in regenerated shoots, which was confirmed by the reduction in relative water content (RWC) from 93.33 % to 80.33. Silver ions reduced H2O2 content by 50 % and increased photosynthetic pigments (chlorophyll a, b and carotenoids) by 50 %, which contributed to the formation of healthy regenerated shoots, with opened stomata compared to closed in hyperhydric shoots. Compared with those of hyperhydric shoots, the gene expression patterns of 1-aminocyclopropane-1-carboxylase synthase (ACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1) decreased after the retroversion of shoots to 15 mg/L PHAgNP medium. The relative gene expression profiles of ACS1 and ACO1 in hyperhydric shoots were 8.8- and 1.8-fold greater than those in normal shoots, respectively. Profuse root growth was observed in MS basal media supplemented with IBA (1.0 mg/L) and PHAgNPs (10.0 mg/L), which resulted in the greatest number of roots (12.33), with an average root length of 4.33 cm and a response of 89.33 %. The genotoxic effect of PHAgNPs evaluated using RAPD and SCoT molecular markers, showed that the addition of low levels of silver nanoparticles to the medium did not cause any genetic variation. The efficient regeneration protocol standardized in this study provides a solution to solve the problem of hyperhydricity in watermelon regeneration.

Varying Tolerance to Diesel Toxicity Revealed by Growth Response Evaluation of Petunia grandiflora Shoot Lines Regenerated after Diesel Fuel Treatment

Continuous efforts are required to find ways to protect crop production against the toxicity of petroleum hydrocarbons, such as diesel, and contamination of soils. There is a need for identification of candidate plants that are tolerant to diesel toxicity that might also have the potential for remediation of diesel-contaminated soils. In this study, petunia, a popular ornamental plant and a model experimental plant in research on phytoremediation of environmental pollutants, was used to evaluate a novel method for rapidly assessing diesel toxicity based on the tolerance of shoots generated through in vitro plant cell culture selection. Petunia shoot lines (L1 to L4) regenerated from diesel-treated callus were compared with those from non-diesel-treated callus (control). Significant morphological differences were observed among the tested lines and control, notably with L1 and L4 showing superior growth. In particular, L4 exhibited remarkable adaptability, with increased root development and microbial counts in a diesel-contaminated potting mix, suggesting that the shoots exhibited enhanced tolerance to diesel exposure. Here, this rapid bioassay has been shown to effectively identify plants with varying levels of tolerance to diesel toxicity and could therefore assist accelerated selection of superior plants for phytoremediation. Further research is needed to understand the genetic and physiological mechanisms underlying tolerance traits, with potential applications beyond petunias to other environmentally significant plants.

Correction to: Production of betalains in plant cell and organ cultures: a review

Correction to: Production of betalains in plant cell and organ cultures: a review

185th anniversary of the cell theory and modern cell technologies

The article briefly reviews the history of the development of the cell theory in the 19th century and analyses the contribution of its authors Theodor Schwann, Matthias Schleiden, and Rudolf Virchow. Particular attention is paid to the development of the cell theory and its practical significance, in particular, the invention in the 20th century and further development of cell technologies. The main characteristics of cultured cells and tissues in vitro as a new experimentally created biological system are presented, and the peculiarities of the establishment of strains of cultured plant cells are discussed. Using plants as an example, the genomic variability of somatic cells is analysed both during ontogeny and in the course of their culturing in vitro. It is assumed that any somatic cell with a living (functionally active) nucleus, when isolated and subsequently cultured in vitro can restore in its offspring, including regenerated plants, the genetic polymorphism inherent in this plant species as a result of dedifferentiation and “somaclonal” variability (the latter is assumed to occur according to the N. I. Vavilov’s law of homologous series in variation). It ispostulated that a plant is a system of cell populations characterised by the plasticity of its gene pool, which is based on genome plasticity of somatic cells, which, in interaction with cellular selection, ensures the adaptability of the plant as an integral organism and creates the possibility of inheritance (transmission to offspring) of adaptive genomic changes acquired during ontogeny. It is concluded that the adaptive traits of an organism are determined by the adaptive changes in the cells, which composed it. The author also examines the main directions of modern cell technologies and their application in crop production, in medicine for the treatment and creation of new organs using stem cells, as well as in the food industry on the example of the development of technologies for the production of artificial meat using animal stem cells, etc. It is concluded that the issue of cell adaptations remains one of the most important and still insufficiently understood issues of modern biology.

Role of 6mA in the Regulation of Metabolic Biosynthesis in Sorghum Callus.

Plant cell culture technology helps to obtain natural plant-derived metabolites. The callus of sorghum, a prominent cereal crop, possesses various metabolites with potential health benefits. However, the epigenetic mechanism regulating metabolic biosynthetic capabilities in sorghum remains unknown. Therefore, we conducted N6-methyladenine (6mA) methylome analysis using transcriptome profiling and metabolome analysis to investigate the role of 6mA alterations in two calluses having different biosynthetic capacities, which were derived from immature sorghum embryos. Our findings indicate that the 6mA upregulation within gene bodies is crucial in transcriptional activity potentially mediated by the DNA demethylase SbALKBH1. Furthermore, 6mA was significantly enriched in genes involved in the biosynthesis of flavonoids and isoflavonoids. This could serve as a novel source of bioactive compounds for human health. Thus, 6mA could play an essential role in flavonoid biosynthesis in the sorghum callus.

Initiation of Cassava Callus Culture and Its Prospect for Starch Production: A Systematic Mapping Study

Mass propagation of cassava on several hectares of arable land due to increasing demand for its starch is not feasible due to land availability, pests and disease invasion, and long cultivation period. Plant cell culture technology is a promising solution despite the scarcity of cassava callus culture for starch production applications. Therefore, a systematic mapping study (SMS) was performed to identify the applications of cassava tissue culture and its prospects in starch production and investigate the important parameters for cassava callus culture initiation. The SMS began with formulating research questions (RQs), conducting searches on various databases, collecting and screening related articles, and extracting and mapping the selected articles. A total of 56 of 589 articles in the initial searching phase were chosen to be used as references to answer each RQ. The extracted data indicates that cassava tissue culture was mostly used for micropropagation, while starch production from its tissue culture is still limited. Basal medium and plant growth regulators influence cassava callus culture initiation most. The findings of the SMS offer a better understanding of cassava tissue culture and the prospects of producing cassava starch.

A rationally optimised batch bioreactor cultivation of Viola odorata plant cells for sustainable production of its key bioactive principles

A rationally optimised batch bioreactor cultivation of Viola odorata plant cells for sustainable production of its key bioactive principles

Production of betalains in plant cell and organ cultures: a review

Production of betalains in plant cell and organ cultures: a review

The optimised method of HPLC analysis of glutathione allows to determine the degree of oxidative stress in plant cell culture

Redox regulations and antioxidant defence play a central role in the acclimation of plants to their environment. Glutathione represents an essential component of the cellular antioxidant defence system, which keeps levels of reactive oxygen species (ROS) under control. High-performance liquid chromatography (HPLC) separation with fluorescence detection is a sensitive method that enables analysis of reduced and oxidised glutathione levels in small samples of plant tissues or plant cell culture. We aimed to optimise the method to obtain more accurate information about the total level of glutathione and the proportion of the reduced form (GSH) by choosing the most suitable reduction reagent and the conditions under which the reduction occurs. The applicability of the developed method was verified by analysing tobacco cells treated with hydrogen peroxide, which caused a decrease in the GSH/total glutathione ratio. Significant changes in the level of glutathione as well as in the GSH/total glutathione ratio were also observed during tobacco cell culture development.

Verazine biosynthesis from simple sugars in engineered Saccharomyces cerevisiae

Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

Influence of Culture Conditions on Growth and Daidzein and Genistein Production in Hairy Root Cultures of Pueraria candollei var. mirifica

Pueraria candollei var. mirifica produces and accumulates various phytoestrogen compounds in its tuberous roots, including daidzein and genistein. Plant cell culture methods have been established to alleviate the problems associated with producing valuable phytochemicals from natural or field-cultivated plants, and hairy root culture is one of the most promising methods for the in vitro production of plant secondary metabolites. Thus, this study aimed to produce daidzein and genistein from hairy root cultures of P. candollei var. mirifica. The influences of cultivation parameters, including the culture medium, light conditions, sugar content in the culture medium, incubation temperature, and agitation speed, on biomass and daidzein and genistein production in hairy root cultures of this medicinal plant were investigated. The results revealed that the optimal cultivation conditions for biomass and bioactive compound production were Murashige & Skoog (MS) medium, a sucrose concentration of 30 g/L, a 16/8 h light/dark photoperiod, an incubation temperature of 26 °C, and an agitation speed of 90 rpm. The highest biomass and daidzein and genistein contents achieved in this study were 17.76 g/L, 6.85 mg/g DW, and 0.96 mg/g DW, respectively. Interestingly, the daidzein and genistein contents obtained from hairy roots were approximately 45.7- and 12.0-fold greater than those obtained from normal roots, respectively, suggesting that hairy root culture is a suitable method for the sustainable production of phytoestrogen, daidzein, and genistein from this medicinal plant.

Reactive oxygen species and depolarization the inner mitochondrial membrane under effect of synthetic auxin herbicides in plant cell culture of Arabidopsis thaliana L.

Reactive oxygen species and depolarization the inner mitochondrial membrane under effect of synthetic auxin herbicides in plant cell culture of Arabidopsis thaliana L.

Metabolome and transcriptome integration explored the mechanism of browning in Glycyrrhiza uralensis Fisch cells.

Glycyrrhiza uralensis Fisch, a traditional Chinese medicinal herb known for its diverse pharmacological effects including heat-clearing, detoxification, phlegm dissolving, and cough relief, has experienced an exponential increase in demand due to its expanding clinical use and development prospects. Currently, large-scale cell culture stands out as one of the most promising biotechnological approaches for producing bioactive compounds from medicinal plants. However, the problem of cell browning represents a significant bottleneck in industrial applications of cell culture. This study focuses on the Glycyrrhiza uralensis Fisch cells from the Ordos plateau, aiming to elucidate the enzymatic browning process during plant cell culture. Key substrates and genes involved in enzymatic browning were identified by metabolome and transcriptome analysis of normal and browning cells. Metabolome analysis reveals significant changes in the levels of chalcone, isoflavone, imidazole-pyrimidine, purine nucleosides, organic oxides, carboxylic acids and their derivatives, benzene and its derivatives, flavonoids, 2-arylated benzofuran flavonoids, diazanaphthalenes and fatty acyls within browning cells. In particular, chalcones, isoflavones, and flavones compounds account for a higher proportion of these changes. Furthermore, these compounds collectively show enrichment in four metabolic pathways: Isoflavone biosynthesis pathway; Cutin suberine and wax biosynthesis pathway; Aminoacyl-tRNA biosynthesis pathway; Isoquinoline alkaloid biosynthesis pathway; Transcriptome analysis revealed that the MYB transcription factor is a key regulator of flavonoid synthesis during the browning process in cells. In addition, 223 differentially expressed genes were identified, including phenylpropane, shikimic acid, glycolysis, and pentose phosphate pathways. Among these genes, 23 are directly involved in flavonoid biosynthesis; qPCR validation showed that eight genes (GlPK, GlPAL, Gl24CL, Gl1PDT, Gl3CHI, GlC4H, Gl2F3'H, and Gl2CCR) were up-regulated in browning cells compared to normal cells. These findings corroborate the sequencing results and underscore the critical role of these genes in cellular browning. Consequently, modulation of their expression offers promising strategies for effective control of cellular browning issues.