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Isoflavones, a class of substances with high biological activity, are abundant in soybeans. This study investigated isoflavone biosynthesis in soybean cell suspension cultures under UV-B radiation. UV-B radiation enhanced the transcription level and activity of key enzymes involved in isoflavone synthesis in cell suspension cultures. As a result, the isoflavone contents significantly increased by 19.80% and 91.21% in hypocotyl and cotyledon suspension cultures compared with the control, respectively. Meanwhile, a significant difference was observed in the composition of isoflavones between soybean hypocotyl and cotyledon suspension cultures. Genistin was only detected in hypocotyl suspension cultures, whereas glycitin, daidzein, and genistein accumulated in cotyledon suspension cultures. Therefore, UV-B radiation exhibited tissue-specific regulation of isoflavone biosynthesis in soybean cell suspension cultures. The combination of suspension cultures and abiotic stress provides a novel technological approach to isoflavone accumulation.

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Protoplasts are plant cells from which the pectocellulosic cell wall has been removed, thus keeping the plasma membrane intact. For plant secondary metabolites research, this system is a powerful tool to study the metabolites' dynamics inside the cells, such as the subcellular localization of proteins, characterization of gene function, transcription factors involved in metabolite pathways, protein transport machinery, and to perform single-cell omics studies. Due to its lack of a cell wall, better images of the interior of the cell can be obtained compared to the whole tissue. This allows the identification of specific cell types involved in the accumulation of specialized metabolites, such as alkaloids, given their autofluorescence properties. Here is a simplified protocol to obtain protoplasts from leaves and in vitro cell cultures from Argemone mexicana, which produces the pharmacologically important alkaloids berberine and sanguinarine.

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Plants reprogramme their proteome to alter cellular metabolism for effective stress adaptation. Intracellular proteomic responses have been extensively studied, and the extracellular matrix stands as a key hub where peptide signals are generated/processed to trigger critical adaptive signal transduction cascades inaugurated at the cell surface. Therefore, it is important to study the plant extracellular proteome to understand its role in plant development and stress response. This study examined changes in the soluble extracellular sub-proteome of sorghum cell cultures exposed to a combination of sorbitol-induced osmotic stress and heat at 40 °C. The combined stress significantly reduced metabolic activity and altered protein secretion. While cells treated with osmotic stress alone had elevated proline content, the osmoprotectant in the combined treatment remained unchanged, confirming that sorghum cells exposed to combined stress utilise adaptive processes distinct from those invoked by the single stresses applied separately. Reactive oxygen species (ROS)-metabolising proteins and proteases dominated differentially expressed proteins identified in cells subjected to combined stress. ROS-generating peroxidases were suppressed, while ROS-degrading proteins were upregulated for protection from oxidative damage. Overall, our study provides protein candidates that could be used to develop crops better suited for an increasingly hot and dry climate.

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Plant tissue culture has evolved in the last decades with several types of cultures being developed to promote a more sustainable food production system. Moreover, these cultures can be applied for the production of relevant metabolites with medicinal potential, thus contributing to nutrition and healthcare. Importantly, plant micropropagation has enabled agricultural expansion and tissue culture has emerged as a promising production alternative for several plants and their metabolites in the food, cosmetic, and pharmaceutical industries. Plant tissue cultures present several advantages over conventional propagation techniques as they are season independent, enabling a continuous supply of the plants/compounds of interest, with the guarantee of high phytosanitary quality. In addition, genetic uniformity is generally maintained, thus reducing chemical variability that can compromise safety and efficacy. Nevertheless, despite their undeniable potential, with many researchers focusing on new strategies to improve production yield in cell cultures, such as with the use of elicitors or resorting to metabolomics engineering, an effective and lucrative large-scale production has yet to be obtained. Indeed, only a few compounds with market value are produced in this regard and several limitations such as contaminations, low culture yield and production costs still need to be overcome in order to take advantage of the full potential of these techniques.

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Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

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Anaerobic digestion of organic waste produces effluent (ADE) that requires further treatment. Biofilm-based microalgal cultivation is a favoured approach to ADE treatment. This study compared Chlorella sp. MUR 268 and Scenedesmus sp. MUR 269 in biofilm and suspension cultures to treat anaerobic digestate food effluent (ADFE). Chlorella sp. MUR 268 biofilm had significantly higher biomass (50.38 g m-2) than Scenedesmus sp. biofilm (9.39 g m-2). Conversely, Scenedesmus sp. yielded 1.5 times more biomass (1.2 g L-1) than Chlorella sp. in suspension. Chlorella sp. biofilm had 49.3 % higher areal productivity than suspension, while Scenedesmus sp. showed 87.3 % higher areal growth in suspension. Chlorella sp. MUR 268 and Scenedesmus sp. MUR 269 significantly removed nutrients in ADFE. In suspension, COD, ammoniacal nitrogen, and phosphate were reduced to 94.9, 5.2, and 5.98 mg L-1 for Chlorella sp. MUR 268, and 245, 2.89, and 3.22 mg L-1 for Scenedesmus sp. MUR 269, respectively. In biofilm, Chlorella sp. MUR 268 achieved reductions to 149.9, 1.16, and 3.57 mg L-1, while Scenedesmus sp. MUR 269 achieved 100.2, 6.9 and 2.07 mg L-1. Most of these values are below the recommended effluent discharge standard, highlighting the efficacy of this system in ADFE treatment. Biofilm cultures fixed 68-81 % of removed nitrogen in biomass, while in suspension, only 55-71 % ended in the biomass. Chlorella sp. MUR 268 biofilm fixed 88 % of removed phosphorus, while Scenedesmus sp. MUR 269 suspension fixed more phosphorus (55 %) than the biofilm counterpart (34 %). This biofilm design offers advantages like simplified, cost-effective operation, easy biomass recovery, and reduced water usage.

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Hybanthus enneaspermus (L.)F.Muell. is a highly indispensable medicinal herb yielding L-Dopa, deemed the gold standard drug among the therapeutic options for Parkinson's disease. This investigation is the first attempt to evaluate the eliciting influence of carboxylic acid functionalized multi-walled carbon nanotube (MWCNT-COOH) on the biosynthesis of L-Dopa and on biomass aggregation and antioxidant metabolites in H. enneaspermus cell suspension cultures. Suspension cells were accomplished from friable calli generated from the nodal segments of H. enneaspermus in Murashige and Skoog (MS) liquid medium infused with 2 mg L-1 2, 4-Dichlorophenoxyacetic acid (2, 4-D), and 0.3 mg L-1meta-Topolin (mT). The influence of MWCNTs on L-Dopa synthesis, biomass accumulation, and biochemical parameters was examined on the basis of the exposure time and in a concentration-dependent manner of MWCNTs. The inclusion of 30 mg L-1 MWCNTs increased the biomass and the L-Dopa level by 2.00 and 16.37-folds, respectively, compared with that of the control. Furthermore, the effect of MWCNTs on physiological parameters such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase (APX), hydrogen peroxide (H2O2), malondialdehyde (MDA) content, 2-diphenylpicrylhydrazyl (DPPH), and ferric-reducing ability of plasma (FRAP) was examined over the elicited cells. Among the antioxidant enzymatic activities, CAT enhanced 8.0 fold compared with that of the control. MDA and DPPH content enhanced 2.60 and 1.12 folds, respectively, compared with that of the control. The current study showed that MWCNTs offer new possibilities for their usage over in vitro by acting as potential innovative plant metabolite elicitors and stress-protecting entities.

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Inducing the differentiation of specific cell type(s) synchronously and on-demand is a great experimental system to understand the sequential progression of the cellular processes, their timing and their resulting properties for distinct isolated plant cells independently of their tissue context. The inducible differentiation in cell suspension cultures, moreover, enables to obtain large quantities of distinct cell types at specific development stage, which is not possible when using whole plants. The differentiation of tracheary elements (TEs) - the cell type responsible for the hydro-mineral sap conduction and skeletal support of plants in xylem tissues - has been the most studied using inducible cell suspension cultures. We herein describe how to establish and use inducible pluripotent suspension cell cultures (iPSCs) in Arabidopsis thaliana to trigger on-demand different cell types, such as TEs or mesophyll cells. We, moreover, describe the methods to establish, monitor, and modify the sequence, duration, and properties of differentiated cells using iPSCs.

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Drought stress adversely affects plant growth, often leading to total crop failure. Upon sensing soil water deficits, plants switch on biosynthesis of abscisic acid (ABA), a stress hormone for drought adaptation. Here, we used exogenous ABA application to dark-grown sorghum cell suspension cultures as an experimental system to understand how a drought-tolerant crop responds to ABA. We evaluated intracellular and secreted proteins using isobaric tags for relative and absolute quantification. While the abundance of only ~ 7% (46 proteins) intracellular proteins changed in response to ABA, ~32% (82 proteins) of secreted proteins identified in this study were ABA responsive. This shows that the extracellular matrix is disproportionately targeted and suggests it plays a vital role in sorghum adaptation to drought. Extracellular proteins responsive to ABA were predominantly defense/detoxification and cell wall-modifying enzymes. We confirmed that sorghum plants exposed to drought stress activate genes encoding the same proteins identified in the in vitro cell culture system with ABA. Our results suggest that ABA activates defense and cell wall remodeling systems during stress response. This could underpin the success of sorghum adaptation to drought stress.

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BACKGROUND: Rehmannia glutinosa is a rich source of terpenoids with a high medicinal reputation. The present study compared dedifferentiated cells (DDCs) and cambial meristematic cells (CMCs) cell cultures of R. glutinosa for terpenoid (catalpol) and indole alkaloid (IA) biosynthesis. In this regard, we used widely targeted metabolomics and transcriptome sequencing approaches together with the comparison of cell morphology, cell death (%), and catalpol production at different time points. RESULTS: We were able to identify CMCs based on their morphology and hypersensitivity to zeocin. CMCs showed higher dry weight content and better catalpol production compared to DDCs. The metabolome analysis revealed higher concentrations of IA, terpenoids, and catalpol in CMCs compared to DDCs. The transcriptome sequencing analysis showed that a total of 27,201 genes enriched in 139 pathways were differentially expressed. The higher catalpol concentration in CMCs is related to the expression changes in genes involved in acetyl-CoA and geranyl-PP biosynthesis, which are precursors for monoterpenoid biosynthesis. Moreover, the expressions of the four primary genes involved in monoterpenoid biosynthesis (NMD, CYP76A26, UGT6, and CYP76F14), along with a squalene monooxygenase, exhibit a strong association with the distinct catalpol biosynthesis. Contrarily, expression changes in AADC, STR, and RBG genes were consistent with the IA biosynthesis. Finally, we discussed the phytohormone signaling and transcription factors in relation to observed changes in metabolome. CONCLUSIONS: Overall, our study provides novel data for improving the catalpol and IA biosynthesis in R. glutinosa.

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Sageretia thea is used in the preparation of herbal medicine in China and Korea; this plant is rich in various bioactive compounds, including phenolics and flavonoids. The objective of the current study was to enhance the production of phenolic compounds in plant cell suspension cultures of Sageretia thea. Optimum callus was induced from cotyledon explants on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 mg L-1), naphthalene acetic acid (NAA, 0.5 mg L-1), kinetin (KN; 0.1 mg L-1) and sucrose (30 g L-1). Browning of callus was successfully avoided by using 200 mg L-1 ascorbic acid in the callus cultures. The elicitor effect of methyl jasmonate (MeJA), salicylic acid (SA), and sodium nitroprusside (SNP) was studied in cell suspension cultures, and the addition of 200 µM MeJA was found suitable for elicitation of phenolic accumulation in the cultured cells. Phenolic and flavonoid content and antioxidant activity were determined using 2,2 Diphenyl 1 picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethybenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP) assays and results showed that cell cultures possessed highest phenolic and flavonoid content as well as highest DPPH, ABTS, and FRAP activities. Cell suspension cultures were established using 5 L capacity balloon-type bubble bioreactors using 2 L of MS medium 30 g L-1 sucrose and 0.5 mg L-1 2,4-D, 0.5 mg L-1 NAA, and 0.1 mg L-1 KN. The optimum yield of 230.81 g of fresh biomass and 16.48 g of dry biomass was evident after four weeks of cultures. High-pressure liquid chromatography (HPLC) analysis showed the cell biomass produced in bioreactors possessed higher concentrations of catechin hydrate, chlorogenic acid, naringenin, and other phenolic compounds.

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This study aimed to establish a cryopreservation protocol for G. chacoensis embryogenic cultures (ECs) and to investigate the role of antioxidant enzymes activities during cryopreservation. The growth dynamics of cell suspensions were also investigated, followed by a phytotoxicity test to assess the ECs' ability to tolerate the use of cryoprotective solutions for different incubation times (0, 30, 60, 120, and 240 min). We evaluated the EC redox state in three steps of cryopreservation: after incubation in cryoprotection solution, after thawing, and 60 days after regrowth. Our results showed that the ECs support the use of cryoprotective solution until 120 min, showing phytotoxic effects with 240 min of incubation. This study reports a 100% survival of the cultures and a 10% increase ratio in fresh material for both incubation times tested (60 and 120 min). Increased malonaldehyde content was identified after incubation in the cryoprotective solution. An increase in the activities of catalase and ascorbate peroxidase was also identified in the subsequent steps, suggesting that the activation of antioxidant enzymes is essential for maintaining cell homeostasis during cryopreservation.

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Adipose-derived mesenchymal stromal cells (MSC(AT)) display immunomodulatory and angiogenic properties, but an improved understanding of quantitative critical quality attributes (CQAs) that inform basal MSC(AT) fitness ranges for immunomodulatory and/or angiogenic applications is urgently needed for effective clinical translation. We constructed an in vitro matrix of multivariate readouts to identify putative CQAs that were sensitive enough to discriminate between specific critical processing parameters (CPPs) chosen for their ability to enhance MSC immunomodulatory and angiogenic potencies, with consideration for donor heterogeneity. We compared 3D aggregate culture conditions (3D normoxic, 3D-N) and 2D hypoxic (2D-H) culture as non-genetic CPP conditions that augment immunomodulatory and angiogenic fitness of MSC(AT). We measured multivariate panels of curated genes, soluble factors, and morphometric features for MSC(AT) cultured under varying CPP and licensing conditions, and we benchmarked these against two functional and therapeutically relevant anchor assays - in vitro monocyte/macrophage (MΦ) polarization and in vitro angiogenesis. Our results showed that varying CPP conditions was the primary driver of MSC(AT) immunomodulatory fitness; 3D-N conditions induced greater MSC(AT)-mediated MΦ polarization toward inflammation-resolving subtypes. In contrast, donor heterogeneity was the primary driver of MSC(AT) angiogenic fitness. Our analysis further revealed panels of putative CQAs with minimum and maximum values that consisted of twenty MSC(AT) characteristics that informed immunomodulatory fitness ranges, and ten MSC(AT) characteristics that informed angiogenic fitness ranges. Interestingly, many of the putative CQAs consisted of angiogenic genes or soluble factors that were inversely correlated with immunomodulatory functions (THBS1, CCN2, EDN1, PDGFA, VEGFA, EDIL3, ANGPT1, and ANG genes), and positively correlated to angiogenic functions (VEGF protein), respectively. We applied desirability analysis to empirically rank the putative CQAs for MSC(AT) under varying CPP conditions and donors to numerically identify the desirable CPP conditions or donors with maximal MSC(AT) immunomodulatory and/or angiogenic fitness. Taken together, our approach enabled combinatorial analysis of the matrix of multivariate readouts to provide putative quantitative CQAs that were sensitive to variations in select CPPs that enhance MSC immunomodulatory/angiogenic potency, and donor heterogeneity. These putative CQAs may be used to prospectively screen potent MSC(AT) donors or cell culture conditions to optimize for desired basal MSC(AT) immunomodulatory or angiogenic fitness.

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Glucosinolates, a group of sulfur-containing specialized metabolites of the Brassicales, have attracted a lot of interest in nutrition, medicine and agriculture due to their positive health effects and their involvement in plant defense. Their biological activities and the extensive knowledge of their biosynthesis have inspired research into development of crops with enhanced glucosinolate contents as well as their biotechnological production in homologous and heterologous systems. Here, we provide proof-of-concept for transgenic suspension cultures of carrot (Daucus carota, Apiacae) as a scalable production platform for plant specialized metabolites using benzylglucosinolate as a model. Two T-DNAs carrying in total six genes of the benzylglucosinolate biosynthesis pathway from Arabidopsis thaliana as well as NPTII and BAR as selectable markers were transferred to carrot cells by Agrobacterium tumefaciens-mediated transformation. Putative transformants selected based on their kanamycin and BASTA resistances were subjected to HPLC-MS analysis. Of 79 putative transformants, 17 produced benzylglucosinolate. T-DNA-integration was confirmed for the five best producers. Callus from these transformants was used to establish suspension cultures for quantitative analysis. When grown in 60-ml-cultures, the best transformants produced roughly 2.5 nmol (g fw)-1 benzylglucosinolate, together with up to 10 nmol (g fw)-1 desulfobenzylglucosinolate. Only one transformant produced more benzylglucosinolate than desulfobenzylglucosinolate. The concentration of sulfate in the medium was not a major limiting factor. High production seemed to be associated with poor growth and vice versa. Therefore, future research should try to optimize medium and cultivation process and to separate growth and production phase by using an inducible promoter.

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BACKGROUND AND AIMS: Rhamnogalacturonan-II (RG-II) is a domain of primary cell-wall pectin. Pairs of RG-II domains are covalently cross-linked via borate diester bridges, necessary for normal cell growth. Interpreting the precise mechanism and roles of boron bridging is difficult because there are conflicting hypotheses as to whether bridging occurs mainly within the Golgi system, concurrently with secretion or within the cell wall. We therefore explored the kinetics of RG-II bridging. METHODS: Cell-suspension cultures of Rosa and arabidopsis were pulse-radiolabelled with [14C]glucose, then the boron bridging status of newly synthesized [14C]RG-II domains was tracked by polyacrylamide gel electrophoresis of endo-polygalacturonase digests. KEY RESULTS: Optimal culture ages for 14C-labelling were ~5 and ~1 d in Rosa and arabidopsis respectively. De-novo [14C]polysaccharide production occurred for the first ~90 min; thereafter the radiolabelled molecules were tracked as they 'aged' in the wall. Monomeric and (boron-bridged) dimeric [14C]RG-II domains appeared simultaneously, both being detectable within 4 min of [14C]glucose feeding, i.e. well before the secretion of newly synthesized [14C]polysaccharides into the apoplast at ~15-20 min. The [14C]dimer : [14C]monomer ratio of RG-II remained approximately constant from 4 to 120 min, indicating that boron bridging was occurring within the Golgi system during polysaccharide biosynthesis. However, [14C]dimers increased slightly over the following 15 h, indicating that limited boron bridging was continuing after secretion. CONCLUSIONS: The results show where in the cell (and thus when in the 'career' of an RG-II domain) boron bridging occurs, helping to define the possible biological roles of RG-II dimerization and the probable localization of boron-donating glycoproteins or glycolipids.

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Shake flasks remain one of the most widely used cultivation systems in biotechnology, especially for process development (cell line and parameter screening). This can be justified by their ease of use as well as their low investment and running costs. A disadvantage, however, is that cultivations in shake flasks are black box processes with reduced possibilities for recording online data, resulting in a lack of control and time-consuming, manual data analysis. Although different measurement methods have been developed for shake flasks, they lack comparability, especially when changing production organisms. In this study, the use of online backscattered light, dissolved oxygen, and pH data for characterization of animal, plant, and microbial cell culture processes in shake flasks are evaluated and compared. The application of these different online measurement techniques allows key performance indicators (KPIs) to be determined based on online data. This paper evaluates a novel data science workflow to automatically determine KPIs using online data from early development stages without human bias. This enables standardized and cost-effective process-oriented cell line characterization of shake flask cultivations to be performed in accordance with the process analytical technology (PAT) initiative. The comparison showed very good agreement between KPIs determined using offline data, manual techniques, and automatic calculations based on multiple signals of varying strengths with respect to the selected measurement signal.

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Forage and turf grasses are widely grown and contribute significantly to sustainable agriculture. This chapter describes a protocol for protoplast transformation and plant regeneration for major forage and turf grass species, including tall fescue, red fescue, meadow fescue, perennial ryegrass, and Italian ryegrass. Embryogenic calli induced from caryopsis were used to establish embryogenic cell suspension cultures. Protoplasts were isolated from embryogenic suspension cultures and used for direct gene transfer. Chimeric genes were introduced into protoplasts by polyethylene glycol treatment. Upon selection with antibiotics or herbicide, resistant calli were obtained and transgenic plants were regenerated from these calli.

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Fluorescence-activated cell sorting (FACS) allows for the enrichment of specific plant cell populations after protoplasting. In this book chapter, we describe the transformation and protoplasting of an Arabidopsis thaliana cell suspension culture (PSB-D, derived from MM2d) that can be used for the evaluation of CRISPR vectors in a subpopulation of cells. We also describe the protoplasting of Arabidopsis thaliana cells from the roots and stomatal lineage for the evaluation of tissue-specific gene editing. These protocols allow us to rapidly and accurately quantify various CRISPR systems in plant cells.

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The glucose oligosaccharide-derived cyclodextrins (CDs) are used for improving bioactive compound production in plant cell cultures because, in addition to their elicitation activity, CDs promote product removal from cells. However, despite these advantages, the industrial application of CDs is hampered by their high market price. A strategy to overcome this constraint was recently tested, in which reusable CD polymers coated with magnetic Fe3O4 nanoparticles were harnessed in Vitis vinifera cell cultures to produce t-resveratrol (t-R). In this study, we applied hydroxypropyl-ß-CDs (HPCD) and HPCDs coated with magnetic nanoparticles (HPCD-EPI-MN) in methyl jasmonate (MJ)-treated transgenic Silybum marianum cultures ectopically expressing either a stilbene synthase gene (STS) or a chalcone synthase gene (CHS), and compared their effects on the yields of t-R and naringenin (Ng), respectively. HPCD-EPI-MN at 15 g/L stimulated the accumulation of metabolites in the culture medium of the corresponding transgenic cell lines, with up to 4 mg/L of t-R and 3 mg/L of Ng released after 3 days. Similar amounts were produced in cultures treated with HPCD. Concentrations higher than 15 g/L of HPCD-EPI-MN and prolonged incubation periods negatively affected cell growth and viability in both transgenic cell lines. Reutilization of HPCD-EPI-MN was possible in three elicitation cycles (72 h each), after which the polymer retained 25-30% of its initial efficiency, indicating good stability and reusability. Due to their capacity to adsorb metabolites and their recyclability, the application of magnetic CD polymers may reduce the costs of establishing efficient secondary metabolite production systems on a commercial scale. KEY POINTS: • Long-term transgenic S. marianum suspensions stably produce transgene products • t-R and Ng accumulated extracellularly in cultures elicited with HPCD and HPCD-EPI-MN • The recyclability of HPCD-EPI-MN for metabolite production was proven.

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