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In vitro plant cultures are able to remove and metabolise xenobiotics, making them promising tools for decontamination strategies. In this work, we evaluated Brassica napus hairy roots (HRs) to tolerate and remove high concentrations of the azo dye Naphthol Blue-Black (NBB). Experiments were performed using both growing and resting culture systems at different pHs. Reuse of HRs biomass was evaluated in successive decolourisation cycles. Proteomics was applied to understand the molecular responses likely to be involved in the tolerance and removal of NBB. The HRs tolerated up to 480 µg mL-1 NBB, and 100 % removal was achieved at 180 µg mL-1 NBB after 10 days using both culture systems. Interestingly, the HRs are robust enough to be reused, showing 55-60 % removal even after three reuse cycles. The highest dye removal rates were achieved during the first 2 days of incubation, as initial removal is mainly driven by passive processes. Active mechanisms are triggered later by regulating the expression of proteins with different biological functions, mainly those related to xenobiotic metabolism, such as hydrolytic and redox enzymes. These results suggest that B. napus HRs are a robust tool that could make a significant contribution to textile wastewater treatment.
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Tetrandrine, a bioactive active compound mainly found in the roots of Stephania tetrandra, exhibits various pharmacological properties. In vitro hairy root (HR) culture may serve as a promising solution for the extraction of tetrandrine, overcoming the limitations of natural cultivation. The present study describes the consistent production of tetrandrine from S. tetrandra hairy roots induced by different strains of Agrobacterium rhizogenes. Cultivation in woody plant medium (WPM) resulted in the highest HR biomass (0.056â¯g/petri-dish) and tetrandrine content (7.28â¯mg/L) as compared to other media. The maximum HR biomass (6.95â¯g dw/L) and tetrandrine production (68.69â¯mg/L) were obtained in the fifth week of cultivation. The presence of ammonium nitrate (800â¯mg/L), calcium nitrate (1156â¯mg/L), sucrose (20â¯g/L) and casein (2â¯g/L) enhanced the tetrandrine production. Moreover, the fed-batch cultivation demonstrated that the NH4NO3 (1200â¯mg/L) was an important growth limiting factor that yielded the highest tetrandrine amount (119.59â¯mg/L). The cultivation of hairy roots in a mist trickling bioreactor for eight weeks was less (26.24â¯mg/L) than in the flask. Despite a lower tetrandrine yield observed in bioreactors compared to flask cultures, refining the growth medium and fine-tuning bioreactor operations hold promise for boosting tetrandrine yield.
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The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.
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Agrobacterium's journey has been a roller coaster, from being a pathogen to becoming a powerful biotechnological tool. While A. tumefaciens has provided the scientific community with a versatile tool for plant transformation, Agrobacterium rhizogenes has given researchers a Swiss army knife for developing many applications. These applications range from a methodology to regenerate plants, often recalcitrant, to establish bioremediation protocols to a valuable system to produce secondary metabolites. This chapter reviews its discovery, biology, controversies over its nomenclature, and some of the multiple applications developed using A. rhizogenes as a platform.
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Agrobacterium , Biotecnologia , Agrobacterium/genética , Biotecnologia/métodos , Transformação Genética , História do Século XX , História do Século XXI , Plantas Geneticamente Modificadas/genética , Plantas/microbiologia , Plantas/genéticaRESUMO
Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However, its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has been less studied in this plant because an efficient transformation method has not been established yet. In this study, we established a rapid and efficient hairy root transformation method by infecting the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using different infection methods. Among these transformation methods, a higher transformation efficiency was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that the seedling age significantly affected the transformation efficiency, either using whole or rootless seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken together, our study lays the foundation for future study and for genetically modifying wood traits in the future.
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BACKGROUND: One of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and biotic elicitors on the growth, key biosynthetic genes expression, antioxidant capacity, and phenolic compounds content in Rhizobium (Agrobacterium) rhizogenes-induced hairy roots cultures of Ficus carica cv. Siah. METHODS: The elicitors included methyl jasmonate (MeJA) as abiotic elicitor, culture filtrate and cell extract of fungus Piriformospora indica as biotic elicitors were prepared to use. The cultures of F. carica hairy roots were exposed to elicitores at different time points. After elicitation treatments, hairy roots were collected, and evaluated for growth index, total phenolic (TPC) and flavonoids (TFC) content, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH and ferric ion reducing antioxidant power, FRAP assays), expression level of key phenolic/flavonoid biosynthesis genes, and high-performance liquid chromatography (HPLC) analysis of some main phenolic compounds in comparison to control. RESULTS: Elicitation positively or negatively affected the growth, content of phenolic/flavonoid compounds and DPPH and FRAP antioxidant activities of hairy roots cultures in depending of elicitor concentration and exposure time. The maximum expression level of chalcone synthase (CHS: 55.1), flavonoid 3'-hydroxylase (F3'H: 34.33) genes and transcription factors MYB3 (32.22), Basic helix-loop-helix (bHLH: 45.73) was induced by MeJA elicitation, whereas the maximum expression level of phenylalanine ammonia-lyase (PAL: 26.72) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT: 27.57) genes was obtained after P. indica culture filtrate elicitation. The P. indica elicitation also caused greatest increase in the content of gallic acid (5848 µg/g), caffeic acid (508.2 µg/g), rutin (43.5 µg/g), quercetin (341 µg/g), and apigenin (1167 µg/g) phenolic compounds. CONCLUSIONS: This study support that elicitation of F. carica cv. Siah hairy roots can be considered as an effective biotechnological method for improved phenolic/flavonoid compounds production, and of course this approach requires further research.
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Acetatos , Ciclopentanos , Ficus , Oxilipinas , Fenóis , Raízes de Plantas , Oxilipinas/metabolismo , Ciclopentanos/metabolismo , Acetatos/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo , Fenóis/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Antioxidantes/metabolismo , Basidiomycota , Reguladores de Crescimento de Plantas/metabolismo , AgrobacteriumRESUMO
KEY MESSAGE: AcEXPA1, an aluminum (Al)-inducible expansin gene, is demonstrated to be involved in carpetgrass (Axonopus compressus) root elongation under Al toxicity through analyzing composite carpetgrass plants overexpressing AcEXPA1. Aluminum (Al) toxicity is a major mineral toxicity that limits plant productivity in acidic soils by inhibiting root growth. Carpetgrass (Axonopus compressus), a dominant warm-season turfgrass widely grown in acidic tropical soils, exhibits superior adaptability to Al toxicity. However, the mechanisms underlying its Al tolerance are largely unclear, and knowledge of the functional genes involved in Al detoxification in this turfgrass is limited. In this study, phenotypic variation in Al tolerance, as indicated by relative root elongation, was observed among seventeen carpetgrass genotypes. Al-responsive genes related to cell wall modification were identified in the roots of the Al-tolerant genotype 'A58' via transcriptome analysis. Among them, a gene encoding α-expansin was cloned and designated AcEXPA1 for functional characterization. Observed Al dose effects and temporal responses revealed that Al induced AcEXPA1 expression in carpetgrass roots. Subsequently, an efficient and convenient Agrobacterium rhizogenes-mediated transformation method was established to generate composite carpetgrass plants with transgenic hairy roots for investigating AcEXPA1 involvement in carpetgrass root growth under Al toxicity. AcEXPA1 was successfully overexpressed in the transgenic hairy roots, and AcEXPA1 overexpression enhanced Al tolerance in composite carpetgrass plants through a decrease in Al-induced root growth inhibition. Taken together, these findings suggest that AcEXPA1 contributes to Al tolerance in carpetgrass via root growth regulation.
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Alumínio , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Raízes de Plantas , Plantas Geneticamente Modificadas , Alumínio/toxicidade , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Adaptação Fisiológica/genética , Adaptação Fisiológica/efeitos dos fármacos , Poaceae/genética , Poaceae/efeitos dos fármacosRESUMO
KEY MESSAGE: LeBAHD56 is preferentially expressed in tissues where shikonin and its derivatives are biosynthesized, and it confers shikonin acylation in vivo. Two WRKY transcriptional factors might regulate LeBAHD56's expression. Shikonin and its derivatives, found in the roots of Lithospermum erythrorhizon, have extensive application in the field of medicine, cosmetics, and other industries. Prior research has demonstrated that LeBAHD1(LeSAT1) is responsible for the biochemical process of shikonin acylation both in vitro and in vivo. However, with the exception of its documented in vitro biochemical function, there is no in vivo genetic evidence supporting the acylation function of the highly homologous gene of LeSAT1, LeBAHD56(LeSAT2), apart from its reported role. Here, we validated the critical acylation function of LeBAHD56 for shikonin using overexpression (OE) and CRISPR/Cas9-based knockout (KO) strategies. The results showed that the OE lines had a significantly higher ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than the control. In contrast, the KO lines had a significantly lower ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than controls. As for its detailed expression patterns, we found that LeBAHD56 is preferentially expressed in roots and callus cells, which are the biosynthesis sites for shikonin and its derivatives. In addition, we anticipated that a wide range of putative transcription factors might control its transcription and verified the direct binding of two crucial WRKY members to the LeBAHD56 promoter's W-box. Our results not only confirmed the in vivo function of LeBAHD56 in shikonin acylation, but also shed light on its transcriptional regulation.
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Regulação da Expressão Gênica de Plantas , Lithospermum , Naftoquinonas , Proteínas de Plantas , Plantas Geneticamente Modificadas , Naftoquinonas/metabolismo , Lithospermum/genética , Lithospermum/metabolismo , Acilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Sistemas CRISPR-Cas , AntraquinonasRESUMO
Lead (Pb) affects gene transcription, metabolite biosynthesis and growth in plants. The tung tree (Vernicia fordii) is highly adaptive to adversity, whereas the mechanisms underlying its response to Pb remain uncertain. In this work, transcriptomic and metabolomic analyses were employed to study tung trees under Pb stress. The results showed that the biomass of tung seedlings decreased with increasing Pb doses, and excessive Pb doses resulted in leaf wilting, root rot, and disruption of Pb homeostasis. Under non-excessive Pb stress, a significant change in the expression patterns of flavonoid biosynthesis genes was observed in the roots of tung seedlings, leading to changes in the accumulation of flavonoids in the roots, especially the upregulation of catechins, which can chelate Pb and reduce its toxicity in plants. In addition, Pb-stressed roots showed a large accumulation of VfWRKY55, VfWRKY75, and VfLRR1 transcripts, which were shown to be involved in the flavonoid biosynthesis pathway by gene module analysis. Overexpression of VfWRKY55, VfWRKY75, and VfLRR1 significantly increased catechin concentrations in tung roots, respectively. These data indicate that Pb stress-induced changes in the expression patterns of those genes regulate the accumulation of catechins. Our findings will help to clarify the molecular mechanism of Pb response in plants.
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Catequina , Chumbo , Transcriptoma , Chumbo/toxicidade , Chumbo/metabolismo , Catequina/metabolismo , Metabolômica , Regulação da Expressão Gênica de Plantas , Poluentes do Solo/toxicidade , Estresse Fisiológico , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Flavonoides/metabolismoRESUMO
Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14â¯mgâ¯l-1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75â¯mgâ¯g-1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11â¯mgâ¯g-â¯1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63â¯mgâ¯g-1 DW of withanolides and 15.66 ± 0.92â¯mgâ¯g-1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids.
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Agrobacterium , Hidroxibenzoatos , Raízes de Plantas , Withania , Vitanolídeos , Withania/metabolismo , Withania/genética , Withania/crescimento & desenvolvimento , Hidroxibenzoatos/metabolismo , Vitanolídeos/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Agrobacterium/genética , Agrobacterium/metabolismo , Transformação GenéticaRESUMO
The medicinal plants of the Asteraceae family are a valuable source of bioactive secondary metabolites, including polyphenols, phenolic acids, flavonoids, acetylenes, sesquiterpene lactones, triterpenes, etc. Under stressful conditions, the plants develop these secondary substances to carry out physiological tasks in plant cells. Secondary Asteraceae metabolites that are of the greatest interest to consumers are artemisinin (an anti-malarial drug from Artemisia annua L.-sweet wormwood), steviol glycosides (an intense sweetener from Stevia rebaudiana Bert.-stevia), caffeic acid derivatives (with a broad spectrum of biological activities synthesized from Echinacea purpurea (L.) Moench-echinacea and Cichorium intybus L.-chicory), helenalin and dihydrohelenalin (anti-inflammatory drug from Arnica montana L.-mountain arnica), parthenolide ("medieval aspirin" from Tanacetum parthenium (L.) Sch.Bip.-feverfew), and silymarin (liver-protective medicine from Silybum marianum (L.) Gaertn.-milk thistle). The necessity to enhance secondary metabolite synthesis has arisen due to the widespread use of these metabolites in numerous industrial sectors. Elicitation is an effective strategy to enhance the production of secondary metabolites in in vitro cultures. Suitable technological platforms for the production of phytochemicals are cell suspension, shoots, and hairy root cultures. Numerous reports describe an enhanced accumulation of desired metabolites after the application of various abiotic and biotic elicitors. Elicitors induce transcriptional changes in biosynthetic genes, leading to the metabolic reprogramming of secondary metabolism and clarifying the mechanism of the synthesis of bioactive compounds. This review summarizes biotechnological investigations concerning the biosynthesis of medicinally essential metabolites in plants of the Asteraceae family after various elicitor treatments.
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Asteraceae , Metabolismo Secundário , Asteraceae/metabolismo , Asteraceae/crescimento & desenvolvimento , Biomassa , Plantas Medicinais/metabolismo , Plantas Medicinais/crescimento & desenvolvimentoRESUMO
Realizing the full potential of plant synthetic biology both to elucidate the relationship between genotype and phenotype and to apply these insights to engineer traits requires rapidly iterating through design-build-test cycles. However, the months-long process of transgenesis, the long generation times, and the size-based limitations on experimentation have stymied progress by limiting the speed and scale of these cycles. Herein, we review a representative sample of recent studies that demonstrate a variety of rapid prototyping technologies that overcome some of these bottlenecks and accelerate progress. However, each of them has caveats that limit their broad utility. Their complementary strengths and weaknesses point to the intriguing possibility that these strategies could be combined in the future to enable rapid and scalable deployment of synthetic biology in plants.
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Plantas , Biologia Sintética , Plantas/genéticaRESUMO
This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2 mg/L and kinetin 2 mg/L; for shoot initiation 6-benzylaminopurine 2 mg/L, kinetin 0.2 mg/L, and indole-3-acetic acid 0.2 mg/L; for shoot multiplication 6-benzylaminopurine 3 mg/L and indole-3-acetic acid 0.2 mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1 mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.
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Compostos de Benzil , Fagopyrum , Purinas , Cinetina , Raízes de Plantas/genética , Melhoramento Vegetal , SacaroseRESUMO
The bioreactor is the centerpiece of the upstream processing in any biotechnological production process. Its design, the cultivation parameters, the production cell line, and the culture medium all have a major influence on the efficiency of the process and the result of the cultivation. Disposable bioreactors have been used for the past 20 years, playing a major role in process development and commercial production of high-value substances at medium scales.Our review deals with scalable, disposable bioreactors that have proven to be useful for the cultivation of plant cell and tissue cultures. Based on the definitions of terms and a categorization approach, the most commonly used, commercially available, disposable bioreactor types are presented below. The focus is on wave-mixed, stirred, and orbitally shaken bioreactors. In addition to their instrumentation and bioengineering characteristics, cultivation results are discussed, and emerging trends for the development of disposable bioreactors for plant cell and tissue cultures are also addressed.
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Reatores Biológicos , Células Vegetais , Células Vegetais/metabolismo , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodos , Equipamentos DescartáveisRESUMO
Herein we report on the generation of hairy root lines of P. scaberrima able to produce hernandulcin (HE), a non-caloric sweetener with nutraceutical properties. From ten different lines analyzed, three synthesized up to 100â mg â L-1 HE under the batch culture conditions standardized in this investigation. Adding elicitors (salicylic acid, chitin, Glucanex, polyethylene glycol) and biosynthetic precursors (farnesol and (+)-epi-alpha-bisabolol) significantly altered HE accumulation. Chitin and Glucanex enhanced HE production from 130 to 160â mg â L-1 , whereas farnesol and (+)-epi-alpha-bisabolol from 165 to 200â mg â L-1 without dependence on biomass accumulation. Improved batch cultures containing liquid Murashige & Skoog medium (MS; pHâ 7), added with 4 % sucrose, 0.5â mg â L-1 naphthaleneacetic acid, 100â mg â L-1 Glucanex, 150â mg â L-1 chitin, 250â mg â L-1 farnesol, and 150â mg â L-1 (+)-epi-alpha-bisabolol at 25 °C (12â h light/12â h darkness), triggered HE accumulation to 250â mg â L-1 in 25â days. The efficiency of each recombinant line is discussed.
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Farneseno Álcool , Sesquiterpenos Monocíclicos , Sesquiterpenos , Edulcorantes , Edulcorantes/análise , Farneseno Álcool/análise , Suplementos Nutricionais , Quitina/análise , Raízes de Plantas/químicaRESUMO
Secondary metabolites derived from plants are recognized as valuable products with several successful applications in the pharmaceutical, cosmetic, and food industries. The major limitation to the broader implementation of these compounds is their low manufacturing efficiency. Current efforts to overcome unprofitability depend mainly on biotechnological methods, especially through the application of plant in vitro cultures. This concept allows unprecedented bioengineering opportunities for culture system modifications with in situ product removal. The silica-based xerogels can be used as a novel, porous biomaterial characterized by a large surface area and high affinity to lipophilic secondary metabolites produced by plant tissue. This study aimed to investigate the influence of xerogel-based biomaterials functionalized with methyl, hydroxyl, carboxylic, and amine groups on Rindera graeca transgenic root growth and the production of naphthoquinone derivatives. The application of xerogel-based scaffolds functionalized with the methyl group resulted in more than 1.5 times higher biomass proliferation than for reference untreated culture. The naphthoquinone derivatives' production was noted exclusively in culture systems supplemented with xerogel functionalized with methyl and hydroxyl groups. Applying chemically functionalized xerogels as in situ adsorbents allowed for the enhanced growth and productivity of in vitro cultured R. graeca transgenic roots, facilitating product isolation due to their selective and efficient accumulation.
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Methyl jasmonate (MJA), a signaling molecule in stress pathways, can be used to induce secondary metabolite synthesis in plants. The present study examines its effects on the growth of Salvia viridis hairy roots, and the accumulation of bioactive compounds, and correlates it with the expression of genes involved in the phenylpropanoid pathway. To our knowledge, this study represents the first exploration of elicitation in S. viridis culture and the first comprehensive analysis of MJA's influence on such a wide array of genes within the polyphenol metabolic pathway in the Salvia genus. Plants were treated with 50 and 100 µM MJA, and samples were collected at intervals of one, three, five, and seven days post-elicitation. HPLC analysis revealed that MJA stimulated the accumulation of all tested compounds, with a 30% increase (38.65 mg/g dry weight) in total polyphenol content (TPC) on day five. Quantitative real-time polymerase chain reaction (RT-PCR) analysis demonstrated a significant increase in the expression of the phenylpropanoid pathway genes-TAT (tyrosine aminotransferase), HPPR (4-hydroxyphenylpyruvate reductase), PAL (phenylalanine ammonia-lyase), C4H (cinnamic acid 4-hydroxylase), 4CL (4-coumarate-CoA ligase), and RAS (rosmarinic acid synthase)-following MJA treatment. For the majority of the genes, this increase was observed after the first day of treatment. Importantly, our present results confirm strong correlations of the analyzed gene expression with polyphenol biosynthesis. These findings support the notion that hairy roots provide a promising biotechnological framework for augmenting polyphenol production. Additionally, the combination of elicitor treatment and transgenic technology emerges as a viable strategy to enhance the biosynthesis of these valuable metabolites.
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Acetatos , Biotecnologia , Ciclopentanos , Oxilipinas , Acetatos/farmacologia , Cromatografia Líquida de Alta Pressão , Expressão GênicaRESUMO
The accumulation of ginsenosides (triterpenic saponins) was determined in Panax quinquefolium hairy root cultures subjected to an elicitation process using carvacrol at 5, 10, 25, 50, 100, 250, and 500 µM concentrations during 24 and 72 h exposure. This study was the first one in which carvacrol was applied as an elicitor. The content of eight ginsenosides, Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, and Re, was determined using HPLC analysis. Moreover, the quantitative RT-PCR method was applied to assess the relative expression level of farnesyl diphosphate synthase, squalene synthase, and dammarenediol synthase genes in the studied cultures. The addition of carvacrol (100 µM) was an effective approach to increase the production of ginsenosides. The highest content and productivity of all detected saponins were, respectively, 20.01 mgâg-1 d.w. and 5.74 mgâL-1âday-1 after 72 h elicitation. The production profile of individual metabolites in P. quinquefolium cultures changed under the influence of carvacrol. The biosynthesis of most examined protopanaxadiol derivatives was reduced under carvacrol treatment. In contrast, the levels of ginsenosides belonging to the Rg group increased. The strongest effect of carvacrol was noticed for Re metabolites, achieving a 7.72-fold increase in comparison to the control. Saponin Rg2, not detected in untreated samples, was accumulated after carvacrol stimulation, reaching its maximum concentration after 72 h exposure to 10 µM elicitor.
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Ginsenosídeos , Panax , Saponinas , Panax/genética , Saponinas/farmacologia , Cimenos , Fármacos do Sistema Nervoso CentralRESUMO
Anthocyanins are widely distributed pigments in flowering plants with red, purple or blue colours. Their properties in promoting heath make anthocyanins perfect natural colourants for food additives. However, anthocyanins with strong colour and stability at neutral pH, suitable as food colourants are relatively rare in nature. Acylation increases anthocyanin stability and confers bluer colour. In this study, we isolated two anthocyanin regulators SbMyb75 and SbDel from S. baicalensis, and showed that constitutive expression of the two TFs led to accumulation of anthocyanins at high levels in black carrot hairy roots. However, these hairy roots had severe growth problems. We then developed a ß-estradiol inducible system using XVE and a Lex-35S promoter, to initiate expression of the anthocyanin regulators and induced this system in hairy roots of black carrot, tobacco and morning glory. Anthocyanins with various decorations were produced in these hairy roots without any accompanying side-effects on growth. We further produced highly acylated anthocyanins with blue colour in a 5 L liquid culture in a bioreactor of hairy roots from morning glory. We provide here a strategy to produce highly decorated anthocyanins without the need for additional engineering of any of the genes encoding decorating enzymes. This strategy could be transferred to other species, with considerable potential for natural colourant production for the food industries.
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Antocianinas , Nicotiana , Antocianinas/genética , Nicotiana/genética , Reatores Biológicos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genéticaRESUMO
Plant-derived natural products are a specific class of active substances with numerous applications in the medical, energy, and industrial fields. Many of these substances are in high demand and have become the fundamental materials for various purposes. Recently, the use of synthetic biology to produce plant-derived natural products has become a significant trend. Plant chassis, in particular, offer unique advantages over microbial chassis in terms of cell structure, product affinity, safety, and storage. The development of the plant hairy root tissue culture system has accelerated the commercialization and industrialization of synthetic biology in the production of plant-derived natural products. This paper will present recent progress in the synthesis of various plant natural products using plant chassis, organized by the types of different structures. Additionally, we will summarize the four primary types of plant chassis used for synthesizing natural products from plant sources and review the enabling technologies that have contributed to the development of synthetic biology in recent years. Finally, we will present the role of isolated and combined use of different optimization strategies in breaking the upper limit of natural product production in plant chassis. This review aims to provide practical references for synthetic biologists and highlight the great commercial potential of plant chassis biosynthesis, such as hairy roots.