Xanthones Production in Gentiana dinarica Beck Hairy Root Cultures Grown in Simple Bioreactors.
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ABSTRACT:
SUBMITTER: Vinterhalter B
PROVIDER: S-EPMC8401843 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background:Tanshinones are diterpenoid compounds that are used to treat cardiovascular diseases. As current extraction methods for tanshinones are inefficient, there is a pressing need to improve the production of these bioactive compounds to meet increasing demand. Results:Overexpression of SmMDS (2-c-methyl-d-erythritol 2,4-cyclodiphosphate synthase, a tanshinone biosynthesis gene) in transgenic Salvia miltiorrhiza hairy roots significantly increased the tanshinone yield compared to the control, and total tanshinone content in SmMDS-overexpressing lines increased after elicitor treatment. Total tanshinones increased to 2.5, 2.3, and 3.2 mg/g DW (dry weight) following treatment with Ag+, YE (yeast extract), and MJ (methyl jasmonate), respectively, compared with the non-induced transgenic line (1.7 mg/g DW). Also, qRT-PCR analysis showed that the expression levels of two pathway genes was positively correlated with increased accumulation of tanshinone. Conclusions:Our study provides an effective strategy for increasing the content of tanshinones and other natural compounds using a combination of genetic engineering and elicitor treatment.
| S-EPMC6532201 | biostudies-literature
Project description:Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g-1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.
| S-EPMC8781555 | biostudies-literature
Project description:Stilbenes are defense molecules produced by grapevine in response to stresses including various elicitors and signal molecules. Together with their prominent role in planta, stilbenes have been the center of much attention in recent decades due to their pharmaceutical properties. With the aim of setting up a cost-effective and high purity production of resveratrol derivatives, hairy root lines were established from Vitis vinifera cv Pinot Noir 40024 to study the organ-specific production of various stilbenes. Biomass increase and stilbene production by roots were monitored during flask experiments. Although there was a constitutive production of stilbenes in roots, an induction of stilbene synthesis by methyl jasmonate (MeJA) after 18 days of growth led to further accumulation of ?-viniferin, ?-viniferin, resveratrol and piceid. The use of 100 µM MeJA after 18 days of culture in the presence of methyl-?-cyclodextrins (MCDs) improved production levels, which reached 1034µg/g fresh weight (FW) in roots and 165 mg/L in the extracellular medium, corresponding to five-and 570-foldincrease in comparison to control. Whereas a low level of stilbene excretion was measured in controls, addition of MeJA induced excretion of up to 37% of total stilbenes. The use of MCDs increased the excretion phenomenon even more, reaching up to 98%. Our results demonstrate the ability of grapevine hairy roots to produce various stilbenes. This production was significantly improved in response to elicitation by methyl jasmonate and/or MCDs. This supports the interest of using hairy roots as a potentially valuable system for producing resveratrol derivatives.
| S-EPMC6273728 | biostudies-literature
Project description:Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.
| S-EPMC5979590 | biostudies-literature
Project description:BackgroundThe therapeutic and health promoting role of highly unsaturated fatty acids (HUFAs) from fish, i.e. eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are well known. These same benefits may however be shared by some of their precursors, the polyunsaturated fatty acids (PUFAs), such as stearidonic acid (SDA, 18:4 n-3). In order to obtain alternative sources for the large-scale production of PUFAs, new searches are being conducted focusing on higher plants oils which can contain these n-3 and n-6 C18 precursors, i.e. SDA and GLA (18:3n-6, ?-linolenic acid).ResultsThe establishment of the novel Echium acanthocarpum hairy root cultures represents a powerful tool in order to research the accumulation and metabolism of fatty acids (FAs) in a plant particularly rich in GLA and SDA. Furthermore, this study constitutes the first example of a Boraginaceae species hairy root induction and establishment for FA studies and production. The dominant PUFAs, 18:2n-6 (LA, linoleic acid) and 18:3n-6 (GLA), accounted for about 50% of total FAs obtained, while the n-3 PUFAs, 18:3n-3 (ALA, ?-linolenic acid) and 18:4n-3 (SDA), represented approximately 5% of the total. Production of FAs did not parallel hairy root growth, and the optimal productivity was always associated with the highest biomass density during the culture period. Assuming a compromise between FA production and hairy root biomass, it was determined that sampling times 4 and 5 gave the most useful FA yields. Total lipid amounts were in general comparable between the different hairy root lines (29.75 and 60.95 mg/g DW), with the major lipid classes being triacylglycerols. The FAs were chiefly stored in the hairy roots with very minute amounts being released into the liquid nutrient medium.ConclusionsThe novel results presented here show the utility and high potential of E. acanthocarpum hairy roots. They are capable of biosynthesizing and accumulating a large range of polyunsaturated FAs, including the target GLA and SDA fatty acids in appreciable quantities.
| S-EPMC3114721 | biostudies-literature
Project description:Artemisinin and its derivatives are still one of the most effective drugs for the treatment of malaria. Artemisia pallens commonly known as Dhavanam, is an aromatic herb belonging to the family Asteraceae. Increasing the artemisinin content of A. pallens by genetic engineering would improve the availability of this much needed drug. In the present study, Agrobacterium rhizogenes (strain NCIM 5140) mediated genetic transformation of Artemisia pallens were carried out for hairy root induction. The effect of different media (Half MS, MS, MS along with BAP 0.5 mg/l and MS along with Kinetin 0.5 mg/l) and type of explants (leaf and stem) on hairy root induction and culture were also studied. Maximum transformation efficiency (70.0 %) was observed in case of stem explants when it was co-cultivated with Agrobacterium rhizogenes and kept on half strength MS media. Artesunate is a derivative of artemisinin, was quantified using HPLC from dried aerial extract and hairy roots. The content of artesunate in hairy roots was increased up to twofold as compared to aerial part of Artemisia pallens. The maximum amount of artesunate found in hairy roots was 5.62 ± 0.16 ?g/g of dry weight. Apart from artesunate the other phytochemicals like alkaloids, polyphenols, and flavonoids are important because they impart the medicinal properties in this plant. Therefore, we have also quantified total alkaloids, flavonoids and polyphenolic content in the aerial part of the plants. The total alkaloids and flavonoids content were found 1.72 ± 0.00 mg/g dry weight in aqueous extract and 3.8 ± 0.00 mg/g in methanolic extract in terms of colchicine and rutin equivalents, respectively. Similarly, total phenolic content is 3.70 ± 0.01 mg/g in ethanolic extract in terms of tannic acid equivalent.
| S-EPMC5002272 | biostudies-literature
Project description:Cichorium intybus is rich in inulin and has several pharmacological applications. Hairy roots culture is a valuable biotechnological tool used to produce plant secondary metabolites. Agrobacterium rhizogenes-mediated genetic transformation of chicory to hairy roots was investigated using Agrobacterium Strains A4, A13, A7, and ATCC15834. Several hairy roots were tested, from which 17 lines were selected based on their fast-growing characteristics. Results of PCR analysis revealed foreign DNA integration into the selected transgenic hairy root lines. Finally, four Adventitious roots that contained the highest ratio of total sugar to total weight (µg/gr DW), were selected. This study investigated the effects of various levels of minerals and sucrose on the production of inulin in Cichorium hairy root culture. Different levels of sucrose, phosphate (Pi) and Iron (Fe) were evaluated, separately. It was found that an increase in sucrose levels from 3 to 5% could decrease the root growth; however, 60 g/l sucrose remarkably enhanced the inulin production rate in all the examined lines. The highest biomass was achieved by applying 3.75 mM Pi but it ended in the decreasing the inulin content per unit weight. In contrast, the highest inulin accumulation and the lowest amount of biomass were observed in 0.5 mM Pi. Fe starvation caused the biomass decrease and a significant increase in inulin accumulation. Results of this study suggest a successfully optimized culture medium to initiate the induction of Cichorium intybus hairy root cells to produce inulin as a valuable medicinal secondary metabolite.
| S-EPMC8310654 | biostudies-literature
Project description:BACKGROUND: Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. RESULTS: A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). CONCLUSIONS: This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.
| S-EPMC3199266 | biostudies-literature
Project description:Hairy root cultures are genetically and biochemically stable, and they regularly possess the same or better biosynthetic capabilities for specialized (secondary) metabolite production compared to the intact plant. Ononis species are well-known herbal remedies in ethnopharmacology and rich sources of isoflavonoids. Besides isoflavones, less prevalent isoflavones and pterocarpans with valuable biological effects can be found in Ononis species as well. As these plants are only collected but not cultivated, biotechnological methods could play a role in the larger-scale extraction of Ononis isoflavonoids. Regarding this information, we aimed to establish Ononis spinosa and Ononis arvensis hairy root cultures (HRCs) and analyze the isoflavonoid profile of hairy root cultures qualitatively and quantitatively, in order to define their capacity to produce biologically valuable isoflavonoids. During the qualitative description, beside isoflavonoids, two new phenolic lactones, namely, bulatlactone 2″-O-β-D-glucoside and ononilactone, were isolated, and their structures were characterized for the first time. Altogether, 29 compounds were identified by the means of UPLC-Orbitrap-MS/MS. Based on UHPLC-UV-DAD measurements, the isoflavonoid spectrum of the Ononis HRCs differed markedly from wild-grown samples, as they produce a limited range of the scaffolds. The most abundant compounds in the HRCs were medicarpin glucoside and sativanone glucoside. The overall isoflavonoid production of the cultures was comparable to wild-grown O. arvensis and approximately twice as high as in wild-grown O. spinosa samples. As the overall content of wild-grown samples include more isoflavonoid derivatives, the HRCs contain structurally less divergent isoflavonoids but in higher quantity.
| S-EPMC7874045 | biostudies-literature
Project description:Turnip (Brassica rapa ssp. rapa) is an important vegetable crop producing glucosinolates (GSLs) and phenolic compounds. The GSLs, phenolic compound contents and transcript levels in hairy root cultures, as well as their antioxidant, antimicrobial and anticancer activity were studied in turnip. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR. GSLs levels (glucoallysin, glucobrassicanapin, gluconasturtiin, glucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin and 4-hydroxyglucobrassicin) and their gene expression levels (BrMYB28, BrMYB29, BrMYB34, BrMYB51, BrMYB122, CYP79 and CYP83) significantly increased in hairy roots compared with that in non-transformed roots. Furthermore, hairy roots efficiently produced several important individual phenolic compounds (flavonols, hydroxybenzoic and hydroxycinnamic acids). Colorimetric analysis revealed that the highest levels of total phenol, flavonoid contents, and their gene expression levels (PAL, CHI and FLS) in hairy roots than non-transformed roots. Our study provides beneficial information on the molecular and physiological active processes that are associated with the phytochemical content and biosynthetic gene expression in turnip. Moreover, antioxidant activity, as measured by DPPH scavenging activity, reducing potential, phosphomolybdenum and ferrous ion chelating ability assays was significantly higher in hairy roots. Hairy root extracts exhibited higher antimicrobial activity against bacterial and fungal species. The extract of hairy roots showed inhibition of human breast and colon cancer cell lines.
| S-EPMC4992476 | biostudies-literature