Ginseng Adventitious Roots Research Articles

Histone deacetylase inhibitors improve MeJA-induced ginsenoside production in ginseng adventitious roots

Post-translational histone modifications such as histone acetylation play essential roles in plant growth, development, and stress responses by modulating gene expression. However, little is known about epigenetic regulation in plant secondary metabolism. To analyze the involvement of histone acetylation in elicitor-induced metabolite production, ginseng adventitious roots were treated with methyl jasmonate (MeJA) together with histone deacetylase (HDAC) inhibitors. Western blot results revealed that HDAC inhibitors cause hyperacetylation of histone H3, especially acetylation of histone H3 lysine 18, under MeJA treatment. In addition, the HDAC inhibitor-induced increase in the transcript levels of MeJA-induced ginsenoside biosynthetic genes resulted in enhancement of MeJA-induced ginsenoside production, indicating that HDAC activity serves as a negative factor in MeJA-induced transcriptional induction of ginsenoside biosynthesis pathways. In addition, we identified 25 HDAC genes from the ginseng genome, which suggested that MeJA-induced PgHDACs (PgHDA05, PgHDA10, PgHDA16, PgHDA17, PgHDA18, PgHDA19, and PgHDA20) might be key factors controlling MeJA-induced ginsenoside production in ginseng adventitious roots. This unique information regarding histone acetylation and ginsenoside production would be helpful in understanding epigenetic regulation in plant secondary metabolism.

Production of ginsenoside by Chaetomium sp. and its effect on enhancing the contents of ginsenosides in Panax ginseng adventitious roots

Using endophytic fungi elicitors to enhance the yield of valuable secondary metabolites in plant tissue culture is an effective biotechnology strategy. In this study, we isolated an endophytic fungus Chaetomium sp. from cultivated Panax ginseng root and detected a high concentration of Rg3 in its mycelium. By comparing the effects of different treatments of Chaetomium sp. on ginsenoside production in Panax ginseng adventitious roots (PGARs), the immobilized spores of Chaetomium sp. (ISC) were proved to be the best elicitor. An initial pH of 6.5 and cocultivation time of 24 h yielded the highest ginsenosides content (56.29 ± 0.62 mg/g) in ISC-treated PGARs, increasing ginsenosides production by 3.2-fold compared with the blank group (17.56 ± 0.49 mg/g). Moreover, we show that ISC could stimulate the temporary accumulation of defense-related signal molecules, intensify the expression of defense genes in PGARs, and upregulate biosynthetic genes involved in the biosynthetic pathway of ginsenosides. Besides, the expression of the biosynthetic gene FPS (farnesyl diphosphate synthase) was deemed the most relevant factor for ginsenosides production during the elicitation period, and the L-aspartic acid secreted by Chaetomium sp. was proven to play a key role in the elicitation process. In conclusion, this study provides an accessible way of improving pharmacologically active compounds production in medicinal plants.

Curcubinoyl flavonoids from wild ginseng adventitious root cultures

Wild ginseng (Panax ginseng) adventitious root cultures were prepared by elicitation using methyl jasmonate and investigated further to find new secondary metabolites. Chromatographic fractionation of wild ginseng adventitious root cultures led to the isolation of eleven compounds. The chemical structures of isolated compounds were identified as four known flavanone derivatives (1–4), one new curcubinoyl derivative, jasmogin A (5) and six new curcubinoyl-flavanone conjugates, jasmoflagins A-F (6–11) by extensive spectroscopic analysis. Newly isolated curcubinoyl derivatives showed inhibitory activity against lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. Therefore, our present study suggested that elicitor stimulated plant cell cultures might contribute to the production of new metabolites.

산삼 부정배양근의 진세노사이드 함량 증진과 성분 변환

Background: Culturing wild ginseng adventitious root using plant factory technology provides genetic safety and high productivity. This production technology is drawing attention in the fields of functional raw materials and product development. The cultivation method using elicitors is key technology for controlling biomass and increasing secondary metabolites.BRMethods and Results: Elicitor treatments using methyl jasmonate, pyruvic acid, squalene, β-sistosterol were performed to amplify total ginsenosides (Rb1, Rc, Rb2, Rb3, and Rd) of cultured wild ginseng adventitious root. Thereafter, fermentation and steaming processes were performed to convert total ginsenosides into minor molecular ginsenosides (Rg3, Rk1, and Rg5). The result indicated that methyl jasmonate minimizes the reduction in fresh weight of cultured wild ginseng adventitious root and maximizes total ginsenosides (sum of Rb1, Rc, Rb2, Rb3, and Rd). Ginsenoside conversion results showed a maximum degree of conversion of 131 ㎎/g.BRConclusions: In this study, we demonstrated that the optimal elicitor treatment method increased the content of total ginsenosides, while the steaming and fermentation processing method increased the content of minor ginsenosides.

Overexpression of the squalene epoxidase gene (PgSE1) resulted in enhanced production of ginsenosides and phytosterols in transgenic ginseng

Squalene epoxidase (also called squalene monooxygenase) catalyses the conversion of squalene into 2,3-oxidosqualene by epoxidation and is regarded as the rate-limiting enzyme for sterol and saponin biosynthesis. However, the role of the squalene epoxidase gene in saponin biosynthesis in plants is not yet well understood. Here, we investigated the effects of overexpression of a Panax ginseng squalene epoxidase gene (PgSE1) on the production of phytosterols and ginsenoside saponins in ginseng adventitious roots. For the functional complementation test, the two squalene epoxidase sequences (PgSE1 and PgSE2) of P. ginseng were expressed in a yeast erg1 mutant (ergosterol auxotroph). The yeast mutant expressing PgSE1 or PgSE2 can restore growth on medium lacking ergosterol. Transgenic ginseng roots overexpressing the PgSE1 gene were constructed by Agrobacterium-mediated genetic transformation. The transgenic ginseng roots resulted in the enhanced production of both ginsenosides (ginsenoside Rg1, Re, Rf, Rc, Rb1, Rb2, and Rd) and phytosterols (campesterol, stigmasterol, and β-sitosterol). qPCR analysis revealed that overexpression of PgSE1 in transgenic ginseng roots clearly enhanced the expression of dammarenediol-II synthase (PgDDS) and cycloartenol synthase (PgPNX), which are key enzymes for ginsenoside and phytosterol biosynthesis in P. ginseng. This result indicates that the P. ginseng squalene epoxidase gene (PgSE1) encodes an efficient enzyme responsible for not only phytosterol production but also ginsenoside production in P. ginseng.

Involvement of Putrescine, Nitric Oxide, and Hydrogen Peroxide in Methyl Jasmonate-Induced Ginsenoside Synthesis in Adventitious Root Cultures of Panax ginseng C.A. Meyer

The adventitious root (AR) culture of Panax ginseng C.A. Meyer is an alternative route for mass production of ginsenosides. During the AR culture of P. ginseng, methyl jasmonate (MeJA) is frequently used as an abiotic elicitor for improving the ginsenoside production. However, knowledge on the signaling molecules involved in MeJA-induced ginsenoside synthesis is lacking to date. Therefore, the present study used MeJA to treat 30-day-old bioreactor-cultured ARs and determined the changes of ginsenosides and three signaling molecules, including putrescine (Put), nitric oxide (NO), and hydrogen peroxide (H2O2). Meanwhile, the effects of Put [(d-(-)-arginine, (d-Arg)] and nitric oxide synthase (NOS) [NG-nitro-l-arginine methyl ester (l-NAME) and S,S′-1,3-phenylenebis (1,2-ethanediyl)-bis-isothiourea (PBITU)] inhibitors and the H2O2 [catalase (CAT)] scavenger on the three signaling molecules and ginsenoside production in ARs were investigated. Results showed that MeJA promoted ginsenoside accumulation, and the maximum content of total ginsenosides was reached after 8 days of MeJA elicitation. The Put, NO, and H2O2 production increased with MeJA treatment. The Put and NO contents peaked at 12 h, whereas the H2O2 content peaked at 24 h. No obvious change (p < 0.05) was found in the MeJA-untreated control group, demonstrating that the change pattern of Put, NO, and H2O2 contents was the specificity of MeJA elicitation. In MeJA-stimulated ARs, Put and ginsenoside contents were significantly decreased with d-Arg treatment. The NOS activity and NO content and ginsenoside contents were also decreased with l-NAME and PBITU treatments. The H2O2 and ginsenoside contents were decreased with CAT treatment. These results demonstrated the enhancement effects of MeJA on the production of Put, NO, H2O2, and ginsenosides, but the increase of ginsenosides was eliminated by the inhibitor or scavenger of the signaling molecules, which proved that Put, NO, and H2O2 involved in MeJA-induced ginsenoside synthesis of P. ginseng ARs.

Penicillium sp. YJM-2013 induces ginsenosides biosynthesis in Panax ginseng adventitious roots by inducing plant resistance responses

ObjectiveFusarium oxysporum is a common pathogenic fungus in ginseng cultivation. Both pathogens and antagonistic fungi have been reported to induce plant resistance responses, thereby promoting the accumulation of secondary metabolites. The purpose of this experiment is to compare the advantages of one of the two fungi, in order to screen out more effective elicitors. The mechanism of fungal elicitor-induced plant resistance response is supplemented. MethodsA gradient dilution and the dural culture were carried out to screen strains. The test strain was identified by morphology and 18 s rDNA. The effect of different concentrations (0, 50, 100, 200, 400 mg/L) of Penicillium sp. YJM-2013 and F. oxysporum on fresh weight and ginsenosides accumulation were tested. Signal molecules transduction, expression of transcription factors and functional genes were investigated to study the induction mechanism of fungal elicitors. ResultsAntagonistic fungi of F. oxysporum was identified as Penicillium sp. YJM-2013, which reduced root biomass. The total ginsenosides content of Panax ginseng adventitious roots reached the maximum (48.95 ± 0.97 mg/g) treated with Penicillium sp. YJM-2013 at 200 mg/L, higher than control by 2.59-fold, in which protopanoxadiol-type ginsenosides (PPD) were increased by 4.57 times. Moreover, Penicillium sp. YJM-2013 activated defense signaling molecules, up-regulated the expression of PgWRKY 1, 2, 3, 5, 7, 9 and functional genes in ginsenosides synthesis. ConclusionCompared with the pathogenic fungi F. oxysporum, antagonistic fungi Penicillium sp. YJM-2013 was more conducive to the accumulation of ginsenosides in P. ginseng adventitious roots. Penicillium sp. YJM-2013 promoted the accumulation of ginsenosides by intensifying the generation of signal molecules, activating the expression of transcription factors and functional genes.

Expression analysis of transcription factor ERF gene family of Panax ginseng

Ethylene responsive factor(ERF), one of the largest families of transcriptional factors in plants, plays a key role in se-condary metabolism of herbal plants. To analyze the expression of ERF family genes, the heat map clustering method was used by analyzing the ginseng transcriptomes of different parts and different growth years. The contents of ginsenosides Rg_1, Re and Rb_1 in various concentrations of MeJA-treated ginseng adventitious roots were determined by UPLC-MS/MS method. The expression of key genes of ginsenoside biosynthesis(DDS, CYP716A47, CYP716A53v2) and ERF family genes in MeJA-treated ginseng adventitious roots were determined by using real-time quantitative PCR. Pearson correlation was adopted to analyze the gene expression pattern of DDS, CYP716A47, CYP716A53v2 gene and ERF family. The results showed that the content of ginseng diol ginsenoside Rb_1 in ginseng adventitious roots treated with different concentrations of MeJA increased, and the content of ginseng triol ginsenoside Rg_1 and Re decreased. It is consistent with the increase of DDS and CYP716A47 expression and the decrease of CYP716A53v2 gene expression. The expression of ERF003, ERF118 and ERF012 genes was significantly positively correlated with CYP716A53v2, but negatively correlated with DDS. While the expression of ERF1B was significantly negatively correlated with CYP716A47.It is proved that ERF003, ERF118 and ERF012 were likely to inhibit the expression of DDS and promote the expression of CYP716A53v2, and ERF1B was likely to inhibit CYP716A47. This work could provide theoretical basis of ERF functional verification of regulating the biosynthesis of ginsenosides.

Effect of nutritional factors on the accretion of secondary metabolites in Malaysian ginseng adventitious root cultures

In this study, we aimed to verify the effect of nutritional factors on the accretion of secondary metabolites in the adventitious root (AR) cultures of Malaysian ginseng (Eurycoma longifolia Jack) grown in small-scale bioreactors. AR were induced from leaf explants and cultured in different types of media including Murashige and Skoog (MS) medium, Driver Kuniyuki Walnut (DKW) medium, Gamborg’s B5 medium, Woody Plant Medium (WPM), and ¾ MS medium. Among these media, the MS and Gamborg’s B5 media induced lateral root development from initial inoculum, which accounted for the increase in AR biomass accretion. By contrast, the DKW and WPM media did not induce lateral root formation from the cultured explants. The ¾ MS medium was optimal for the growth of AR and accretion of secondary metabolites, after 7 weeks of culture, the biomass of AR increased by 8.6-fold in ¾ MS medium, and the total phenolic and flavonoid contents reached 5.23 and 2 mg g−1 of tissue dry weight, respectively. Analysis of mineral elements in the spent medium revealed that ¾ MS medium was most suitable for nutrient supply to developing AR. LC–MS analysis showed the accretion of eurycomanone, a therapeutically useful metabolite, in the AR of Malaysian ginseng.

Promotion of ginsenosides production in a co-cultivation system of Panax ginseng adventitious roots and immobilized Aspergillus niger

Biotic elicitor is an important biotechnology strategy to promote metabolite production in adventitious root culture. In this study, Panax ginseng adventitious roots (PGARs) were co-cultured with sterilized mycelium powders (SMPAN), immobilized mycelium (IMAN), and immobilized spores of Aspergillus niger (ISAN) respectively for the enhancement of ginsenosides accumulation. In this co-cultivation system, ISAN was selected as the best inducer. The maximum ginsenosides content (26.43 ± 0.49 mg/g) were obtained in the ISAN spore concentration of 102 spores/mL, the volume ratio of 4%, and co-culture time of 24 h, which was 3.09 times higher than non-treated control (8.55 ± 0.78 mg/g). Besides, the lactic acid secreted by A. niger was identified as the main component inducing ginsenosides accumulation. Moreover, this study revealed that lactic acid could trigger the instantaneous accumulation of signaling molecules and enhance the expression of transcription factors and defense genes in PGARs, thus resulting in the up-regulated expression of functional genes involved in ginsenosides biosynthetic pathway. Therefore, co-culture of ISAN and PGARs is a very promising method to increase ginsenosides production. It provides a new way to promote the accumulation of secondary metabolites in plants.

Modification of the droplet-vitrification method of cryopreservation to enhance survival rates of adventitious roots of Panax ginseng

Panax ginseng Meyer is an important medicinal plant producing bioactive compounds. A droplet-vitrification method was developed for cryopreserving adventitious root cultures of mountain ginseng, and variations of this procedure were tested to determine their effects on regrowth rates and osmotic stress responses. Root regrowth rates were examined after exposing root segments to two pre-culture treatments, three loading solutions, and two vitrification solutions with different exposure periods. Pre-culturing excised segments of adventitious roots with 0.3 M sucrose produced the highest rate of regrowth after cryopreservation. Loading for 20 min with 17.5% (w/v) sucrose and 17.5% (w/v) glycerol at room temperature increased the regrowth rate of cryopreserved adventitious roots fourfold, compared with non-loaded samples. Treatments involving different vitrification solutions and exposure periods were compared, and the highest rate of regrowth (15%) after cryopreservation was achieved by incubating adventitious roots in modified plant vitrification solution 3 containing 40% (w/v) glycerol and 40% (w/v) sucrose for 10 min at room temperature, suggesting that ginseng adventitious root tips were sensitive to osmotic stress. Further study is necessary to develop optimal vitrification solutions that enhance the survival rate of cryopreserved adventitious roots of mountain ginseng.

Quality evaluation of Panax ginseng adventitious roots based on ginsenoside constituents, functional genes, and ferric-reducing antioxidant power.

In the study, six adventitious root lines of Panax ginseng have been successfully established. HPLC-ESI-MS analysis showed that 20 ginsenosides were identified in root lines, notoginsenoside Fa and notoginsenoside R2 were not found in AR lines. In AR lines, the highest accumulation of total ginsenosides was obtained in five-year main AR (24.87mg/g). Principal component analysis classified root lines into three groups. Five-year ginseng was mostly similar with five-year main AR, five-year rootlet AR, and four-year rootlet AR in ginsenosides composition of group 1. Besides, gene expressions were consistent with the production of total ginsenosides, and correlation analysis revealed that total ginsenosides biosynthesis was significantly positively correlated with the gene expression of dammarenediol synthase. Five-year rootlet AR showed the highest activity on ferric-reducing antioxidant power test among samples. It provides a scientific evidence for the further exploitation and large-scale production of P. ginseng. PRACTICAL APPLICATIONS: This study provides valuable information for the commercial scale culture of ginseng adventitious roots. This report combines morphology, ginsenoside composition and content, gene expression, and ferric-reducing antioxidant power test to evaluate the quality of P. ginseng adventitious root, and combined with principal component analysis to screen out the high yield and stable ginseng adventitious roots. It would be profitable to use adventitious root culture of P. ginseng instead of field cultivation.

PgMYB2, a MeJA-Responsive Transcription Factor, Positively Regulates the Dammarenediol Synthase Gene Expression in Panax Ginseng.

The MYB transcription factor family members have been reported to play different roles in plant growth regulation, defense response, and secondary metabolism. However, MYB gene expression has not been reported in Panax ginseng. In this study, we isolated a gene from ginseng adventitious root, PgMYB2, which encodes an R2R3-MYB protein. Subcellular localization revealed that PgMYB2 protein was exclusively detected in the nucleus of Allium cepa epidermis. The highest expression level of PgMYB2 was found in ginseng root and it was significantly induced by plant hormones methyl jasmonate (MeJA). Furthermore, the binding interaction between PgMYB2 protein and the promoter of dammarenediol synthase (DDS) was found in the yeast strain Y1H Gold. Moreover, the electrophoretic mobility shift assay (EMSA) identified the binding site of the interaction and the results of transiently overexpressing PgMYB2 in plants also illustrated that it may positively regulate the expression of PgDDS. Based on the key role of PgDDS gene in ginsenoside synthesis, it is reasonable to believe that this report will be helpful for the future studies on the MYB family in P. ginseng and ultimately improving the ginsenoside production through genetic and metabolic engineering.

Effect of temperature on morphology, ginsenosides biosynthesis, functional genes, and transcriptional factors expression in Panax ginseng adventitious roots.

This study researched the effect of temperature on growth and ginsenosides accumulation in adventitious root cultures of Panax ginseng. Results showed that the ginseng adventitious roots growth and differentiation ability could be affected faced with different incubation temperatures (15, 20, 25, and 30°C for 35days). Besides, the research also demonstrated that low-temperature stimulation could promote the accumulation of ginsenosides and the content of total ginsenosides increased by 2.53 times at 10°C-7d (10°C for 7days and then transferred to 25°C for 28days) compared with that at 25°C. Moreover, the transcriptional levels of functional genes and PgWRKYs were analyzed by this study and the correlation analysis showed that GPS, SS, CYP716A47, CYP716A53v2, UGT74AE2, UGT94Q2, PgWRKY1, PgWRKY3, and PgWRKY8 were significantly correlated with total ginsenosides content. Furthermore, HPLC-ESI-MSn analyzed that Malonyl-Rb1 only existed in 10°C-7d group. PRACTICAL APPLICATIONS: The survey showed that after a certain time of stimulating P. ginseng adventitious roots at low temperature, the accumulation of ginsenosides could be enhanced as their expression of related genes were regulated. It provides a theoretical foundation for the mass production of ginsenosides by controlling the temperature conditions of P. ginseng adventitious roots.

Antioxidant effects and tyrosinase and elastase inhibitory activities of mountain ginseng adventitious roots extracts at different ethanol concentrations

Antioxidant effects and tyrosinase and elastase inhibitory activities of mountain ginseng adventitious roots extracts at different ethanol concentrations

Quality, safety and efficacy profiling of ginseng adventitious roots produced in vitro.

Ginseng (Panax ginseng C. A. Meyer, Family Araliaceae) is one of the major medicinal and nutraceutical plants, which is native to oriental region. It is used worldwide as a popular herbal medicine because of its pharmacological effects like anti-oxidative, anti aging, anti-cancer, adaptogenic, and other health-improving activities. Chief components of ginseng identified till date are ginsenosides, a group of saponins with triterpenoid structure. Ginseng is cultivated under controlled conditions, and for harvesting of fully grown roots of the plant, the cultivation takes long duration of about 5-7years and cultivated ginseng roots are inferior in quality and ginsenoside content. Wild Mountain ginseng is superior in quality and ginsenoside content but is scarce in nature. Therefore, for obtaining the useful compounds of this plant at commercial scale, cell and organ cultures especially adventitious roots have been established by using superior clones of wild mountain ginseng, ginseng biomass is produced by applying large scale bioreactors. In this paper, an effort has been made to shed light on the scientific literature and to decipher the evidences for quality, safety, and efficacy of ginseng adventitious roots produced from in vitro cultures.

Biological activity investigation of supercritical fluid extract of fermented mountain ginseng adventitious root

Biological activity investigation of supercritical fluid extract of fermented mountain ginseng adventitious root

Optimization of Extraction Condition of Methyl Jasmonate-treated Wild Ginseng Adventitious Root Cultures using Response Surface Methodology

Optimization of Extraction Condition of Methyl Jasmonate-treated Wild Ginseng Adventitious Root Cultures using Response Surface Methodology

Expression analysis of ginsenoside biosynthesis-related genes in methyl jasmonate-treated adventitious roots of Panax ginseng via DNA microarray analysis

Many ginsenoside biosynthesis-related genes have been identified in Panax ginseng. However, there is no report of the analysis of changes in gene expression induced methyl jasmonic acid (MeJA) using DNA microarray analysis. To identify the genes related to ginsenoside biosynthesis, we harvested P. ginseng adventitious roots at 0, 24, 72 and 120 h after MeJA treatment. At 120 h after MeJA elicitation, the contents of all ginsenosides increased compared to the control. We analyzed gene expression patterns in ginseng adventitious roots treated with MeJA using DNA microarray analysis and selected candidate genes related to ginsenoside biosynthesis, including genes encoding squalene synthase (SQS), squalene epoxidase (SE), dammarenediol-II synthase (DS), cytochrome P450 oxidase (CYP) and glycosyltransferase (GT). The expression patterns of these genes in MeJA-treated ginseng adventitious roots obtained by quantitative RT-PCR were consistent with those obtained by microarray analysis. Therefore, DNA microarray analysis is an efficient tool for selecting candidate genes associated with secondary metabolite biosynthesis in plants.

Endophytic Bacteria Isolated from Panax ginseng Improves Ginsenoside Accumulation in Adventitious Ginseng Root Culture.

Ginsenoside is the most important secondary metabolite of ginseng. Natural sources of wild ginseng have been overexploited. Although root culture could reduce the length of the growth cycle of ginseng, the number of ginsenosides is fewer and their contents are lower in adventitious roots of ginseng than that in ginseng cultivated in the field. In this study, we investigated the effects of endophytic bacterial elicitors on biomass and ginsenoside production in adventitious roots cultures of Panax ginseng. Endophyte LB 5-3 as an elicitor could increase biomass and ginsenoside accumulation in ginseng adventitious root culture. After 6 days elicitation with a 10.0 mL of strain LB 5-3, the content of total ginsenoside was 2.026 mg g−1 which was four times more than that in unchallenged roots. The combination of methyl jasmonate and strain LB 5-3 had a negative effect on ginseng adventitious root growth and ginsenoside production. The genomic DNA of strain LB 5-3 was sequenced, and was found to be most closely related to Bacillus altitudinis (KX230132.1). The challenged ginseng adventitious root extracts exerted inhibitory effect against the HepG2 cells, which IC50 value was 0.94 mg mL−1.