Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in rolC transgenic cell cultures of Vitis amurensis

Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.     

Influence of thidiazuron on callus induction and crocin production in corm and style explants of <Emphasis Type="Italic">Crocus sativus</Emphasis> L.

Crocus sativus L., mostly famous as saffron, has gained more attention due to its crocin (crocetin ester) pigment responsible for its extensive pharmaceutical properties. In this study, we established two different callus cultures from corm and style explants of saffron to find out the best explant as a suitable source for crocin production. Comparative analyses of total phenolic, flavonoid, carotenoid and anthocyanin contents were also performed in the two callus cultures. For callus induction, different combinations of MS medium with name thidiazuron (TDZ), benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination were tested. Of the used media, all the combinations containing TDZ and NAA gave 100% callus induction. HPLC-DAD and HPLC–ESI-MS analysis were used for identification of crocin esters in established callus cultures. The highest amount of 0.35 mg g^?1 DW crocin was detected in style originated calli grown on the medium containing 3 mg L^?1 NAA?+?1 mg L^?1 TDZ while the corm calli showed the most abundant total carotenoid (0.73 mg g^?1 DW), phenolic (15.04 mg gallic acid equivalent g^?1 DW) and flavonoid (0.76 mg rutin equivalent g^?1 DW) contents. In general, style-derived calli showed longer time survival with a fine texture and good quality compared to corm-derived calli.     

咖啡酸代谢物的抗肾炎活性分析

在研究治疗肾功能紊乱植物的细胞培养时, 建立了一个Eritrichium sericeum的E-4愈伤组织株系, 发现此株系可产生大量的咖啡酸代谢物、(–)-rabdosiin (1.8%干重)和迷迭香酸(4.6%干重), 通过诱导(–)-rabdosiin的含量提高至4.1% (干重)。将E-4愈伤组织喂服Masugi肾炎大鼠, 结果发现, 与对照组(未喂服E-4愈伤组织的Masugi肾炎大鼠)相比, 处理组(喂服E-4愈伤组织)中的大鼠出现如下症状: 尿多、排泄物中肌氨酸酐降低、尿蛋白水平降低; 当对照组中所有大鼠都出现疼痛症状时, 处理组中仍有约/4的大鼠表现出健康状况良好。以上结果表明, E-4株系具有缓解肾炎症状的潜在功能。此外, 利用富含多酚的rolC转基因的细胞株系, 研究了愈伤组织中咖啡酸代谢物的诱导合成机制。结果发现,在rloC转基因的E. sericeum愈伤组织中,咖啡酸代谢物的高产与迷迭香酸生物合成中的关键基因CYP98A3的高表达有关。     

The rolC gene increases caffeoylquinic acid production in transformed artichoke cells

Caffeoylquinic acids are found in artichokes, and they are currently considered important therapeutic or preventive agents for treating Alzheimer’s disease and diabetes. We transformed artichoke [the cultivated cardoon or Cynara cardunculus var. altilis DC (Asteraceae)] with the rolC gene, which is a known inducer of secondary metabolism. High-performance liquid chromatography with UV and high-resolution mass spectrometry (HPLC-UV-HRMS) revealed that the predominant metabolites synthesized in the transgenic calli were 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and chlorogenic acid. The rolC-transformed calli contained 1.5 % caffeoylquinic acids by dry weight. The overall production of these metabolites was three times higher than that of the corresponding control calli. The enhancing effect of rolC remained stable over long-term cultivation.     

Light and temperature conditions affect bioflavonoid accumulation in callus cultures of Cyclopia subternata Vogel (honeybush)

Callus cultures of the endemic South-African legume Cyclopia subternata were cultivated under varying light and temperature conditions to determine their influence on biomass growth and bioflavonoids accumulation. Experimental modifications of light included complete darkness, light of different spectral quality (white, red, blue and yellow) and ultraviolet C (UVC) irradiation. The calli were also subjected to elevated temperature or cold stress. Among the tested light regimes, cultivation under blue light resulted in the highest levels of hesperidin (H)—118.00 mg 100 g^?1 dry weight (DW) on 28 days of experiment, as well as isoflavones: 7-O-β-glucosides of calycosin (CG), pseudobaptigenin (PG) and formononetin (FG)—28.74, 19.26 and 10.32 mg 100 g^?1 DW, respectively, in 14-days old calli. UVC irradiation applied on 20 days stimulated the accumulation of H (204.14 mg 100 g^?1 DW), CG (31.84 mg 100 g^?1 DW) and PG (18.09 mg 100 g^?1 DW) in 28 days culture by 140, 46 and 165 %, respectively, without negatively influencing callus growth. Low temperature (13 °C) increased CG content by over 1,500 % (235.29 mg 100 g^?1 DW) when applied during the whole 28-days growth cycle, at the same time causing 95 % decrease in culture growth in comparison to reference calli maintained at 24 °C. On the contrary, elevated temperature (29 °C) applied during the second half of the culture period resulted in over 300 and 500 % increase in CG and PG content (61.76 and 58.89 mg 100 g^?1, respectively) while maintaining relatively high biomass yield.     

Effects of 5-azacytidine induced DNA demethylation on methyltransferase gene expression and resveratrol production in cell cultures of Vitis amurensis

DNA becomes methylated in vivo through the action of a specific group of enzymes known as methyltransferases or methylases. Plants are known to possess the methyltransferases (Met), chromo methyltransferases (CMT), and domainrearranged methyltransferases (DRM) methylase families, which affect cytosine methylation within different contexts. DNA methylation has been proposed to play a role in secondary plant metabolism, but there is a lack of valid data connecting these two processes. In this study, we treated control and transformed with rolB gene from Agrobacterium rhizogenes cell cultures of Vitis amurensis with the demethylation agent 5-azacytidine (azaC). The purpose of the current investigation was to study effects of induced DNA demethylation on methyltransferase gene expression in connection to resveratrol production, a naturally occurring polyphenol that has a wide range of intriguing biological properties. Using semi-quantitative and real-time PCR, we showed that rolB gene transformation of V. amurensis cells decreased Met and CMT expression, but significantly increased DRM expression. AzaC treatment of the control and the rolB-transgenic calli significantly increased expression of all methylases (excluding Met). Following 3 months of azaC treatment, we detected significantly elevated levels of rolB gene expression in the transgenic calli. In current paper, we discuss how methylase expression may influence resveratrol biosynthesis and rolB transgene expression. Effects of azaC application are discussed.     

Involvement of DNA methylation in the regulation of STS10 gene expression in Vitis amurensis

DNA methylation is known to play an important role in various developmental processes and defense mechanisms in plants and other organisms. However, it is not known whether DNA methylation is implicated in the genetic regulation of plant secondary metabolism, including resveratrol biosynthesis. Resveratrol is a naturally occurring polyphenol that is present in grapes, peanuts, and other plant sources, and it exhibits a wide range of valuable biologically active properties. The transformation of the wild-growing grape Vitis amurensis with the oncogene rolB from Agrobacterium rhizogenes has been demonstrated to considerably increase resveratrol production. To investigate whether DNA methylation regulates resveratrol biosynthesis, we treated both rolB transgenic and empty vector control V. amurensis cell cultures with the DNA demethylation agent 5-azacytosine (azaC). The azaC treatment significantly increased stilbene synthase 10 gene (VaSTS10) expression and resveratrol content in the V. amurensis cell cultures. Using bisulfite sequencing, we examined the methylation status of VaSTS10 in cell cultures under normal conditions and after azaC treatment. Both the promoter and 3′-end of the protein coding region of the VaSTS10 gene were hypermethylated (54–67 %) in the control cell culture. The rolB transgenic cell culture had high levels of resveratrol and lower hypermethylation levels of the VaSTS10 gene (20–47 %). The azaC treatment resulted in reduction in the DNA methylation levels in the promoter and coding regions of the VaSTS10 gene in both cell cultures. These data suggest that the DNA methylation may be involved in the control of resveratrol biosynthesis via the regulation of STS genes expression.     

Transcriptomics analysis investigates sesquiterpenoids accumulation pattern in different tissues of <Emphasis Type="Italic">Atractylodes lancea</Emphasis> (Thunb.) DC. plantlet

Linum usitatissimum: L. is well-known for production of pharmacologically important secondary metabolites. Due to their tremendous beneficial effects on human health, these compounds are receiving greater attention throughout the World, especially in the treatment of various types of cancers. In present study, we have developed an efficient protocol for production of lignans like secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG) and neo-lignans like dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) by exploiting in vitro callus cultures of Flax. These cultures were established from stem and leaf explants, inoculated on Murashige and Skoog (MS) media supplemented with various concentrations of α-naphthalene acetic acid (NAA), thidiazuron (TDZ) and 6-benzyl adenine (BA). Results revealed that the leaf-derived calli (1.0 mg/l NAA) accumulated highest levels of biomass (DW; 15.7 g/l) and antioxidant activity, while highest production of total phenolics (111.09 mg/l) and flavonoids (45.02 mg/l) were observed in stem-derived calli (1.0 mg/l NAA). The high-performance liquid chromatography (HPLC) analysis revealed that the stem-derived calli (1.0 mg/l NAA) accumulated optimum concentrations of SDG (2.7?±?0.021 mg/g DW), LDG (9.8?±?0.062 mg/g DW) and DCG (13.8?±?0.076 mg/g DW), while leaf-derived calli (1.0 mg/l NAA) showed optimum accumulation of GGCG (3.8?±?0.022 mg/g DW) as compared to all other treatments. These results provided definite evidence that the NAA differentially influence the production of lignans and neo-lignans in callus culture of Flax. This study opens new dimensions to devise strategies to enhance the production of these valuable metabolites.     

Biosynthesis of precious metabolites in callus cultures of <Emphasis Type="Italic">Eclipta alba</Emphasis>

Eclipta alba (False daisy) is an important medicinal plant with well-known antihepatotoxic activity. However, no previous in vitro studies are available for its callus culture for increased production of antioxidant secondary metabolites. Herein, we maintained a competent protocol for callus culture of E. alba using stem and leaf explants grown on MS medium containing various concentrations of thidiazuron, 6-benzylaminopurine (BAP) either alone or in association with α-naphthalene acetic acid (NAA). Among all the applied plant growth regulators, BAP along with NAA resulted in maximal dry biomass of 18.0 and 13.8 g/l for stem and leaf explants, respectively. Furthermore, the highest production of phenolics (375.7 mg/l for stem-associated callus and 298 mg/l for leaf-associated callus) and flavonoids (62.0 and 52.3 mg/l for stem- and leaf-associated callus, respectively) were found to be present in optimized callus culture. Antioxidant activity was also elucidated for both stem and leaf derived calli. The highest antioxidant activities (~?93.5%) were witnessed for stem and leaf associated calli at set concentrations of 3.0 mg/l BAP?+?1.0 mg/l NAA and 4.0 mg/l BAP, respectively. High-performance liquid chromatography analyses revealed optimum accumulation of coumarin (1.98 mg/g DW) and wedelolactone (49.63 mg/g DW) in leaf associated callus and desmethylwedelolactone (69.96 mg/g DW), β-amyrin (0.8179 mg/g DW) and eclalbatin (0.3202 mg/g DW) in stem associated callus at optimized concentration.     

Accumulation of hydroxybenzoic acids and other biologically active phenolic acids in shoot and callus cultures of Aronia melanocarpa (Michx.) Elliott (black chokeberry)

Phenolic acids are plant metabolites important in phytotherapy and also in cosmetology. In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog (L-S) medium containing different levels of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), ranging from 0.1 to 3.0 mg l^?1. Methanolic extracts from the biomass of these cultures and from the fruits of soil-grown plants were used to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. Out of a total of twelve analyzed compounds, all of the extracts contained four of them: caffeic acid, p-hydroxybenzoic acid, syringic acid, and vanillic acid. Moreover, shoot extracts also contained salicylic acid (o-hydroxybenzoic acid), while callus extracts contained p-coumaric acid. On the other hand, fruit extracts also contained both salicylic acid and p-coumaric acid. The total amount of the analyzed compounds in extracts from both shoot and callus cultures depended on the L-S medium used, and varied between 103.05 and 150.95 mg 100 g^?1 dry weight (DW), and between 50.23 and 81.56 mg 100 g^?1 DW, respectively. Both types of culture contained higher levels of phenolic acids than the fruit extracts (32.43 mg 100 g^?1 DW). In shoot cultures, p-hydroxybenzoic acid and salicylic acid were the predominant metabolites (reaching 55.14 and 78.25 mg 100 g^?1 DW, respectively), while in callus cultures, p-hydroxybenzoic acid (25.60 mg 100 g^?1 DW) and syringic acid (41.20 mg 100 g^?1 DW) were the main compounds. In fruit extracts, salicylic acid (15.60 mg 100 g^?1 DW) and p-hydroxybenzoic acid (5.29 mg 100 g^?1 DW) were predominant.     

Activation of stilbene synthesis in cell cultures of <Emphasis Type="Italic">Vitis amurensis</Emphasis> by calcium-dependent protein kinases <Emphasis Type="Italic">VaCPK1</Emphasis> and <Emphasis Type="Italic">VaCPK26</Emphasis>

Stilbenes, including trans-resveratrol (3,4′,5-trihydroxy-trans-stilbene), are known to exert beneficial health effects and contribute to plant biotic stress resistance. Much remains to be discovered about the cell signaling pathways regulating stilbene biosynthesis. It has recently been shown that overexpression of the calcium-dependent protein kinase VaCPK20 gene considerably increased t-resveratrol accumulation in cell cultures of Vitis amurensis. In this study, we analyzed the involvement of other CDPK family members, VaCPK1 and VaCPK26, on stilbene synthesis and biomass production by cell cultures of V. amurensis. We showed that overexpression of the VaCPK1 and 26 genes induced production of stilbenes by 1.7–4.6-fold (for VaCPK1) and by 2.5–6.2-fold (for VaCPK26) in several independently established cell lines compared to the empty vector-transformed control. Using HPLC-UV-MS, we detected five stilbenes in the grape cells: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε- and δ-viniferin. The VaCPK1- and VaCPK26-transformed calli were capable of producing 1.4–3.1 and 1.8–4.9 mg/l of t-resveratrol, respectively (up to 0.4 for and 0.6 mg/g of dry weight for VaCPK26 and VaCPK1, respectively), while the control line synthesized only 0.5 mg/l of t-resveratrol (0.07 mg/g DW). The up-regulation of t-resveratrol production in the VaCPK1- and VaCPK26-overexpressing grape calli correlated with a significant up-regulation of stilbene synthase (STS) gene expression, especially VaSTS7. The data indicate that VaCPK1 and 26 genes, which are close homologues of VaCPK20, are positive regulators of stilbene biosynthesis in grapevine.     

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.     

Production of biologically active phenolic acids in Aronia melanocarpa (Michx.) Elliott in vitro cultures cultivated on different variants of the Murashige and Skoog medium

Phenolic acids, both benzoic and cinnamic acid derivatives, are plant metabolites with high therapeutic and cosmetic values. Methanolic extracts from the biomass of shoot and callus cultures of Aronia melanocarpa growing on seven variants of the Murashige and Skoog (MS) medium with different concentrations of plant growth regulators, BA and NAA, ranging from 0.1 to 3.0 mg l^?1, were examined for the production of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. The extracts from the shoot and callus cultures were confirmed to contain five of the twelve compounds tested for: caffeic, p-coumaric, p-hydroxybenzoic, syringic and vanillic acids. The shoot extracts contained additionally salicylic acid. Both the total amounts and the amounts of individual compounds in either the shoot or callus extracts were dependent on the concentration of cytokinin and auxin in the MS medium variants. The total amounts in the shoot and callus cultures were in the range from 93.52 to 217.00 mg 100 g^?1 DW and from 47.11 to 83.83 mg 100 g^?1 DW, respectively. The amounts of individual compounds showed wide variation, from 1.31 to 91.86 mg 100 g^?1 DW in the shoot extracts, and from 2.58 to 40.16 mg 100 g^?1 DW in the callus extracts. Salicylic acid (max. 91.86 mg 100 g^?1 DW), p-coumaric acid (max. 62.39 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 50.66 mg 100 g^?1 DW) dominated in the shoot extracts, while syringic acid (max. 40.16 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 23.59 mg 100 g^?1 DW) were the main metabolites in the callus extracts. This is the first report on the quantitative analysis of benzoic and cinnamic acid derivatives in shoot and callus cultures of A. melanocarpa growing on MS-based media with different concentrations of selected plant growth regulators—BA and NAA. The obtained maximum amounts of some metabolites are of interest from a practical perspective.     

Agrobacterium-mediated transformation of Lolium rigidum Gaud.

Lolium rigidum Gaud. is an annual grass grown for forage but also an economically damaging crop weed. A single genotype somatic embryogenic callus line, VLR1-60, was identified from a herbicide susceptible L. rigidum population, VLR1, and proved to be amenable to Agrobacterium tumefaciens-mediated transformation. Somatic embryogenic calli were continuously induced from the meristematic region of VLR1-60 plants multiplied in vitro and the basic tolerance level of VLR1-60 to hygromycin B was determined. A hygromycin phosphotransferase gene was used as a selectable marker for hygromycin B selection. Somatic embryogenic calli derived from in vitro grown vegetative tillers were co-cultivated with the A. tumefaciens strain EHA105 harbouring binary vector carrying reporter genes and selectable marker in the presence of acetosyringone for 3 days. Inoculated calli were recovered on callus proliferation medium containing Timentin? but lacking hygromycin and were then subcultured onto media with hygromycin concentrations increased progressively through time for selection of transformed plant cells. Putative transgenic plants were recovered and integration of transgenes was confirmed by Southern hybridization analysis and by detection of DsRed or GUS activity in transgenic plants. The frequency of plant transformation was 1.3 %. The ability to transform L. rigidum will provide opportunities for functional characterization of genes to improve forage quality and increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make L. rigidum a damaging crop weed.