Response of Saponaria officinalis L. hairy roots to the application of TiO2 nanoparticles in terms of production of valuable polyphenolic compounds and SO6 protein.

Saponaria officinalis L. is a perennial plant from the Caryophyllaceae family whose various parts are used in traditional medicine as the treatment agent of skin diseases, blood purifier, diuretic, sudorific, and bile purifier. The cultivation system of hairy roots is a proper alternative for improving the valuable pharmaceutical compounds production compared to other in-vitro methods. The extensive nanotechnology applications in hairy roots cultivation is a sustainable production foundation to produce such active elements. In this study, the effect of various concentrations of titanium dioxide nanoparticles (TiO2 NPs) (0, 10, 20, 30, 50 mg L-1) with two treatments (24 and 48 h) was examined on the growth level, antioxidant capacity, total phenol and flavonoid contents, antioxidant enzyme activities, certain polyphenol compounds and SO6 protein in hairy roots of S. officinalis. According to the results, the maximum (3.09 g) and minimum (0.96 g) fresh weight (FW) of hairy roots were observed in treated culture media with 10 and 20 mg L-1 of TiO2 NPs after 24 and 48 h of exposure times, respectively. The highest rate of total phenol (9.79 mg GLA g-1 FW) and total flavonoid contents (1.06 mg QE g-1 FW) were obtained in the treated hairy roots with 50 and 30 mg L-1 of nano elicitor in 24 and 48 h of treatments, respectively. The maximum level of most polyphenols, such as rosmarinic acid, cinnamic acid, and rutin, was produced in 24 h of treatment. The use of TiO2 NP for 48 h with 50 mg L-1 concentration showed the highest production level of SO6 protein.

Effect of Polyploidy Induction on Natural Metabolite Production in Medicinal Plants.

Polyploidy plays an important role in plant diversification and speciation. The ploidy level of plants is associated with morphological and biochemical characteristics, and its modification has been used as a strategy to alter the quantitative and qualitative patterns of secondary metabolite production in different medicinal plants. Polyploidization can be induced by many anti-mitotic agents, among which colchicine, oryzalin, and trifluralin are the most common. Other variables involved in the induction process include the culture media, explant types, and exposure times. Due to the effects of polyploidization on plant growth and development, chromosome doubling has been applied in plant breeding to increase the levels of target compounds and improve morphological characteristics. Prompted by the importance of herbal medicines and the increasing demand for drugs based on plant secondary metabolites, this review presents an overview of how polyploidy can be used to enhance metabolite production in medicinal plants.

Improved tropane alkaloid production and changes in gene expression in hairy root cultures of two Hyoscyamus species elicited by silicon dioxide nanoparticles.

Species of Hyoscyamus are rich sources of medicinally important tropane alkaloids, which have anticholinergic, antispasmodic and sedative effects and are competitive inhibitors of acetylcholine. The application of nanotechnology and nanomaterials for elicitation is rapidly expanding and recent research indicates that silicon dioxide nanoparticles (SiO2 NPs) can be used as an efficient elicitor to increase the production of hyoscyamine and scopolamine in Hyoscyamus species. Thus, in this work, the effect of SiO2 NPs (0, 25, 50, 100 and 200 mg L-1) with two treatment times (24 and 48 h) on the growth rate, total phenol and flavonoid content (TPC, TFC), antioxidant enzyme activity, tropane alkaloid yield and pmt (putrescine N-methyltransferase) and h6h (hyoscyamine 6-hydroxylase) gene expression levels in hairy roots of two Hyoscyamus species (H. reticulatus and H. pusillus) was investigated. The highest TPC and TFC accumulation was obtained in H. reticulatus elicited by SiO2 NPs (100 and 200 mg L-1), respectively, at 24 h of treatment. High-performance liquid chromatography (HPLC) revealed the highest amount of hyoscyamine (140.15 µg g-1 FW) and scopolamine (67.71 µg g-1 FW) accumulated in H. reticulatus transformed roots treated with 100 mg L-1 SiO2 NPs at 24 h, with a respective increase of 1212% and 272% compared to non-treated roots. In H. pusillus, the highest hyoscyamine (7.42 µg g-1 FW) and scopolamine (15.56 µg g-1 FW) production (about 82% and 241% higher, respectively, compared to the lowest amounts) was achieved with 25 and 100 mg L-1 SiO2 NPs, respectively, at 48 h of treatment. Semi-quantitative RT-PCR analysis determined the highest expression level of pmt and h6h genes in H. reticulatus transformed roots supplemented with 100 mg L-1 SiO2 NPs.

Fruit phytochemical composition and color parameters of 21 accessions of five Rosa species grown in North West Iran.

BACKGROUND: The genus Rosa comprises economically important horticultural plants belonging to the family Rosaceae. Recently, the use of different Rosa species has increased owing to their multipurpose properties (ornamental, food and medicinal uses). In this study, 21 accessions of Rosa genotypes were compared for fruit phytochemical composition and color parameters. RESULTS: The highest antioxidant activity (37.86 mg AAE g-1 FW) and total phenolic (8.17 mg GAE g-1 FW), total flavonoid (2.53 mg QUE g-1 FW), total carotenoid (20.21 mg g-1 FW) and ascorbic acid (84.27 mg g-1 FW) contents were observed in G20 (R. canina), G8 (R. canina), G9 (R. canina), G5 (R. damascena) and G10 (R. moschata) respectively. Chlorogenic acid and gallic acid were found as the main phenolic constituents of Rosa fruits. High amounts of apigenin, rutin, quercetin, p-coumaric acid, cinnamic acid, chlorogenic acid, caffeic acid and gallic acid were obtained in fruit extracts of G6, G14, G6, G8, G19, G9, G19 and G12 respectively. Moreover, the level of color parameters also varied among genotypes. The highest values of a*, b*, L* and chroma were obtained in G4 (R. canina). Based on hierarchical clustering analysis with heat-map, five groups of accessions were identified. CONCLUSION: Different Rosa genotypes are rich in certain phytochemical compounds, with significant variations in their levels being observed. Hence evaluation of Rosa genetic resources can supply valuable data for screening accessions containing high levels of individual phenolics, antioxidants and other bioactive compounds for use in breeding programs and food and pharma industries. © 2019 Society of Chemical Industry.

Sodium nitroprusside stimulated production of tropane alkaloids and antioxidant enzymes activity in hairy root culture of Hyoscyamus reticulatus L.

Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson's or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 µM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 µM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures.

Enhanced production of hyoscyamine and scopolamine from genetically transformed root culture of Hyoscyamus reticulatus L. elicited by iron oxide nanoparticles.

The medicinal plant Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with Agrobacterium rhizogenes and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L-1) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L-1 FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L-1 FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production.

High-frequency in vitro plantlet regeneration from apical bud as a novel explant of Carum copticum L.

OBJECTIVE: To develop an in vitro regeneration system to increase the recovery of Carum copticum L. plantlets as a part of developing a metabolic engineering program. METHODS: The efficacy of different concentrations and combinations of 6-benzyladenine, indole-3-acetic acid and indole butyric acid on direct shoot regeneration and rooting of ajowan from apical bud explants were assessed. All explants were cultured on Murashige and Skoog (MS) medium supplemented with different combinations of 6-benzyl amino purine (BAP) (0, 2.2, 4.4, 8.8 µmol/L) and indole-3-acetic acid (IAA) (0, 0.5, 1.1, 2.2 µmol/L). RESULTS: The maximum shoot regeneration frequency (97.5%) and the highest number of shoots produced from apical buds (34 shoots per explant) were obtained on MS medium fortified with BAP (4.4 µmol/L) and IAA (0.5 µmol/L). Low shoot regeneration frequency was observed in BAP free treatments. The effects of different strengths of MS medium and various concentrations of IAA and indole-3- butyric acid on rooting rate, length and average number of roots were also investigated. Application of indole-3- butyric acid (6 µmol/L) in full-strength MS medium, was more effective than IAA and resulted in highest shoot regeneration frequency with the rooting rate of 100% and highest mean number of roots per shoot (41.8). The rooted plantlets were acclimatized successfully in greenhouse conditions with a survival rate of 90%. CONCLUSION: In this study, a simple and reliable regeneration and acclimatization protocol for Carum copticum has been presented. This protocol can be found very advantageous for a variety of purposes, including mass multiplication of Carum species, medicinal plant breeding studies and transgenic plant production.

Overproduction of valuable methoxylated flavones in induced tetraploid plants of Dracocephalum kotschyi Boiss.

BACKGROUND: Ploidy manipulation is considered an efficient method to increase production potential of medicinally important compounds. Dracocephalum kotschyi Boiss. is an endangered medicinal plant of Iran. Various concentrations of colchicine (0.05, 0.10, 0.20, and 0.50% w/v) were applied to shoot apical meristems of D. kotschyi seedlings in two and four-leaf stages to induce tetraploidy. RESULTS: According to the results, 0.5% (w/v) of colchicine can be effective for polyploidy induction in D. kotschyi. Putative tetraploids were selected by morphological and microscopic characteristics and their ploidy level was confirmed by flow cytometry analysis and chromosome counting. The chromosome number of original diploid plant was confirmed to be 2n = 2× = 20 whereas that of the tetraploid plant was 2n = 4× = 40. Tetraploid and mixoploid plants showed different morphological, physiological and microscopic characteristics from those of diploid counterparts. The total content of flavonoids was increased from 1583.28 in diploids to 1890.07 (µg/g DW) in stable tetraploids. CONCLUSION: High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) confirmed over accumulation of methoxylated hydroxyflavones in solid tetraploid plants of D. kotschyi.