Salicylic acid‐enhanced biosynthesis of pharmacologically important lignans and neo lignans in cell suspension culture of Linum ussitatsimum L

Linum usitatsimum L. (flax) is a perennial herb with magnitude of medicinal and commercial applications. In the present study, we investigated the effects of salicylic acid (SA) on biosynthesis of lignans (secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG)) and neolignans (dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol‐β‐coniferyl alcohol ether glucoside (GGCG)) in cell cultures of flax. Moderate concentration of SA (50 μM) enhanced biomass accumulation (10.98 g/L dry weight (DW)), total phenolic content (37.81 mg/g DW), and antioxidant potential (87.23%) to two‐fold than their respective controls after 72 h of exposure. However, higher levels of total flavonoid content (5.32 mg/g DW) were noted after 48 h of exposure to 50 μM of SA. HPLC analyses revealed that 50 μM SA, significantly enhanced biosynthesis of SDG (7.95 mg/g DW), LDG (7.52 mg/g DW), DCG (54.90 mg/g DW), and GGCG (16.78 mg/g DW), which was almost 2.7, 1.8, 3.88, and 3.98 fold higher than their respective controls after 72 h of exposure time, respectively. These results indicated that moderate concentrations of SA had significant effects on biosynthesis and productivity of lignans and neolignans in cell culture of L. usitatissimum.     

Effects of yeast extract on the production of phenylpropanoid metabolites in callus culture of purple basil (Ocimum Basilicum L. var purpurascens) and their in-vitro evaluation for antioxidant potential

Ocimum basilicum L. var. purpurascens is an enriched reservoir of pharmaceutically important compounds with plenty of health and therapeutic attributes such as phenolic acids and anthocyanins. However, the inefficient production of aforementioned metabolites in wild has restricted its commercial utilization. Herein, commercially viable phytochemicals have been enhanced through elicitation of in-vitro cultures of O. basilicum using yeast extract.The impact of various concentrations (YE 1 mg/L,YE 10 mg/L, YE 25 mg/L, YE 50 mg/L, YE 100 mg/L, YE 200 mg/L and YE 400 mg/L) of yeast extract on biomass accumulation, phytochemical production, and antioxidant activities were assessed in callus cultures. Moderate concentration of yeast extract (100 mg/L) enhanced biomass accumulation i.e. fresh weight (FW 216.28 g/L) and dry weight (DW 15.49 g/L) up to 1.5 folds as compared to control (FW 167.14 g/L and DW 10.25 g/L). Similarly, yeast extract (100 mg/L) increased total phenolic and flavonoid contents as well as enhanced antioxidant activities such as ABTS (2,2 azinobis 3-ethylbenzthiazoline-6-sulphonic acid), FRAP (ferric reducing antioxidant power) and DPPH (2,2-diphenyl-1-picryhydrazyl). High performance liquid chromatography (HPLC) analysis was elucidated for further phytochemical investigation. HPLC analysis showed an increase of almost 1.9 folds as compared to control in rosmarinic acid (15.19 mg/g DW), chicoric acid (2.13 mg/g DW), peonidin (2.70 mg/g DW) and cyanidin (1.57 mg/g DW). Likewise, 1.8 fold and 2.4 folds increase was observed in eugenol essential oils (0.25 mg/g DW) and chavicol (0.037 mg/g DW), respectively. For cellular antioxidant activity, reactive oxygen specie or reactive nitogen specie (ROS/RNS) was induced in yeast cells and the effect of O. basilicum callus culture was further investigated in stressed yeast cells. A positive correlation exists between the antioxidant activities, TPC and TFC analysis. In short, these results showed that yeast extract could act as an efficient elicitor to enhance pharmacologically important metabolites in callus cultures of Ocimum basilicum.

Graphical abstract

     

Simultaneous heat shock and in situ adsorption enhance plumbagin production in Plumbago indica root cultures

Plumbago indica L. is an important source of plumbagin, a commercially valuable bioactive compound. However, the uses of plumbagin are limited due to its low supply as well as low yields and slow growth of the plant sources. This study evaluated the use of a simple, easy, and low‐cost approach using heat shock (HS) and ultrasound (US), and an in situ adsorption using a nonpolar copolymer adsorbent styrene‐divynilbenzene resin (Diaion^® HP‐20) to enhance plumbagin production in Plumbago indica root cultures. Treatment with HS (60°C) for 10 min significantly increased the production of plumbagin (5.51 mg/g DW) by up to five‐fold, compared to the level in untreated root cultures (1.14 mg/g DW). In contrast, treatments with US alone or with HS treatment produced no satisfactory increase of plumbagin production. However, combined treatment of a 20‐day‐old root culture with HS (60°C, for 10 min) in the presence of Diaion^® HP‐20 (10 g/L) markedly increased the production up to 20.28 mg/g DW of plumbagin that was almost 14‐fold higher, compared to the level in an untreated root culture. Such an increase would be sufficient for commercial applications of this method to produce plumbagin.     

Elicitation with methyl jasmonate combined with cultivation in the Plantform™ temporary immersion bioreactor highly increases the accumulation of selected centellosides and phenolics in Centella asiatica (L.) Urban shoot culture

Centella asiatica (L.) Urban is an important pharmacopoeial plant used not only in medicine but also in cosmetology. C. asiatica agitated shoot cultures were established to study the influence of ethephon, methyl jasmonate, L ‐phenylalanine (Eth 50 µM, MeJa 50 µM, L‐Phe 2.4 g/L of medium, respectively; seven variants of the supplementation) on the accumulation of secondary metabolites: the main centellosides (asiaticoside and madecassoside) and selected phenolic acids, and flavonoids in the biomass. Microshoots were harvested two and six days after the supplementation. Secondary metabolites were analyzed in methanolic extracts by UPLC‐MS/MS (centellosides) and by HPLC‐DAD (phenolics). In comparison with the reference cultures, the concentrations of individual secondary metabolites increased as follows: centellosides up to 5.6‐fold (asiaticoside), phenolic acids up to 122‐fold (p‐coumaric acid) and flavonoids up to 22.4‐fold (kaempherol). The highest production increase of individual compounds was observed for different variants of supplementation. Variant C (50 µM MeJa), the most optimal for centellosides and flavonoid accumulation, was selected for the experiment with bioreactors. Bioreactor Plantform?, compared to RITA^® system and agitated cultures, appeared to be the most advantageous for secondary metabolites production in C. asiatica shoot cultures. The phenolic acid, flavonoid, centelloside, and total secondary metabolite productivity in Plantform? system is 1.8‐fold, 1.7‐fold, 2.8‐fold, 2.1‐fold, respectively, higher than in MeJa elicitated agitated cultures, and 4.3‐fold, 7.3‐fold, 12.2‐fold, 7.2‐fold, respectively, higher than in control agitated cultures.     

Stimulating effect of both 4’‐O‐methylnorbelladine feeding and temporary immersion conditions on galanthamine and lycorine production by Leucojum aestivum L. bulblets

Bulb cultures of Leucojum aestivum and L. aestivum ‘Gravety Giant’ were subcultured in medium containing the precursor 4’‐O‐methylnorbelladine (MN) at various concentrations [0 (control), 0.15 and 0.3 g/L]. The cultures were conducted in bioreactor RITA® and lasted for 15, 30, 40 and 50 days. The growth rate and the alkaloid accumulation in bulblets were studied. For this latter purpose, a purification method was developed. It comprised a highly selective solid phase extraction using on the one hand, UPTI‐CLEAN SI and SCX cartridges for plant extracts and on the other hand, 2H cartridges for culture media. Pure alkaloidal fractions were, thus, analyzed by LC‐ESI‐MS allowing the quantitative evaluation of galanthamine and lycorine from culture extracts. Precursor feeding along with temporary immersion conditions was found to significantly improve the accumulation of both galanthamine and lycorine. The maximal concentrations of galanthamine (0.81 mg/g DW) and lycorine (0.54 mg/g DW) in L. aestivum bulblets were reached, respectively, after 40 days of culture with 0.15 g/L of precursor and after 30 days of culture with 0.3 g/L of precursor. In L. aestivum ‘Gravety Giant’ bulb cultures, 0.3 g/L of precursor was the best condition for both galanthamine (0.6 mg/g DW after 50 days) and lycorine (1.13 mg/g DW after 30 days).     

Seed Polyphenolic Profile,Antioxidative Activity,and Fatty Acids Composition of Wild and Cultivated Carthamus Species

Total flavonoid content (TFC) and cyanidin‐3‐glucoside (Cyd‐3‐glu) of seed and seed coat extract of 16 genotypes from five species of Carthamus were studied, and their major polyphenolic compounds and antioxidant activity of the seed coat extracts were determined using HPLC analysis and DPPH assay, respectively. Additionally, fatty acids composition of the seed oil was analyzed by GC. In general, TFC and Cyd‐3‐glu content of seed coat extracts were higher than those of seed extracts. A novel breeding line with black seed coat (named A82) depicted lower TFC (3.79 mg QUE/g DW) but higher Cyd‐3‐glu (24.64 mg/g DW) compared to the white and other seed‐pigmented genotypes. DPPH radical scavenging activity showed a strong association with Cyd‐3‐glu content (r = 0.84), but no correlation with TFC (r = ?0.32). HPLC analysis of seed coat extracts revealed that four compounds were dominant constituents including rutin (7.23 – 117.95 mg/100 g DW), apigenin (4.37 – 64.88 mg/100 g DW), quercetin (3.09 – 14.10 mg/100 g DW), and ferulic acid (4.49 – 30.41 mg/100 g DW). Interestingly, the genotype A82 with an appropriate polyunsaturated/saturated fatty acids index (5.46%) and a moderate linoleic fatty acid content (64.70%) had higher nutritional and pharmaceutical value than all the other genotypes.     

Cell line selection through gamma irradiation combined with multi-walled carbon nanotubes elicitation enhanced phenolic compounds accumulation in Salvia nemorosa cell culture

The current study focused on improving the production of phenolic acids in the Woodland Sage cell suspension culture (CSC) through attaining high-yielding cell lines and carboxyl functionalized multi-walled carbon nanotubes (MWCNT-COOH) elicitation. The leaf-derived callus was irradiated at different doses of gamma irradiation 10 to 100 Gy. The maximum content of rosmarinic acid (RA), salvianolic acid B (SAB), ferulic acid (FA), and cinnamic acid (CA) was recorded in callus cultures irradiated with 70 Gy, which was 18.53, 5.21, 1.9, and 7.59 mg/g DW, respectively. The CSC that established from 70 Gy γ-irradiated calli accumulated 1.7-fold RA more higher irradiated callus culture. The CSC elicited with various concentrations of MWCNT-COOH in range 25 to 100 mg/l significantly increased fresh weight (FW), dry weight (DW), and phenolic acid contents of cells. The highest FW with 268.47 g/l and DW with 22.17 g/l was obtained in 100 mg/l MWCNT-COOH treatment. The RA, SAB, CA and FA content of CSC treated with 100 mg/l MWCNT-COOH were 13-fold, 14.2-fold, 20-fold, and 3- fold higher than wild S. nemorosa plant at flowering stage, respectively. The antioxidant activity of cultures significantly enhanced with both gamma and MWCNT-COOH based on DPPH and FRAP assay. Our results showed that the combination of cell line selection and MWCNT-COOH elicitation significantly improved the production of secondary metabolites in Woodland Sage, which is useful for large-scale production of phenolic compounds.

     

Effects of donor plants and plant growth regulators on naphthoquinone production in root cultures of <Emphasis Type="Italic">Impatiens balsamina</Emphasis>

The effects of type of explant (leaves and roots), donor plants, and plant growth regulators on naphthoquinone (NQ) production of Impatiens balsamina L. root cultures were evaluated. The root cultures were initiated in liquid Gamborg’s B5 medium supplemented with 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ kinetin (Kn) and 1.0 mg l⁻¹ 6-benzyladenine (BA). The present investigation indicated that the root cultures established from the leaf explants produced higher total NQ content [1.01 ± 0.046 mg/g dry weight (DW)] than those established from the root explants (0.62 ± 0.023 mg/g DW). The leaf explants of four I. balsamina strains including white flower plant (IbW), pink flower plant (IbP), violet flower plant (IbV) and red flower plant (IbR) were used to establish the root cultures. Based on HPLC analysis, IbP strain produced the highest total NQ content (3.39 ± 0.072 mg/g DW), while IbR strain produced the lowest one (1.45 ± 0.055 mg/g DW). The root cultures established from the IbP explant were capable of producing higher content of total NQs (2.76 ± 0.093 mg/g DW) than those established from the other strains. The results suggest that the tissue cultures initiated from the high-yielding donor plants should be capable of producing higher content of secondary compounds than those initiated from low-yielding donor plants. In addition, plant growth regulator manipulation exhibited that a combination of 0.1 mg l⁻¹ NAA, 1.0 mg l⁻¹ Kn and 2.0 mg l⁻¹ BA is capable of increasing NQ production (2.97 ± 0.072 mg/g DW) in I. balsamina root cultures.     

Variation in polyphenolic profile and in vitro antioxidant activity of eastern teaberry (Gaultheria procumbens L.) leaves following foliar development

Seasonal dynamics in the polyphenolic composition, antioxidant activity, and their relationships during plant development were evaluated for eastern teaberry (Gaultheria procumbens L.) leaves, a traditional herbal medicine of North American natives. With the complementary UHPLC-PDA-ESI-MS³, HPLC-PDA-fingerprint, Folin-Ciocalteau, and n-butanol/HCl assays of methanol-water (75:25, v/v) extracts, the dried leaf samples harvested monthly across the growing season under Polish climate conditions were found rich in structurally diverse polyphenols (149.2–210.7 mg/g DW) including the dominating salicylates (64.6–107.5 mg/g DW), proanthocyanidins (53.0–66.8 mg/g DW), and flavonoids (17.3–25.3 mg/g DW), and the accompanying chlorogenic acid isomers (2.4–4.4 mg/g DW) and simple phenolic acids (0.9–1.1 mg/g DW). Among 28 detected analytes, gaultherin (64.6–107.5 mg/g DW), miquelianin (14.6–21.1 mg/g DW), procyanidin A-type trimer (5.5–9.5 mg/g DW), and (–)-epicatechin (5.8–7.8 mg/g DW) were the most abundant. The phenolic levels and antioxidant activity parameters in the DPPH (EC₅₀, 15.0–18.2 μg DW/mL; 0.95–1.16 mmol Trolox equivalents/g DW) and FRAP (2.3–3.4 mmol Fe ²⁺/g DW; 0.86–1.26 mmol Trolox equivalents/g DW) assays showed parallel seasonal trends with maxima in September and October. As the subsequent correlation studies confirmed the determinative impact of polyphenols on the leaf antioxidant activity and its seasonal fluctuations, the Fall season could be recommended as optimal for harvesting the plant material for medicinal purposes and cost-effective production of natural health products.     

Phytochemical Compounds from the Crop Byproducts of Tunisian Globe Artichoke Cultivars

The phytochemical composition in two Tunisian globe artichoke cultivars (bracts, leaves, and floral stems) was evaluated in the plant byproducts. The results indicated that the bracts contain the highest levels of total phenols, o‐diphenols, and flavonoids, whereas tannins seem to be more abundant in the leaves. Bracts from the ‘Violet d'Hyères’ cultivar possessed more total phenols (160.8 mg/g DW), flavonoids (64.9 mg/g DW), and anthocyanins (15.3 μg/g DW) than the ‘Blanc d'Oran’ bracts (134.5 mg/g DW, 51.2 mg/g DW, and 8.3 μg/g DW, resp.). Sixty‐four volatile compounds were identified in the headspace of globe artichoke material, particularly in the bracts. The volatile profile showed that sesquiterpene hydrocarbons and non‐terpene derivatives were the main volatiles emitted by the bracts in both cultivars. These results suggest that globe artichoke byproducts might represent a potential source of natural compounds, which could be used as nutraceuticals or as ingredients in the design of functional foods.     

Deacetylation biocatalysis and elicitation by immobilized Penicillium canescens in Astragalus membranaceus hairy root cultures: towards the enhanced and sustainable production of astragaloside IV

A novel biotechnology approach by combining deacetylation biocatalysis with elicitation of immobilized Penicillium canescens (IPC) in Astragalus membranaceus hairy root cultures (AMHRCs) was proposed for the elevated production of astragaloside IV (AG IV). The highest AG IV accumulation was achieved in 36‐day‐old AMHRCs co‐cultured with IPC for 60 h, which resulted in the enhanced production of AG IV by 14.59‐fold in comparison with that in control (0.193 ± 0.007 mg/g DW). Meanwhile, AG IV precursors were almost transformed to AG IV by IPC deacetylation. Moreover, expression of genes involved in AG IV biosynthetic pathway was significantly up‐regulated in response to IPC elicitation. Also, FTIR and SEM showed that cell wall lignification was enhanced following IPC treatment and root surface was likely to be IPC deacetylation site. Overall, dual roles of IPC (biocatalyst and elicitor) offered an effective and sustainable way for the mass production of AG IV in AMHRCs.     

High production of bioactive depsides in shoot and callus cultures of <Emphasis Type="Italic">Aronia arbutifolia</Emphasis> and <Emphasis Type="Italic">Aronia</Emphasis> × <Emphasis Type="Italic">prunifolia</Emphasis>

Methanolic extracts from calluses and shoots of Aronia arbutifolia and Aronia × prunifolia cultivated in vitro were quantitatively analysed for phenolic acids by DAD-HPLC. The cultures were grown on ten variants of Murashige–Skoog medium variants enriched with various concentrations of growth regulators (GRs), BA and NAA, in the concentration range 0.1–3.0 mg/L. The analysed extracts were confirmed to contain from four to six compounds (depsides—chlorogenic acid, neochlorogenic acid, and rosmarinic acid, and also protocatechuic acid, p-hydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid). The total amounts of the metabolites varied considerably, depending on the amounts of the GRs in the tested medium variants, and increased in the callus and shoot extracts, respectively, up to 1.7 and 3.2 times (A. arbutifolia), and 2.2 and 2.7 times (A. × prunifolia). Maximum total amounts were confirmed in shoot extracts of both plants (approx. 200 and 600 mg/100 g DW, respectively). The main compounds in A. arbutifolia cultures were the depsides—chlorogenic acid, rosmarinic acid, and neochlorogenic acid (max. 91.94, 77.03, 32.57 mg/100 g DW, respectively). The same depsides dominated quantitatively in the cultures of A. × prunifolia (max. 131.82, 206.62 and 257.39 mg/100 g DW, respectively).     

Physicochemical Properties,Antioxidant and Anti‐proliferative Capacities of Dried Leaf and Its Extract from Xao tam phan (Paramignya trimera)

Xao tam phan (Paramignya trimera) has been used for the treatment of cancer and cancer‐like aliments. Among different parts of the P. trimera plant, leaf is considered as a residual part after harvesting of the root. This study aimed to determine the physiochemical properties and the antioxidant and anti‐proliferative capacities of P. trimera leaf (PTL) using microwave drying for the preparation of dry sample; MeOH and microwave‐assisted extraction for the preparation of crude extract; and freeze‐drying for the preparation of powdered extract. The results showed that total phenolic, total flavonoid, proanthocyanidin, and saponin contents of PTL prepared by microwave drying at 450 W were 25.4 mg gallic acid equiv. (GAE), 86.3 mg rutin equiv. (RE), 5.6 mg catechin equiv. (CE), and 702.1 mg escin equiv. (EE) per gram dried sample, respectively. Gallic acid, protocatechuic acid, ellagic acid, rutin, and quercetin were identified in the PTL MeOH extract. Dried PTL displayed potent antioxidant activity, while the powdered PTL extract exhibited great anti‐proliferative capacity on various cancer cell lines including MiaPaCa‐2 (pancreas), HT29 (colon), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2‐C (neuroblastoma), MCF‐7 (breast), MCF‐10A (normal breast), and U87, SJ‐G2, and SMA (glioblastoma). Anti‐proliferative capacity on pancreatic cancer cells (MiaCaPa2, BxPc3, and CFPAC1) of PTL extract (200 μg/ml) was significantly higher (P < 0.05) than those of ostruthin (20 μg/ml) and gemcitabine (50 nm ), and to be comparable to the powdered P. trimera root extract and a saponin‐enriched extract from quillajia bark (a commercial product). The findings from this study allow us to conclude that the PTL is a rich source of phytochemicals that possess promising antioxidant and anti‐proliferative activities, therefore it shows potential as lead compounds for application in the nutraceutical, medicinal and pharmaceutical industries.     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.     

Studies on the accumulation of phenolic acids and flavonoids in different in vitro culture systems of Schisandra chinensis (Turcz.) Baill. using a DAD-HPLC method

Different in vitro culture systems of the East-Asian origin medicinal plant species − Schisandra chinensis, were tested in order to investigate their potential for the accumulation of two groups of phenolic compounds. In vitro cultures were maintained on the Murashige and Skoog (MS) medium supplemented with 3 mg/l BA and 1 mg/l NAA in an agar system (30- and 60-day growth cycles), and also in two different liquid systems: stationary and agitated. Stationary liquid cultures were grown in batch (30- and 60-day growth cycles) and fed-batch modes. Of the twenty compounds, seven free phenolic acids and of the eleven compounds, five flavonoids were quantified in methanolic extracts from lyophilized biomass and in the growth media using the RP-HPLC-DAD method. For comparison purposes, phytochemical analyses of leaf and fruit extracts from the parent plant were also conducted. The estimated compounds were not detected in the growth media. The highest total amounts of phenolic acids (71.48 mg/100 g DW) and flavonoids (29.36 mg/100 g DW) were found in extracts from the biomass of agar cultures harvested after 30 days of cultivation. The main metabolites in all the tested systems were: protocatechuic acid (max. 35.69 mg/100 g DW), chlorogenic acid (max. 13.05 mg/100 g DW), and quercitrin (max. 27.43 mg/100 g DW).     

Production of scopadulcic acid B from Scoparia dulcis Linn. using a Luffa sponge bioreactor

Production of Scopadulcic acid B (SDB), a diterpene having antiviral and anti-tumour activity, was accomplished by immobilizing suspension culture-derived cells of Scoparia dulcis on Luffa sponge matrix. The yield of SDB in shake flask cultures was 50.85 mg/g of cells after 30 days of incubation while, in Luffa sponge inoculated columns SDB production was scaled up to 350.57 mg/g of cells by the 19th day of the first batch operation. The trend was maintained up to the 22nd day and the half life period of the reactor was 10 days. The bioreactor with Luffa sponge is a novel system and can be exploited for the production of many pharmaceutically active natural compounds effectively.     

Stimulation of anthocyanin synthesis in grape (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) cell cultures by pulsed electric fields and ethephon

Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes; particularly due to their reported health-promoting properties. In this study, production of anthocyanin in cell suspension culture of Vitis vinifera was evaluated following treatment with either ethephon and/or pulsed electric fields (PEF). Overall, total production of anthocyanin increased in treated cells compared to untreated cells. Treatment of cell suspension with PEF at day 14 of culture resulted in 1.7-fold increase (1.42 mg/g DW) in anthocyanin content when compared to control cells; while, treatment with ethephon resulted in 2.3-fold increase (1.99 mg/g DW) in anthocyanin content. When cells were treated with both ethephon and PEF, 2.5-fold increase in anthocyanin content (2.2 mg/g DW) was observed. These findings demonstrate that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.     

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Summary Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g⁻¹ DW, valtrate (VAL) 10.2mgg⁻¹ DW, and didrovaltrate (DID) 2.9mg g⁻¹ DW were observed in root cultures after 7–8wk of culture.