Elicitor Concentration Research Articles

Influence of exogenous cinnamic acid on the production of chlorogenic acid in Cichorium intybus L cell culture

Cichorium intybusL(C. intybus) is a source for several important secondary metabolites (SM), including 3-O-caffeoylquinic acid (CGA). Isolation of SM from field plants is difficult due to their batch to batch variation in response to various environmental factors. The study aims to circumvent this problem in C. intybus by increasing CGA concentration through optimization of media conditions and elicitors using plant cell culture. An increased callus growth was observed in leaf explant inoculated on MS solid medium supplemented with 2 mg/l 1-napthaleneacetic acid (NAA), 2 mg/l kinetin (Kn), 30 g/l sucrose and 0.7% agar. Optimum callus growth (13 g/l) was obtained with 5% callus (w/v) sub-cultured in MS suspension medium containing 2 mg/l NAA, 2 mg/l Kn, and 50 g/l glucose at pH 5.5. After optimization of culture conditions, elicitors (cinnamic acid, jasmonic acid, chitosan and methyl jasmonate) were added towards the end of log phase to increase CGA content in callus. Among the various concentrations of elicitors, 0.5 mg/l cinnamic acid was found to reduce cell death and increase phenol content in callus as well as the culture media. These results correlated with the TLC-densitometry study, which showed cinnamic acid (0.5 mg/l) treatment to increase CGA content in callus by 0.39- fold in comparison to control callus. The elicitation was confirmed when 3T3-L1 adipocytes treated with 1 ng/ml methanol extract of callus exposed to 0.5 mg/l cinnamic acid showed a significant 0.3-fold increase in glucose uptake compared to 1 ng/ml control callus and field plant methanol extracts. The elicited callus showed a decrease in the levels of hydrogen peroxide, nitric oxide, and phenylalanine ammonia lyase activity, demonstrating the role of cinnamic acid as a precursor for CGA synthesis.

Molecular and Functional Characterization of Elicitor PeBC1 Extracted from Botrytis cinerea Involved in the Induction of Resistance against Green Peach Aphid (Myzus persicae) in Common Beans (Phaseolus vulgaris L.).

Elicitors are biofactors that induce resistance in plants against different insect pests. This in vitro study evaluated the impact of a novel elicitor protein PeBC1, extracted from a necrotrophic fungus Botrytis cinerea, on the development and fecundity parameters of green peach aphid (Myzus persicae) on common beans (Phaseolus vulgaris L.). Three different concentrations of PeBC1 elicitor (i.e., 33.56, 25.43, 19.33 µg mL−1) were applied at three different temperature regimes (i.e., 18, 21, and 25 °C). Elicitor treatments were applied topically on the bean plants at 3-leaf stage and newly emerged (0–6 h old) apterous adult aphids were exposed to these treated leaves. In addition to the biological parameters of aphids, the relative expression levels of key genes associated with jasmonic acid (JA) and salicylic acid (SA) plant defense pathways were also determined through RT-qPCR. Results of bioassays revealed that the application of PeBC1 elicitor protein exhibited pronounced and significant (p < 0.05) sub-lethal effects on green peach aphids. The fecundity was reduced and the nymphal development time was prolonged by different concentrations of PeBC1 elicitor and temperature regimes. Gene expression studies showed that the exogenous application of PeBC1 induced a significant upregulation of the expression levels of JA and SA pathway-associated genes in bean plants. As compared to control, elicitor-treated plants exhibited an induced resistance against aphids. Our findings suggest the potential use of PeBC1 elicitor protein in future bio-intensive management strategies against sap-sucking insect pests such as green peach aphids.

Enhancement of eurycomanone biosynthesis in cell culture of longjack (Eurycoma longifolia) by elicitor treatment

In this study, the effect of elicitors such as yeast extract (YE), methyl jasmonate (MeJA) and salicylic acid (SA) on the accumulation of eurycomanone in Eurycoma longifolia cell cultures were investigated. Suspension cells of E. longifolia was cultured in Murashige and Skoog (MS) medium supplemented with 30 g/L sucrose, 1.25 mg/L naphthaleneacetic acid (NAA) and 1 mg/L kinetin at a shaking speed of 120 rpm. Elicitors were added in the culture at different concentrations and times to stimulate eurycomanone accumulation in the Eurycoma longifolia cells. Eurycomanone content was determined by HPLC with a C18 column, flow rate of 0.8 mL/min, run time of 17.5 min, and a detector wavelength of 254 nm. The stationary phase was silica gel and the mobile phase was acetonitrile: H2O. Non-elicited cells were used as the control. The study showed the effect of different elicitor concentrations, YE at 200 mg/L, MeJA at 20 μM and SA at 20 μM stimulated high production of eurycomanone. In which, treatment of 20 μM MeJA after 4 days of culture resulted in the highest accumulation of this compound (17.36 mg/g dry weight), approximately 10-fold higher than that of untreated cells (1.70 mg/g dry weight).

Production of carbazole alkaloids through callus and suspension cultures in Clausena harmandiana

Carbazole alkaloids are major constituents in Clausena spp. and exhibit a wide range of biological activities. The roots of Clausena harmandiana are a rich source of active carbazole alkaloids. However, its roots take several years to grow to be able to harvest. To obtain an alternative source of carbazole alkaloids, in vitro callus cultures of C. harmandiana were induced, and the formation of two active carbazole alkaloids was investigated. The effects of precursor, concentrations of sucrose, elicitors and light were studied to improve carbazole alkaloids formation. In this study, light had a strong effect on the formation of both carbazole alkaloids. The highest yields of clausine K and 7-methoxymukonal were 4.74 ± 0.26 and 0.92 ± 0.04 mg/g DW, respectively, which have more than 10-fold found in intact roots. According to the results of this study, C. harmandiana callus cultures can be used as an alternative source of carbazole alkaloids for additional biological studies.

Promising strategy to efficiently improve the kinsenoside and polysaccharide production of rhizome cultures of Anoectochilus roxburghii (Wall.) Lindl

Elicitation is an efficient strategy for enhancement of metabolite production during plant cell or organ culture. Salicylic acid (SA) and methyl jasmonate (MeJA) are the wildly used abiotic elicitors. To improve the accumulation of the kinsenoside and polysaccharides in rhizome cultures of Anoectochilus roxburghii (Wall.) Lindl., the present study added SA or MeJA to the culture medium after 30 days of rhizome suspension culture, and the effects of concentration and elicitation time of both elicitors on the kinsensode and polysaccharide accumulation were investigated. SA and MeJA efficiently enhanced contents of the bioactive compounds in concentration- and time-dependent manners. In SA groups, the maximum kinsenoside and polysaccharide contents were determined when rhizomes were treated with 500 μM SA for 12 days; in MeJA groups, kinsenoside and polysaccharide contents reached the highest levels when rhizomes were treated with 550 μM MeJA for 14 days and 16 days, respectively. In addition, the elicitation effects were compared in a large-scale bioreactor system. Kinsenoside and polysaccharide contents were markedly improved by elicitation with SA and MeJA compared with the un-elicited rhizomes and field-grown plants. The SA-treated group produced high kinsenoside (661.4 mg/g DW), which was 1.9-fold more than that from MeJA treatment. No significant difference was found in the polysaccharide content between SA and MeJA treatments. Thus, SA is a promising elicitor to economically improve the industrial production of kinsenoside and polysaccharides during rhizome culture of A. roxburghii.

Control of Alternaria solani by herbal Formulation

The use of natural products for the control of fungal diseases in plants is considered as an interesting alternative to synthetic fungicides due to their less negative impacts on the environment. In the present study various concentration 25%, 50% 75% and 100% of elicitors like neem oilcake and mustard oilcake and binder cow dung were used to find the concentration which is antifungal in nature. 20 gm of elicitors and binders were dissolved in 100 ml of autoclaved water. The mixture was then filtered and use for antifungal activity individually. Maximum activity was observed and 100% concentration of elicitors and binders so that this concentration were used for preparation of herbal formulations with leaf extract of Lawsonia inermis (family-Lythraceae), Polyalthia longifoila (family-Annonaceae), Fruit pulp extract of Cassia fistula (Family-Caesalpiniaceae) and Seed extract of Pongamia pinnaat (family- Fabaceae). 28 herbal formulations were prepared for antifungal activity against Alternaria solani. Antifungal activity was done by poison food technique. Results suggested that formulation no. 2, 16, 20, 28 that is 82.85%, 82%, 81.42% and 80% respectively against A. solani. On the basis of results obtained, best combination of herbal formulation will be used for in vivo experiments. The purpose of this work was to evaluate antifungal activity of herbal formulation which can help to minimize the economical loss of tomato crop as well as free from side effects caused by synthetic chemicals.

Feeding elicitors and precursors enhance colchicine accumulation in morphogenic cultures of Gloriosa superba L.

Morphogenic cultures of Gloriosa superba were initiated on Murashige and Skoog’s medium fortified with 2 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L−1 naphthaleneacetic acid (NAA), 4% sucrose and 0.1% activated charcoal. To enhance the content of the alkaloid colchicine, morphogenic cultures were treated with different concentrations of abiotic elicitors like signalling compounds, metals, biotic elicitors, precursors and a combination of elicitors. Signalling molecules like acetyl salicylic acid (ASA) and sodium nitroprusside improved the production of colchicine. Abiotic elicitors have markedly (p ≤ 0.05 or ≤ 0.01) enhanced the colchicine content either at lower or higher concentrations. Among the metals, the highest amount of 11.67 mg of colchicine g−1 dry wt was noticed at 60 mM rubidium chloride, followed by 60 mM NaCl (11.18 mg g−1). Contrarily, in the presence of biotic elicitors such as Fusarium oxysporum, Alternaria solani, and Saccharomyces cerevisiae, colchicine content ranged only between 2 and 5.32 mg g−1, but Bacillus subtilis repressed it. Among the aromatic amino acids, phenylalanine at 500 mg L−1 influenced the highest accumulation of 19.48 mg g−1 dry tissue, followed by tryptophan (12.47 mg g−1), and tyrosine (9.87 mg g−1), a direct precursor of colchicine biosynthesis, while intact tubers and leaves contained 4.65 and 4.16 mg of colchicine g−1 dry tissue respectively. A combination of 10 µM AlCl3 and 50 µM salicylic acid (SA) registered 17.34 mg g−1 followed by 16.24 mg g−1 tissue in presence of 1 µM HgCl2 and 50 µM SA. The results suggest that the elicitor-stimulated colchicine accumulation was a stress response and can be exploited further for commercial production.

Effect of salicylic acid and methyl jasmonate in the production of phenolic compounds in plant cell suspension cultures of Thevetia peruviana

The objective was to enhance the production of the phenolic compounds in plant cell suspension cultures of T. peruviana at shake flask scale. The effects of salicylic acid (SA), methyl-jasmonate (MeJA) and the combination of both (SA/MeJA) were studied. Elicitor concentration, elicitation time and harvest time of cells were optimized. Phenolic compound content (PCC), flavonoid content (FC) and antioxidant activity (AA) were determined by the folin-ciocalteu method, flavonoid-aluminum complexation method and the ABTS assay, respectively. Differences between intracellular metabolite profiles due to the mentioned treatments were analyzed by Thin-layer chromatography and High-performance liquid chromatography. Highest PCC, FC and AA were obtained under the following treatments: 3 μM MeJA > 3 μM MeJA/300 μM SA > 300 μM SA > control, when elicited on the 4th day and harvested 96-h post-elicitation. It was demonstrated that exposure to 3 μM MeJA increase 1.49-fold of PCC, 1.66-fold of AA and 2.55-fold of FC compared to the control culture.

The effect of biotic and abiotic elicitors on biomass and saponin production of secondary root cultivated in shake flasks Panax vietnamensis adventitious root

Panax vietnamensis Ha et Grushv. is an endemic ginseng of Vietnam which has highly nutraceutical, medicinal and comercial values. Many its special saponins provide resistance to stress, disease and exhaustion. In this study, the biotic elicitors (yeast extract and chitosan) at concentrations from 50 – 200 mg/l and abiotic elicitors (jasmonic acid, abscisic acid and salicylic acid) at concentrations from 50 – 200 µl/l were used to evaluate the possibility of increasing secondary root’s biomass and saponin in Panax vietnamensis adventitious root cultures. This cultures were maintained on Innova 2100 shaker shaker plantform at a speed of 100 rpm, and its modified MS medium (NH4+/NO3-: 7.19/18.5 mM/mM) were supplemented with 7 mg/l IBA, 0.5 mg/l BA and 3% sucrose. Results after 56 days of cultured showed that most elicitors significantly reduced dry biomass of secondary roots as compared to control (exception the addition of 50 – 100 mg/l yeast extract or 50 µl/l salicylic acid). However, all elicitors increased saponin-accumulation, and abiotic elicitor is more effective saponin-accumulation than those biotic elicitors. Jasmonic acid obviously gave the best results; with total amount of 3 saponins at 150 µl/l, Rg1 at 100 µl/l, Rb1 at 50 µl/l and MR2 at 200 µl/l and Rg1 (3.22%), significantly higher than other concentration of elicitor. However, for accumulating saponins and developing secondary root, 150 mg/l YE is the most real effective in all elicitors (0.88 mg).

Methyl jasmonate as a control factor of the synthase squalene gene promoter and ginsenoside production in American ginseng hairy root cultured in shake flasks and a nutrient sprinkle bioreactor

Hairy root cultures of Panax quinquefolium (L.) produce triterpenoid saponins: ginsenosides with broad medical applications. The crucial enzyme in the process of ginsenoside biosynthesis is squalene synthase (SSq). In this study a 741 bp fragment of the P. quinquefolium SSq gene, consisting of a proximal promoter, 5′UTR (5′ untranslated region) and 5′ CDS (coding DNA sequence) was isolated. An analysis of an isolated fragment with in silico tools indicated a lack of tandem repeats, miRNA binding sites and CpG/CpNpG elements. However, the proximal promoter contained potential cis-elements, mediating the response to multiple external stimuli, including light, heat-stress and drought. Among them, two CGTCA motifs could be involved in the response to methyl jasmonate (MeJA) treatment. To evaluate the functional significance of MeJA on P. quinquefolium SSq expression, quantitative RT-PCR experiments were performed at different elicitor concentration. Additionally, the effect of methyl jasmonate on ginsenoside biosynthesis was examined at 5, 50, 100, 250, 500 μM l−1 concentrations. Experiments were performed in shake flasks and a nutrient sprinkle bioreactor offering better oxygenation rate and potential for future scaling-up of the biosynthesis process. The saponin content was determined using HPLC. In shake flask and bioreactor culture, the maximum yield (respectively 27.33 mg g−1 d.w. and 51.0 mg g−1 d.w.) of the sum of six examined ginsenosides was achieved in modified Gamborg B-5 medium containing 250 μM l−1methyl jasmonate after seven days of elicitation. Rb1 (20(S)protopanaxadiol-3-[O-β-d-glucopyranosyl(l → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranosyl(1 → 6)-β-d-glucopyranoside) and Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(l → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) metabolites quantitatively dominated both in shake flask and bioreactor cultures. The level of Rb1 increased 5.6-fold and achieved 14.35 mg g−1 d.w. of hairy roots cultivated in flasks. In bioreactor cultures, this metabolite achieved 24.77 mg g−1 d.w. increasing its content 2.6-fold compared to control. The level of Re increased 1.8-fold and reached 4.92 mg g−1 d.w. in flask cultures. In bioreactor cultures the content of this metabolite was 6.23 mg g−1 d.w. The highest productivity of ginseng saponins (15.37 mg g−1d.w. l−1 day−1) was noted in bioreactor hairy root cultures after 7 days elicitation of 250 μM l−1 MeJA.

Mentha × piperita L. nodal segments cultures and their essential oil production

The aim of the present study was the achievement of sterile shoot cultures from peppermint (Mentha × piperita L. cv. ʻKristinkaʼ), their multiplication and the evaluation of the production of essential oils in in vitro conditions. The study was carried out by culturing the nodal segments both on basal or on media supplemented with different concentrations of growth regulators (2,4-D, kinetin, BAP, ZEA) or elicitors (CoCl2). The growth of nodal segments did not showed differences after subculturing, neither when cultivated on media supplemented with 2,4-D or CoCl2. The supplementation of 0.5 mg L−1 BAP caused an increase in the number of de novo formed shoots. The composition of the essential oils showed remarkable differences, with an enhancement of the percentage of menthofuran after the supplementation of 2,4-D and CoCl2 or of pulegone after application of BAP and ZEA.

Nonhost resistance: Reactive oxygen species (ROS) signal causes DNA damage prior to the induction of PR genes and disease resistance in pea tissue

The defense gene activations that provide non-host disease resistance in plants are signaled by various elicitors. A number of the fungal-derived signal such as chitosan and DNase are uniquely able to promote transcription through direct effects on the DNA of pea chromatin at sites of defense gene transcription. Other biological signals are contained within the reactive oxygen species (ROS) and make substantial changes in DNA in other tissues. The current research probes the ROS elicitor concentrations and the conditions within pea endocarp nuclear material temporally associated with the activation of pathogenesis-related (PR) genes. Within 10 min, ROS accumulate in response to a bean pathogen Fusarium solani f.sp. phaseoli (Fsph) to levels 4-fold those elicited by the pea pathogen Fusarium solani f.sp. pisi (Fspi). Application of Hydrogen peroxide (H2O2) (HP) increases DNA fragmentation and activates the PR genes (DRR206, defensin, PR10, and PR1b) within 50 min and changes the nuclear diameters of the pea host cells in 3 h. Increased levels of the phytoalexin, pisatin and the growth suppression of the pea pathogen, Fspi, were observed within 24 h after H2O2 treatment to the endocarp. Our results suggest that H2O2 contributes to the induction of the defense response in pea endocarp tissue, which is likely mediated by activation of DNA damage responses.

PENAMBAHAN ISOLAT PROTEIN KEDELE DAN SUKROSA SEBAGAI ELISITOR TERHADAP SENYAWA ANTIOKSIDAN DAN RACUN PADA KECAMBAH KORO KOMAK (Lablab purpureus L Sweet)

The germination of “ koro komak ” with elicitation can improve antioxidant substance. Sucrose and ISP on concentration 0, 500, 1000, 1500, 2000 ppm were use as elicitor. The obyective of this research were to know the effect of the kind and the concentration of elicitor to the value of antioxidant substance and the best time of germination produced maximal antioxidant and minimum toxic substance.The result of this research was the kind and the concentration of elicitor influence antioxidant substance and one day is the best time of germination.

Pathogenic fungal elicitors enhance ginsenoside biosynthesis of adventitious roots in Panax quinquefolius during bioreactor culture

Fungi have been widely used as biotic elicitors to promote metabolite production of plant cell, tissue, and organ cultures. Alternaria panax Whetz. and Cylindrocarpon destructans (Zinss) Scholten are the main pathogenic fungi in ginseng cultivation. The present study used extracts of A. panax and C. destructans as elicitors to simulate adventitious roots (ARs) of Panax quinquefolius L. for improvement of ginsenoside accumulation during AR bioreactor culture. To select a suitable elicitation method, the effects of treatment duration and concentration of both fungal extracts were investigated. After 30 d of AR bioreactor culture, the addition of A. panax and C. destructans extracts for 8 d to the culture medium produced higher ginsenoside content and productivity than the other treatment periods. During the elicitation of both fungal extracts, the peak contents of nitric oxide (NO), putrescine (Put), and ginsenoside were observed at 2, 4, and 8 d, respectively. Therefore, NO and Put could be upstream signals to regulate ginsenoside synthesis. The optimal elicitor concentration varied between extracts of A. panax and C. destructans. The maximum total ginsenoside content and productivity were observed with 4mg/L and 20mg/L of the A. panax and C. destructans extracts, respectively. The ginsenoside monomers with the most increase were Rg3, Rh2, and Re for the A. panax extract and Rg3, Rh2, and Rf for the C. destructans extract. In addition, the AR biomass was more inhibited by the C. destructans (20mg/L) extract, although its ginsenoside content (34.1mg/g DW) was higher than that with the A. panax (4mg/L) extract. The maximum ginsenoside production (276.0mg/L) was achieved with the A. panax (4mg/L) extract. Therefore, the A. panax extract was more suitable as an elicitor for ginsenoside production during AR bioreactor culture. The present study confirmed that treating 30-d-old P. quinquefolius ARs with 4mg/L A. panax extract for 8 d during bioreactor culture is the optimal elicitation method; these ARs can be applied to the commercial production of P. quinquefolius products.

Differential fructan accumulation and expression of fructan biosynthesis, invertase and defense genes is induced in Agave tequilana plantlets by sucrose or stress-related elicitors

The effect of short-term treatments with non-toxic concentrations of chemical elicitors of carbon mobilization and/or defense responses on fructan accumulation and complexity was analyzed in Agave tequilana plantlets. These included sucrose (ExSuc), salicylic acid (SA), and methyl jasmonate (MeJA). Methyl viologen (MV), an oxidative-stress elicitor, was also tested. Stems of ExSuc- and SA-treated agaves accumulated fructan with contrasting degree of polymerization (DP), being higher (DP12) in the ExSuc treatment. The difference agreed with 1-SST, 6G-FFT and 6-fructan exohydrolase (FEH) gene expression patterns. Thus, a strong, 6G-FFT expression detected 6days after treatment (DAT), coupled to a systematic repression of FEH genes occurred in Ex-Suc treated agaves. Conversely, SA treatment induced maximum 6G-FFT expression and a transitory induction of FEH genes 2 DAT. MV also induced an accumulation of low-DP fructans 6 DAT. Additionally, it stimulated the highest fructan accumulation in leaves. Contrariwise, MeJA led to a depletion of soluble non-structural carbohydrates (NSCs) and fructan, particularly in leaves. An inverse relationship between high invertase and FEH gene expression levels and minimal NSCs and fructan reserves was observed in response to MeJA. Low DP fructan accumulation by MV could not be attributed to a measurable oxidative stress. Still, high antioxidant enzyme activity, indirectly manifesting oxidative stress, coincided with fructan accumulation in MV-treated agaves. High invertase and FEH expression levels induced by MeJA in leaves, and to a lesser degree by SA and MV, coincided with transcript accumulation of defense-related genes, and were, to a certain extent, in accordance with the “sweet immunity” concept, linking sugar and defense signaling.

Gene expression analysis of the withanolide biosynthetic pathway in hairy root cultures of Withania somnifera elicited with methyl jasmonate and the fungus Piriformospora indica

Withania somnifera, also known as Indian ginseng is known to contain valuable bioactive compounds, called withanolides that structurally resemble ginsenosides of Panax ginseng. These compounds provide the basis of pharmacological relevance in traditional systems of medicine. In the present study, 150 hairy root lines of W. somnifera were induced of which nine fast growing lines were analysed for their growth and withanolide content. Hairy root line W9 was selected due to its high specific growth rate (0.196 ± 0.005 d−1) and high withanolide content. The response to different concentrations of elicitors (methyl jasmonate and P. indica cell homogenate) and various exposure durations was assessed in the W9 hairy root line. The withanolide content as well as the pattern of gene expression from MVA, MEP and sterol pathway, was evaluated using qPCR. Though gene expression and withanolide content were found to be elevated in almost all MeJ and CHP treatments, the exposure of hairy roots to 15 μM MeJ for 4 h gave the maximum withanolide yield. The results suggest that the elicitation potential of methyl jasmonate was higher than that of P. indica cell homogenate for increasing withanolide levels in hairy roots of W. somnifera.

Abiotic elicitors mediated resistance and enhanced defense related enzymes in Capsicum annuum L. against anthracnose disease

The present study was intended to investigate the efficacy of abiotic elicitors (L- isoleucine, L- leucine, L- methionine, L- phenylalanine and L- proline) to induce growth and resistance in chilli against anthracnose disease caused by Colletotrichum capsici. Among the different concentrations of abiotic elicitors tested, L- leucine treatment at 50mM concentration for three hour duration offered a maximum increase in seed germination (89%) and seedling vigor (947.2) followed by other abiotic treatments. Under greenhouse conditions, significant protection against anthracnose disease was recorded by L- leucine (65%) followed by L- proline (62%) and it was also noted that these elicitors required a minimum of four days after challenge inoculation to build up maximum resistance to anthracnose disease. Apart from disease protection, abiotic elicitors were also found effective in enhancing vegetative growth parameters. At the biochemical level, the maximum activity of Phenylalanine ammonia lyase (PAL), Peroxidase (POX) and total phenolic content was observed upon treatment with L- leucine. There was an increase in PAL, POX and total phenolic content up to one to two fold in inducer treated challenge inoculated seedlings when compared to susceptible inoculated seedlings which were similar to that of resistant inoculated seedlings. Increased activity of defense enzymes in inducer treated seedlings are in correlation with the disease protection studies.

New Synthetic Pyrazine Carboxamide Derivatives as Potential Elicitors in Production of Secondary Metabolite in In vitro Cultures.

Background:Silymarin, an active polyphenolic fraction of Silybum marianum, and high flavonoid content of Fagopyrum possess various interesting biological activities. The substituted pyrazine-2-carboxamides were previously used as effective elicitors of studied secondary metabolites.Objective:To study the effect of new synthetic pyrazine carboxamide derivatives, N-(4-chlorobenzyl)-5-tert-butylpyrazine-2-carboxamide (1) and 3-(3-((trifluoromethyl) benzyl) amino) pyrazine-2-carboxamide (2), on flavonolignan and flavonoid production in S. marianum and Fagopyrumes culentum in vitro cultures.Materials and Methods:Callus and suspension cultures were cultured on MS medium containing α-naphtaleneacetic acid or 2,4-D. Three elicitor concentrations for different exposure times were tested. Dried and powdered samples of callus and suspension cultures were extracted with methanol and analyzed by DAD-HPLC.Results:Compound 1 showed as a good elicitor of taxifolin production. The effect on silymarin complex was less visible with a maximum between 24 and 48 h after 3.292 ×10−4 mol/L concentration. The detailed analysis showed that silychristin was the most abundant. Compound 2 was effective in rutin production only in callus culture with maximum 24 h and 168 h after application of 3.3756 ×10−3 mol/L concentration and 48 and 72 h after 3.3756 ×10−4 mol/L concentration.Conclusion:From the results of the performed experiments, it can be concluded that compound 1 shows to be suitable elicitor for enhanced production of taxifolin and silychristin in S. marianum, mainly when 3.292 ×10−4 mol/L concentration was used, and compound 2 is suitable for increase rutin production in callus cultures and less appropriate for suspension cultures of F. esculentum.SUMMARY The influence of two new synthetic pyrazine-2-carboxamidesderivatives on secondary metabolite content of Silybum marianum and Fagopyrum esculentum in vitro cultures was tested.In S. marianum, the derivate N-(4-chlorobenzyl)-5-tert-butylpyrazine-2-carboxamide showed as a good elicitor of taxifolin production and less effective for silymarin complex production with silychristin as the most abundant.The derivate 3-(3-((trifluoromethyl) benzyl) amino) pyrazine-2-carboxamide is suitable for increase rutin production in callus cultures and less appropriate for suspension cultures of F. esculentum.

Enhanced camptothecin production induced by elicitors in the cell suspension cultures of Ophiorrhiza mungos Linn.

Production of camptothecin (CPT), an anticancer compound was enhanced in the cell suspension cultures of Ophiorrhiza mungos Linn. through elicitor treatment. Cell suspension culture was established using the friable callus tissues induced from the field grown leaf explants cultured in MS solid media supplemented with 3 % sucrose, 3 mg L−1 1-Naphthaleneacetic acid (NAA), 1 mg L−1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 mg L−1 kinetin (KIN). The callus tissues were used for establishing cell suspension culture in half-strength MS (1/2X MS) liquid media supplemented with the same hormone concentration. NAA was found to be essential for the prolific growth of O. mungos cells in suspension culture. Influence of different elicitors such as yeast extract (YE) and silver nitrate (AgNO3) on cell growth, CPT accumulation and cell viability was studied and found that YE and AgNO3 caused a significant increase in biomass and CPT yield according to their concentration, incubation time and feeding time. A maximum of 13.3-fold increment in CPT production and threefold increase in cell growth were recorded in cell cultures elicited with 50 mg L−1 YE on the 10th day of incubation. Cell growth and CPT level were found to decrease in the cultures treated with high concentration of elicitors. CPT was estimated using high performance liquid chromatography (HPLC). The results obtained in the present investigation suggest the use of elicitation as a promising alternative method to increase CPT production and cell growth in the cell suspension cultures of O. mungos.

Enhanced plumbagin accumulation in embryogenic cell suspension cultures of Plumbago rosea L. following elicitation

This study focused on enhancing the production of plumbagin, an anticancer compound, in embryogenic cell suspension cultures of Plumbago rosea. Elicitation techniques have been reported to enhance plumbagin production. Cell suspension cultures raised from embryogenic calli induced from in vitro leaf explants were exposed to different concentrations of jasmonic acid, yeast extract and different auxin combinations. Influence of these on cell growth, biomass and plumbagin production was studied. To our knowledge this is the first report on elicitation of embryogenic cell suspension cultures of P. rosea for enhanced plumbagin production. Elicitor treated suspension cultures exhibited decreased culture viability and increased plumbagin synthesis. A maximum of 5.59-fold enhancement of plumbagin production was observed in cultures added with 1 mg L−1 naphthalene acetic acid after 6 days of incubation. Viability of cultures decreased with increased concentration of elicitors and prolonged incubation period. Application of elicitors in cell suspension cultures induces defense related responses which lead to increased secondary metabolite production for making the cells adapt to the situation. If the stressed condition persists or is in intolerable level this will eventually lead to programmed cell death and loss of culture viability.