Optimization of Elicitation Conditions with Methyl Jasmonate and Salicylic Acid to Improve the Productivity of Withanolides in the Adventitious Root Culture of Withania somnifera (L.) Dunal

Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70?g FW) on 30-day-old adventitious root cultures with 150???M SA for 4?h elicitor exposure period resulted in the production of 64.65?mg?g^?l dry weight (DW) withanolide A (48-fold), 33.74?mg?g^?l DW withanolide B (29-fold), 17.47?mg?g^?l DW withaferin A (20-fold), 42.88?mg?g^?l DW withanone (37-fold), 5.34?mg?g^?l DW 12-deoxy withastramonolide (nine fold), 7.23?mg?g^?l DW withanoside V (seven fold), and 9.45?mg?g^?l DW withanoside IV (nine fold) after 10?days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.     

Pyrazinecarboxamides as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro

The effect of new synthetic pyrazinecarboxamide derivatives as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro was investigated. Both tested elicitors increased the production of flavonolignans in S. marianum callus and suspension cultures and flavonoids in O. arvensis callus and suspension cultures. Compound I, 5-(2-hydroxybenzoyl)-pyrazine-2-carboxamide, has shown to be an effective elicitor of flavonolignans and taxifoline production in Silybum marianum culture in vitro. The maximum content of silydianin (0.11%) in S. marianum suspension culture was induced by 24 h elicitor application in concentration of 1.159 × 10?3 mol/L. The maximum content of silymarin complex (0.08%) in callus culture of S. marianum was induced by 168 h elicitor application of a concentration 1.159 × 10?? mol/L, which represents contents of silydianin (0.03%), silychristin (0.01%) and isosilybin A (0.04%) compared with control. All three tested concentrations of compound II, N-(2-bromo-3-methylphenyl)-5-tert-butylpyrazin-2-carboxamide increased the flavonoid production in callus culture of O. arvensis in a statistically significant way. The best elicitation effect of all elicitor concentrations had the weakest c? concentration (8.36 × 10?? mol/L) after 168 h time of duration. The maximum content of flavonoids (about 5,900%) in suspension culture of O. arvensis was induced by 48 h application of c? concentration (8.36 × 10?? mol/L).     

Methyl jasmonate and salicylic acid induced oxidative stress and accumulation of phenolics in Panax ginseng bioreactor root suspension cultures

To investigate the enzyme variations responsible for the synthesis of phenolics, 40 day-old adventitious roots of Panax ginseng were treated with 200 microM methyl jasmonate (MJ) or salicylic acid (SA) in a 5 L bioreactor suspension culture (working volume 4 L). Both treatments caused an increase in the carbonyl and hydrogen peroxide (H2O2) contents, although the levels were lower in SA treated roots. Total phenolic, flavonoid, ascorbic acid, non-protein thiol (NPSH) and cysteine contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical reducing activity were increased by MJ and SA. Fresh weight (FW) and dry weight (DW) decreased significantly after 9 days of exposure to SA and MJ. The highest total phenolics (62%), DPPH activity (40%), flavonoids (88%), ascorbic acid (55%), NPSH (33%), and cysteine (62%) contents compared to control were obtained after 9 days in SA treated roots. The activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, substrate specific peroxidases (caffeic acid peroxidase, quercetin peroxidase and ferulic acid peroxidase) were higher in MJ treated roots than the SA treated ones. Increased shikimate dehydrogenase, chlorogenic acid peroxidase and beta-glucosidase activities and proline content were observed in SA treated roots than in MJ ones. Cinnamyl alcohol dehydrogenase activity remained unaffected by both MJ and SA. These results strongly indicate that MJ and SA induce the accumulation of phenolic compounds in ginseng root by altering the phenolic synthesis enzymes.     

Enhanced daidzin production from jasmonic and acetyl salicylic acid elicited hairy root cultures of Psoralea corylifolia L. (Fabaceae)

Daidzin (7-O-glucoside of daidzein) has several pharmacological benefits in herbal remedy, as antioxidant and shown antidipsotropic activity. Hairy root culture of Psoralea corylifolia L. was developed for biomass and enhanced daidzin production using signalling compounds such as jasmonic acid (JA) and acetyl salicylic acid (ASA). Best response of 2.8-fold daidzin (5.09% DW) with 1 μM JA treatment after second week and 7.3-fold (3.43% DW) with 10 μM JA elicitation after 10th week was obtained from hairy roots compared to untreated control. ASA at 10 μM promoted 1.7-fold increase in daidzin (1.49% DW) content after seventh week compared to control (0.83% DW). Addition of 25 μM ASA resulted in 1.44% DW daidzin (1.5-fold increase) with 0.91% DW in control after fifth week and 1.44% DW daidzin (2.3-fold increase) after eighth week when compared to untreated control (0.62% DW). Reduced biomass with increased daidzin content was facilitated by elicited hairy root cultures.     

Effects of Phytohormones and Jasmonic Acid on Glucosinolate Content in Hairy Root Cultures of Sinapis alba and Brassica rapa

Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species—Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis—were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg?L^?1) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg?L^?1) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.     

Effects of abiotic inducers on sesquiterpene synthesis in hairy root and cell-suspension cultures ofhyoscyomus muticus

The effects of hydrogen peroxide (H₂O₂), salicylic acid (SA), ethanol (EtOH), and methyl jasmonate (MJ) on the lubimin and solavetivone production in hairy root and cell suspension cultures ofHyoscyamus muticus were investigated. H₂O₂ (0.194–1.94) mM did not show any positive effect of the lubimin and solavetivone production. Lubimin production of cellsuspension cultures increased 50% in the presence of 40 ΜM SA. For hairy root cultures, lubimin and solavetivone production increased 5 and 48%, respectively, in the presence of 4 ΜM SA ?EtOH (20 mM) increased the lubimin and solavetivone concentrations 13% for hairy roots suggesting a potential rule for wounding or membrane disruption. MJ (4 ΜM) and 20 mM EtOH increased the lubimin and solavetivone concentrations 25 and 85%, respectively for hairy roots ofH. muticus.     

In Vitro Azadirachtin Production by Hairy Root Cultivation of <Emphasis Type="Italic">Azadirachta indica</Emphasis> in Nutrient Mist Bioreactor

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.     

Studies on mass production of transformed Panax ginseng hairy roots in bioreactor

The growth properties of Panax ginseng hairy roots transformed by Agrobacterium rhizogenes were compared between flask and aerated column or stirred bioreactor. In flask cultures, sucrose, initially 30 g/L, was nearly exhausted after 45 d of culture. The pH of the medium dropped from 5.5 to 4.96 after 10 d, but afterward it gradually increased to 6.4. After 45 d, hairy roots grew about 16-folds. The growth rate of hairy roots in air-bubble column or stirred bioreactor cultures was 1.13 (1.11) to 1.23 (1.20) g fresh wt (dry wt)/(g of cells·d), respectively. For both bioreactors, growth was about three times as high as in the flask cultivation.     

Effect of Elicitation with (+)-Usnic Acid on Accumulation of Phenolic Acids and Flavonoids in Agitated Microshoots of Eryngium alpinum L.

The present work was aimed at studying the potential of elicitation on the accumulation of phenolic compounds in in vitro shoot cultures of Eryngium alpinum L., a protected plant from the Apiaceae family. The study examined the influence of (+)-usnic acid on the biomass growth as well as on the biosynthesis of the desired flavonoids and phenolic acids in the cultured microshoots. The phenolic compound content was determined by HPLC-DAD. The flavonoid of the highest concentration was isoquercetin, and the phenolic acids of the highest amount were rosmarinic acid, caffeic acid and 3,4-dihydroxyphenylacetic acid, both in the non-elicited and elicited biomass. Isoquercetin accumulation was efficiently increased by a longer elicitation with a lower concentration of lichenic compound (107.17 ± 4.67 mg/100 g DW) or a shorter elicitation with a higher concentration of acid (127.54 ± 11.34 and 108.37 ± 12.1 mg/100 g DW). Rosmarinic acid production generally remained high in all elicited and non-elicited microshoots. The highest content of this acid was recorded at 24 h of elicitation with 3.125 µM usnic acid (512.69 ± 4.89 mg/100 g DW). The process of elicitation with (+)-usnic acid, a well-known lichenic compound with allelopathic nature, may therefore be an effective technique of enhancing phenolic compound accumulation in alpine eryngo microshoot biomass.     

Comparison of growth characteristics of Panax ginseng hairy roots in various bioreactors

This study investigated the effects of flask-to-liquid volume ratio on the growth of Panax ginseng hairy root, transformed by Agrobacterium rhizogenes, in flask cultures and compared the characteristics of various bioreactors for scale-up. The flask-to-liquid volume ratio was optimum at 1.5 mL of air/mL of medium in flask cultures, and hairy root growth was not affected above the optimum ratio. In 500-mL flask culture, hairy root showed two growth phases. After the first exponential growth, specific growth rate decreased. The growth characteristics of P. ginseng hairy root in various bioreactors were investigated. Hairy root growth was about 55-fold of inoculum after 39 d in a 5-L bioreactor and about 38-fold of inoculum after 40 d in a 19-L bioreactor. Carbon yield was higher in a 19-L bioreactor than in others, but it did not show any linear relationship to the growth rate of hairy roots in bioreactors.     

Effect of chitosan on peroxidase activity and isoenzyme profile in hairy root cultures of Armoracia lapathifolia

Hairy root cultures of Armoracia lapathifolia established by infection with Agrobacterium rhizogenes LBA 9402 present a level and isoenzyme pattern of peroxidases (POD) comparable to nontransformed roots. Elicitation with chitosan (10, 50, and 100 mg/L) was used in order to improve POD production. Total POD activity increased about 170% after 48h of treatment with chitosan 100 mg/L. Elicitation effect on soluble and ionically cell-wall-bound POD fractions of A. lapathifolia hairy roots was analyzed. POD activity of the ionically cell-wall-bound protein fraction increased in the presence of chitosan in a dose-response manner. No effect on soluble POD fractions was observed, but the isoenzyme pattern analyzed by isoelectrofocusing showed an increase of an acidic isoenzyme (pI=3.4) after the elicitation treatment. The ionically cell-wall-bound protein fraction showed only basic isoenzymes, with an increase of an isoenzyme of pI=8.7, after the elicitation treatment.     

Chitosan Elicitation Impacts Flavonolignan Biosynthesis in Silybum marianum (L.) Gaertn Cell Suspension and Enhances Antioxidant and Anti-Inflammatory Activities of Cell Extracts

Silybum marianum (L.) Gaertn is a rich source of antioxidants and anti-inflammatory flavonolignans with great potential for use in pharmaceutical and cosmetic products. Its biotechnological production using in vitro culture system has been proposed. Chitosan is a well-known elicitor that strongly affects both secondary metabolites and biomass production by plants. The effect of chitosan on S. marianum cell suspension is not known yet. In the present study, suspension cultures of S. marianum were exploited for their in vitro potential to produce bioactive flavonolignans in the presence of chitosan. Established cell suspension cultures were maintained on the same hormonal media supplemented with 0.5 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L NAA (α-naphthalene acetic acid) under photoperiod 16/8 h (light/dark) and exposed to various treatments of chitosan (ranging from 0.5 to 50.0 mg/L). The highest biomass production was observed for cell suspension treated with 5.0 mg/L chitosan, resulting in 123.3 ± 1.7 g/L fresh weight (FW) and 17.7 ± 0.5 g/L dry weight (DW) productions. All chitosan treatments resulted in an overall increase in the accumulation of total flavonoids (5.0 ± 0.1 mg/g DW for 5.0 mg/L chitosan), total phenolic compounds (11.0 ± 0.2 mg/g DW for 0.5 mg/L chitosan) and silymarin (9.9 ± 0.5 mg/g DW for 0.5 mg/L chitosan). In particular, higher accumulation levels of silybin B (6.3 ± 0.2 mg/g DW), silybin A (1.2 ± 0.1 mg/g DW) and silydianin (1.0 ± 0.0 mg/g DW) were recorded for 0.5 mg/L chitosan. The corresponding extracts displayed enhanced antioxidant and anti-inflammatory capacities: in particular, high ABTS antioxidant activity (741.5 ± 4.4 μM Trolox C equivalent antioxidant capacity) was recorded in extracts obtained in presence of 0.5 mg/L of chitosan, whereas highest inhibitions of cyclooxygenase 2 (COX-2, 30.5 ± 1.3 %), secretory phospholipase A2 (sPLA2, 33.9 ± 1.3 %) and 15-lipoxygenase (15-LOX-2, 31.6 ± 1.2 %) enzymes involved in inflammation process were measured in extracts obtained in the presence of 5.0 mg/L of chitosan. Taken together, these results highlight the high potential of the chitosan elicitation in the S. marianum cell suspension for enhanced production of antioxidant and anti-inflammatory silymarin-rich extracts.     

Methyl Jasmonate Induce Enhanced Production of Soluble Biophenols in PANAX GINSENG Adventitious Roots from Commercial Scale Bioreactors

The contents of soluble biophenols such as protocatechuic, gentisic, vanillic, caffeic, syringic, p-coumaric, ferulic, salicylic, and cinnamic acids were screened and quantified from the adventitious roots of Panax ginseng by HPLC-MS. Control adventitious roots which showed the greatest accumulation of ferulic acid (0.09 mg/g DW) were observed in our experiments. An increase in the total soluble biophenol content of adventitious roots was observed 5 days after treatment with elicitor 200 μM/L methyl jasmonate (MeJa) i.e., 35 to 40 days of inoculation. Among the biophenols investigated, the salicylic acid content was higher (0.44 mg/g DW) in MeJa treated adventitious root culture in the bioreactor. MeJa might apparently increase soluble biophenols production in adventitious roots by enhancing the biosynthesis pathway from phenylalanine to salicylic acid and other simple biophenols. __________ Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 551–554, November–December, 2005     

Regulation of vincamine biosynthesis and associated growth promoting effects through abiotic elicitation,cyclooxygenase inhibition,and precursor feeding of bioreactor grown Vinca minor hairy roots

Hydroxylase/acetyltransferase elicitors and cyclooxygenase inhibitor along with various precursors from primary shikimate and secoiridoid pools have been fortified to vincamine less hairy root clone of Vinca minor to determine the regulatory factors associated with vincamine biosynthesis. Growth kinetic studies revealed that acetyltransferase elicitor acetic anhydride and terpenoid precursor loganin significantly reduce the growth either supplemented alone or in combination (GI?=?140.6?±?18.5 to 246.7?±?24.3), while shikimate and tryptophan trigger biomass accumulation (GI?=?440.2?±?31.5 to 540.5?±?40.3). Loganin also downregulates total alkaloid biosynthesis. Maximum flux towards vincamine production (0.017?±?0.001 % dry wt.) was obtained when 20-day-old hairy roots were fortified with secologanin (10 mg/l) along with tryptophan (100 mg/l), naproxen (8.4 mg/l), hydrogen peroxide (20 μg/l), and acetic anhydride (32.4 mg/l). This was supported by RT PCR (qPCR) analysis where 2- and 3-fold increase in tryptophan decarboxylase (TDC; RQ?=?2.0?±?0.09) and strictosidine synthase (STR; RQ?=?3.3?±?0.36) activity, respectively, was recorded. The analysis of variance (ANOVA) for growth kinetics, total alkaloid content, and gene expression studies favored highly significant data (P?<?0.05–0.01). Above treated hairy roots were also up-scaled in a 5-l stirred-tank bioreactor where a 40-day cycle yielded 8-fold increase in fresh root mass.     

Genetic transformation of Echinacea purpurea with Agrobacterium rhizogenes and bioactive ingredient analysis in transformed cultures

A method of the transformed hairy roots cultures of Echinacea purpurea was established by infecting different types of explants with three type strains of Agrobacterium rhizogenes (A4, R1601 and R1000) in this paper. We obtained that the transformed percentage of E. purpurea leaves with A4, R1601 and R1000 were 80%, 60%, 40%, respectively and that of E. purpurea leafstalks were 10%, 30%, 45%, respectively. The contents of polysaccharides and phenolic compounds were measured in transformed hairy roots and non-transformed roots after 2 months in culture. For transformed hairy roots, the contents of polysaccharides and phenolic compounds were 236.0 and 18.9mgg(-1)DW, respectively. While the contents of polysaccharides and phenolic compounds in non-transformed roots were 161.5 and 33.3mgg(-1)DW, respectively.     

Volatile oils from the plant and hairy root cultures of Ageratum conyzoides L

Two lines of hairy root culture of Ageratum conyzoides L. induced by Agrobacterium rhizogenes ATCC 15834 were established under either complete darkness or 16 h light/8 h dark photoperiod conditions. The volatile oil yields from aerial parts and roots of the parent plant, the hairy root culture photoperiod line and the hairy root culture dark line were 0.2%, 0.08%, 0.03% and 0.02%, (w/w), respectively. The compositions of the volatiles from the hairy roots, plant roots and aerial parts were analysed by GC and GC-MS. The main components of the volatiles from the hairy root cultures were β-farnesene, precocene I and β-caryophyllene, in different amounts, depending on light conditions and also on the age of cultures. Precocene I, β-farnesene, precocene II and β-caryophyllene were the main constituents of the volatile oils from the parent plant roots, whereas precocene I, germacrene D, β-caryophyllene and precocene II were the main constituents of the aerial parts of the parent plant. Growth and time-course studies of volatile constituents of the two hairy root lines were compared. Qualitative and quantitative differences were found between the volatile oils from the roots of the parent plant and those from the hairy roots.     

Optimization of Adventitious Root Culture for Production of Biomass and Secondary Metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l^?1) and naphthalene acetic acid (NAA; 1.5 mg l^?1). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5–2.0 mg l^?1). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l^?1 NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l^?1 NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l^?1 NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.     

Production of the Biopesticide Azadirachtin by Hairy Root Cultivation of Azadirachta indica in Liquid-Phase Bioreactors

Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2 g l^?1dry weight and azadirachtin yield of 3.2 mg g^?1 leading to a volumetric productivity of azadirachtin as 1.14 mg l^?1 day^?1. The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97 % at a concentration of 2 % w/v and 83 % at a concentration of 0.05 % (w/v), respectively, in ethanol).