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.     

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.     

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.     

Polyscias filicifolia (Araliaceae) Hairy Roots with Antigenotoxic and Anti-Photogenotoxic Activity

Hairy root cultures are considered as a valuable source of bioactive phytoconstituents with expanding applicability for their production. In the present study, hairy root cultures of Polyscias filicifolia (Araliaceae), a traditional Southeast Asian medicinal plant, were established. The transformation with Agrobacterium rhizogenes ATCC 15834 allowed to obtain 15 root lines. The K-1 line, demonstrating the highest growth capabilities, was subjected to further investigations. To enhance the biosynthetic potential of hairy roots, methyl jasmonate elicitation approach was applied (MeJA; at different doses and exposure time), with subsequent transfer of elicited roots to control medium. This strategy resulted in chlorogenic acid production up to 1.59 mg/g dry weight. HPLC-PDA-ESI-MS analysis demonstrated variation in extracts composition and allowed to identify different caffeic and ferulic acid derivatives. Next, cytotoxic, antigenotoxic, and anti-photogenotoxic properties of hairy roots extracts were determined. None of the tested extracts were cytotoxic. In addition, they demonstrated significant antigenotoxic activity with the highest protective potential; up to 52% and 49% of inhibition of induction ratio (IR) induced by the 2-aminoanthracene was revealed for extracts derived from hairy roots elicited for 3 days with 50 µM MeJA and roots elicited for 7 days with 100 µM MeJA and then transferred for 30 days to control medium, respectively. These same extracts exhibited the highest anti-photogenotoxic potential, up to 36% of inhibition of chloropromazine-induced genotoxicity.     

Enhanced secondary metabolite biosynthesis by elicitation in transformed plant root system

Plants generally produce secondary metabolites in nature as a defense mechanism against pathogenic and insect attack. In this study, we applied several abiotic elicitors in order to enhance growth and ginseng saponin biosynthesis in the hairy roots of Panax ginseng. Generally, elicitor treatments were found to inhibit the growth of the hairy roots, although simultaneously enhancing ginseng saponin biosynthesis. Tannic acid profoundly inhibited the hairy root growth during growth period. Also, ginseng saponin content was not significantly different from that of the control. The addition of selenium at inoculum time did not significantly affect ginseng saponin biosynthesis. However, when 0.5 mM selenium was added as an elicitor after 21 d of culture, ginseng saponin content and productivity increased to about 1.31 and 1.33 times control levels, respectively. Also, the addition of 20μM NiSO₄ resulted in an increase in ginseng saponin content and productivity, to about 1.20 and 1.23 times control levels, respectively, and also did not inhibit the growth of the roots. Sodium chloride treatment inhibited hairy root growth, except at a concentration of 0.3% (w/v). Increases in the amounts of synthesized ginseng saponin were observed at all concentrations of added sodium chloride. At 0.1% (w/v) sodium chloride, ginseng saponin content and productivity were increased to approx, 1.15 and 1.13 times control values, respectively. These results suggest that processing time for the generation of ginseng saponin in a hairy root culture can be reduced via the application of an elicitor.     

Methyl jasmonate influence on silymarin production and plant stress responses in Silybum marianum hairy root cultures in a bioreactor

In this article our aim was to evaluate mass cultivation of S. marianum hairy roots in a bioreactor to produce silymarin. The effects of methyl jasmonate (MJ) elicitation on the accumulation of silymarin and the extent of the MJ-induced oxidative damage were investigated in bioreactor hairy root cultures of S. marianum. The growth rate of the bioreactor hairy root cultures was higher than that of those in a shake flask after 3 weeks. Silymarin accumulation was increased from 0.13?mg?g?1 DW in non-treated hairy roots to 0.22?mg?g?1 DW in hairy roots 72?h after 100?μM?MJ treatment. Guaiacol peroxidase and ascorbate peroxidase were activated by MJ?72?h after treatment, being 3.2- and 1.3-fold higher, respectively, than that of the control. An increase in enzymatic activity suggests increased scavenging of reactive oxygen species, indicating the tolerance to MJ stress. These results suggest that MJ elicitation is beneficial for silymarin production using bioreactor hairy root cultures.     

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).     

GC–MS Studies of Thiophenes in the Supercritical Fluid CO2 and Solvent Extracts of Tagetes patula L.

The intact plant parts and genetically modified hairy root clone #TpA6 of Tagetes patula were extracted with supercritical fluid CO₂ extraction (SFE) and a conventional solvent extraction. SFE optimization included the variation of fluid CO₂ pressure, dynamic time, and the addition of methanol modifier co-solvent. The four characteristic thiophene metabolites, 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT), 2,2′:5′,2″-terthiophene (α-T), 5-(4-acetoxy-1-butynyl)-2,2′-bithienyl (BBTOAc), and 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl [BBT(OAc)₂], were analysed by GC–MS. The proposed SFE method allowed the selective extraction of thiophenes in 60 min dynamic time with supercritical CO₂ without modifier co-solvent, at 30 MPa and 40 °C. The SFE and the reference solvent extraction yielded similar results. The SFE of intact roots and flowers yielded 717 ± 31.3 and 480 ± 26.6 μg g^?1 α-T, respectively, while the leaves did not contain considerable amounts of thiophenes. Remarkable amounts of BBT, BBTOAc, and BBT(OAc)₂ were characteristic of the SFE of hairy root cultures.     

Enhanced Resveratrol Production in Vitis vinifera Cell Suspension Cultures by Heavy Metals Without Loss of Cell Viability

The effects of heavy metal ions (Co²⁺, Ag⁺, Cd²⁺) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.     

Strategies to Overcome Oxygen Transfer Limitations During Hairy Root Cultivation of Azadiracta indica for Enhanced Azadirachtin Production

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O₂ in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20–100% (v/v) O₂ in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O₂ in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3–2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1–4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day).     

The Expression Profiles of the Salvia miltiorrhiza 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase 4 Gene and Its Influence on the Biosynthesis of Tanshinones

Salvia miltiorrhiza is a medicinal plant that synthesises biologically-active tanshinones with numerous therapeutic properties. An important rate-limiting enzyme in the biosynthesis of their precursors is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). This study presents the organ-specific expression profile of the S. miltiorrhiza HMGR4 gene and its sensitivity to potential regulators, viz. gibberellic acid (GA₃), indole-3-acetic acid (IAA) and salicylic acid (SA). In addition, it demonstrates the importance of the HMGR4 gene, the hormone used, the plant organ, and the culture environment for the biosynthesis of tanshinones. HMGR4 overexpression was found to significantly boost the accumulation of dihydrotanshinone I (DHTI), cryptotanshinone (CT), tanshinone I (TI) and tanshinone IIA (TIIA) in roots by 0.44 to 5.39 mg/g dry weight (DW), as well as TIIA in stems and leaves. S. miltiorrhiza roots cultivated in soil demonstrated higher concentrations of the examined metabolites than those grown in vitro. GA₃ caused a considerable increase in the quantity of CT (by 794.2 µg/g DW) and TIIA (by 88.1 µg/g DW) in roots. In turn, IAA significantly inhibited the biosynthesis of the studied tanshinones in root material.     

Investigation of Tropane Alkaloids in Genetically Transformed <Emphasis Type="Italic">Atropa belladonna</Emphasis> L. Cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform – methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile – 30 mM phosphate buffer - methanol 12.2:79.7:8.1(v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K₅ (0,223 m/m%) and in hairy root clone #K₄ (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants.     

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0–4.0 μM) alone or in combination with IBA (0.2–0.6 μM) or 2, 4-D (0.5–1.5 μM). The optimum frequency (94 %) of callus induction was recorded on MS medium supplemented with 3.0 μM Kn and 0.4 μM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0–7.0 μM) of BA or TDZ alone or in combination with NAA (0.2–1.0 μm) was employed. The highest frequency of shoot regeneration (91 %) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 μM BA and 0.7 μM NAA. The shoots were excised and cultured on MS medium with 4.0 μM IBA produced 3.4 roots per shoot in 88 % cultures. Of the 65 plants transferred to soil 54 survived (83 %). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.     

GC–MS Studies of Thiophenes in the Supercritical Fluid CO2 and Solvent Extracts of Tagetes patula L.

The intact plant parts and genetically modified hairy root clone #TpA6 of Tagetes patula were extracted with supercritical fluid CO₂ extraction (SFE) and a conventional solvent extraction. SFE optimization included the variation of fluid CO₂ pressure, dynamic time, and the addition of methanol modifier co-solvent. The four characteristic thiophene metabolites, 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT), 2,2′:5′,2″-terthiophene (α-T), 5-(4-acetoxy-1-butynyl)-2,2′-bithienyl (BBTOAc), and 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl [BBT(OAc)₂], were analysed by GC–MS. The proposed SFE method allowed the selective extraction of thiophenes in 60 min dynamic time with supercritical CO₂ without modifier co-solvent, at 30 MPa and 40 °C. The SFE and the reference solvent extraction yielded similar results. The SFE of intact roots and flowers yielded 717 ± 31.3 and 480 ± 26.6 μg g⁻¹ α-T, respectively, while the leaves did not contain considerable amounts of thiophenes. Remarkable amounts of BBT, BBTOAc, and BBT(OAc)₂ were characteristic of the SFE of hairy root cultures.

     

The Antioxidant,Cytotoxic and Antimicrobial Potential of Phenolic Acids-Enriched Extract of Elicited Hairy Roots of Salvia bulleyana

Hairy root cultures are valuable sources of a range of phytochemicals. Among them, Salvia bulleyana root culture is a promising source of polyphenols, especially rosmarinic acid (RA), a phenolic acid depside with pleiotropic activity and a wide application in medicine and cosmetology. The aim of the study was to enhance the culture productivity by finding suitable elicitation protocol and to determine its biological potential in terms of antioxidant, anticancer and antimicrobial properties. The total content of phenols and the levels of particular constituents in root extracts were analyzed using HPLC-PDA. Among four elicitors tested (yeast extract; methyl jasmonate, MJA; trans-anethol; and cadmium chloride), MJA was found to be the most effective. The greatest boost in phenolic production (up to 124.4 mg/g dry weight) was observed after three-day treatment with MJA at 100 µM, with an almost 100% improvement compared to the controls (non-treated root culture). The hydromethanolic extract from the elicited culture exhibited strong antioxidant activity with IC₅₀ values of 11.1 µg/mL, 6.5 µg/mL and 69.5 µg/mL for DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)) and superoxide anion radical, respectively. Moreover, in concentrations of 0.5–5 mg/mL the extract inhibited the growth of LoVo, AGS and HeLa cell lines, but was safe for the L929 cells up to the concentration of 5 mg/mL. The extract also exhibited moderate antimicrobial activity. Thus, the results confirmed that elicitation can be a beneficial strategy for increase the phenolic acid biosynthesis in hairy roots of S. bulleyana, and that such a highly productive culture can show significant biological potential.     

Determination of lignans,phenolic acids and antioxidant capacity in transformed hairy root culture of Linum usitatissimum

Hairy root culture is a promising alternative method for the production of secondary metabolites. In this study, transformed root of Linum usitatissimum was established using Agrobacterium rhizogenes A4 strain from root cultures for lignans, phenolic acids and antioxidant capacity determination. Total lignin content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55.5% higher in transformed root cultures than in the non-transformed root culture. Secoisolariciresinol was detected in higher concentration (2.107 μmol/g DM) in the transformed root culture than non-transformed culture (1.099 μmol/g DM). Secoisolariciresinol diglucoside and matairesinol were exclusively detected in the transformed root culture, but were not found in the non-transformed root culture. The overall production of phenolic acids in transformed roots was approximately 3.5 times higher than that of the corresponding non-transformed culture. Free radical scavenging DPPH˙ and ABTS˙⁺ assays showed 2.9-fold and 1.76-fold higher anti-oxidant activity in transformed root culture as compared to non-transformed.     

Enhanced Production of Artemisinin by Hairy Root Cultivation of Artemisia annua in a Modified Stirred Tank Reactor

Artemisinin is an important drug commonly used in the treatment of malaria as a combination therapy. It is primarily produced by a plant Artemisia annua, however, its supply from plant is significantly lower than its huge demand and therefore alternative in vitro production routes are sought. Hairy root cultivation could be one such alternative production protocol. Agrobacterium rhizogenes was used to induce hairy roots of A. annua. Statistical optimization of media was thereafter attempted to maximize the biomass/artemisinin production. The growth and product formation kinetics and the significant role of O₂ in hairy root propagation were established in optimized media. Mass cultivation of hairy roots was, thereafter, attempted in a modified 3-L Stirred Tank Bioreactor (Applikon Dependable Instruments, The Netherlands) using optimized culture conditions. The reactor was suitably modified to obtain profuse growth of hairy roots by segregating and protecting the growing roots from the agitator rotation in the reactor using a perforated Teflon disk. It was possible to produce 18 g biomass L^?1 (on dry weight basis) and 4.63 mg L^?1 of artemisinin in 28 days, which increased to 10.33 mg L^?1 by the addition of elicitor methyl jasmonate.     

In vitro biological activity of aromadendrin-4′-methyl ether isolated from root extract of Ventilago madraspatana Gaertn with relevance to anticandidal activity

In this study, antimicrobial and antioxidant activities of the hexane extract of the root of Ventilago madraspatana were evaluated. Based on the significant bioactivity of crude hexane extract, an active compound was purified from the root extract. The active compound was further purified and identified as aromodendrin-4′-methyl ether by the ¹H NMR spectrum. The isolated compound significantly inhibited Staphylococcus epidermidis with the lowest MIC and MBC at 78 μg/mL (P < 0.05). The compound also exhibited significant anticandidal activity with MIC and MBC values of 312 and 625 μg/mL, respectively. The radical scavenging activity of aromodendrin-4′-methyl ether was evident by its lower IC₅₀ values of 60 μg/mL for DPPH scavenging and 3.2 μg/mL for ABTS scavenging. The compound also exhibited ferrous ion chelation and H₂O₂ scavenging activities. The study is an attempt to increase the industrial utility of V. madrasapatana.     

Rapid Determination of Cichoric Acid by Argentation Complex Chromatography and Study on Related Mechanisms of Coordination Simultaneously Used in Rapid Determination of Salvianolic Acid B

Cichoric acid is a water-soluble phenolic acid in Echinacea purpurea and has a high medicinal value. A rapid and novel argentation complex liquid chromatography method has been developed and validated for determination of cichoric acid in E. purpurea extract. The determination of cichoric acid was carried out on a Restek Pinnacle 11 C18 column (250 mm × 4.6 mm, 5 μm), using acetonitrile-deionized water (38:62, v/v, with 6 mmol L^?1 AgNO₃ and 0.8% acetic acid) as the mobile phase at a flow rate of 1.0 mL min^?1 within 10 min. The wavelength was set at 326 nm. It improved the effect of determination by adding AgNO₃ in the mobile phase while cichoric acid coordinated with Ag⁺. The mechanism of coordination between cichoric acid and Ag⁺ has been studied by Fourier transform infrared spectroscopy, electrospray ionization mass spectrometry. The mechanism which improved the effect of determination of cichoric acid is analyzed and applied in the rapid determination of salvianolic acid B (Sal B) in Danshen extract solution which has been stored for half a year. The possible structures of the complex and complex ratio are all provided in this article. The experiments have facilitated the study of cichoric acid–Ag⁺ and Sal B–Ag⁺ complex and provide a theoretical basis for industrialized extraction of cichoric acid and Sal B in the future.     

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.