Enhancement Of Secondary Metabolite Production Research Articles

Influence of gamma radiation and phenylalanine on secondary metabolites in callus cultures of milk thistle (Silybum marianum L.)

BackgroundA useful technique for growing large amounts of plant material is in vitro propagation of important medicinal plants. The present investigation deals with the enhancement of secondary metabolite production via elicitation using gamma (γ)-radiation and phenylalanine (Phe) precursor feeding in callus cultures of Silybum marianum L. ResultsSeeds were exposed to two doses of γ-radiation (25 and 50 Gy) and the calli derived from stem explants obtained from seedlings of these radiated seeds were treated with different concentrations of Phe. The biosynthesis of phenols and flavonoids was evaluated. It was found that callus cultures derived from explants of the seeds exposed to 25 Gy γ-radiation and treated with 4 mg/l Phe accumulated the maximum phenolic content (34.27±0.02 mg/g d.wt.), while the highest flavonoid content (9.56±0.12 mg/g d.wt.) was found in callus cultures derived from explants of seeds radiated with 25 Gy γ-radiation and subjected to 1 mg/l Phe. Similarly, HPLC quantification revealed that the production of flavonoids was highly accumulated (1343.06 μg/mg d.wt.) in callus cultures from explants of seeds exposed to 25 Gy γ-radiation and grown at 1 mg/l Phe compared to the other treatments. In addition, a total of 11 important flavonoids have been determined in all callus cultures, except for acacetin-7-O-rutinoside, which was not found in the callus culture of the control. ConclusionsThese findings suggest that γ-radiation combined with Phe can improve the metabolism of S. marianum L. and could be used to produce such valuable metabolites on a commercial scale.

Responses of in vitro cell cultures to elicitation: regulatory role of jasmonic acid and methyl jasmonate: a review

Plants are regarded as resources to obtain various industrially important compounds such as pharmaceutical, agrochemical and aromatic products which are produced in their roots, stem, leaves and other aerial parts and mostly extracted from field-grown plants. However, the over-exploitation of plants for secondary metabolites often leads to becoming endangered and in threat of extinction. Owing to these constraints, in vitro culture techniques provide an alternative to overcome problems associated with field production and promotes their conservation. The use of elicitation for the enhancement of secondary metabolite production is gaining importance. Elicitation is defined as an increased production of biomass and secondary metabolites in both in vitro and ex vivo grown plants, subjected to the exposure of different elicitors. The process of elicitation is influenced by culture conditions like elicitor concentration, duration, age and composition of culture. So, this review emphasises the role of these in vitro culture conditions on secondary metabolite production and to pave way for their enhanced in vitro production. Also discussed are various elicitor responses on different biochemical and molecular pathways to elucidate the mechanism of action of jasmonic acid and methyl jasmonate in secondary metabolite production.

Modulation of steroidal glycoalkaloid biosynthesis in tomato (Solanum lycopersicum) by jasmonic acid

Jasmonic acid (JA) is a phytohormone involved in plant development and defense. A major role of JA is the enhancement of secondary metabolite production, such as response to herbivory. Systemin is a bioactive plant peptide of 18 amino acids that contributes to the induction of local and systemic defense responses in tomato (Solanum lycopersicum) through JA biosynthesis. The overexpression of systemin (PS-OE) results in constitutive JA accumulation and enhances pest resistance in plants. Conversely, mutant plants affected in linolenic acid synthesis (spr2) are negatively compromised in the production of JA which favors damage and oviposition by insect herbivores. With undirected mass fingerprinting analyses, we found global metabolic differences between genotypes with modified jasmonic acid production. The spr2 mutants were enriched in di-unsaturated fatty acids and generally showed more changes. The PS-OE genotype produced an unidentified compound with a mass-to-charge ratio of 695 (MZ695). Most strikingly, the steroidal glycoalkaloid biosynthesis was negatively affected in the spr2 genotype. Complementation with jasmonic acid could restore the tomatine pathway, which strongly suggests the control of steroidal glycoalkaloid biosynthesis by jasmonic acid. spr2 plants were more susceptible to fungal infection with Fusarium oxysporum f.sp. ciceris, but not to bacterial infection with Clavibacter michiganensis subsp. michiganensis which supports the involvement of steroidal glycoalkaloids in the plant response against fungi.

Diluted Fungal Phytotoxin: An Immerging tool for Boosting Innate Immunity in Plants

Fungal pathogens produce an array of toxin with various chemical structures, specificity, mechanism of action and functions. In recent years fungal toxins have been intensively studied, mainly for enhancing innate immunity in plants. Innate immunity is the only line of defense in plants, whose elicitation by toxin at optimum dilution may trigger the accumulation of various bioactive molecules such as defense enzymes, pathogenesis related proteins and secondary metabolite. Production of these defense related molecules may pave the way for the use of toxic metabolite to counter disease in plants, constituting a potential and cost effective alternative for toxic fungicides. Furthermore, this simple methods might help in enhancement of secondary metabolite production in medicinal plants which eventually be applied in the pharmaceutical industry. Henceforth the present review focus on the role of fungal phytotoxin as an elicitor triggering innate immune responses in plants.

Elicitation: a stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production

Higher plants undergo a variety of stresses and to combat those stresses they acclimatize themselves by producing diverse secondary metabolites. These secondary metabolites also have a wide range of industrial applications and hence they serve as candidates for commercialization. Owing to the constraints faced by natural plant extraction, plant cell/tissue culture has emerged as an alternative platform for the in vitro production of value added bioactive secondary metabolites. Implementation of several productivity enhancement strategies, including elicitation, can overcome the limitations faced by plant cell technology that hampers its extensive commercialization. Elicitation is a technique that involves exogenous addition of elicitors (abiotic or biotic) in the growth medium which consequently triggers stress response with concomitant enhancement in secondary metabolite production. Elicitor induced stress results in the activation of several defense-related genes or inactivation of non-defense-related genes, transient phosphorylation/dephosphorylation of proteins, expression of enzymes whose information can be used to ascertain the biosynthetic pathways of many secondary metabolites. Furthermore, integration of transcriptomics, proteomics and metabolomics with system biology can aid in discovery of novel genes, transcriptional factors and several biosynthetic pathways which in turn can serve as a valuable tool for metabolic engineering and gene manipulation for enhancing the yield and productivity of secondary metabolites.

Elicitor-Induced Cellular and Molecular Events Are Responsible for Productivity Enhancement in Hairy Root Cultures: An Insight Study

A wide range of external stress stimuli triggers a plant cell to undergo a complex network of reactions that ultimately lead to the synthesis and accumulation of secondary metabolites. These secondary metabolites help the plant to survive under stress challenge. The potential of biotic and abiotic elicitors for the induction and enhancement of secondary metabolite production in various culture systems including hairy root (HR) cultures is well-known. The elicitor-induced defense responses involves signal perception of elicitor by a cell surface receptor followed by its transduction involving some major cellular and molecular events including activation of major secondary message signaling pathways. This result in induction of gene expressions escorting to the synthesis of various proteins mainly associated with plant defense responses and secondary metabolite synthesis and accumulation. The review discusses the elicitor-induced various cellular and molecular events and correlates them with enhanced secondary metabolite synthesis in HR systems. Further, this review also concludes that combining elicitation with in-silico approaches enhances the usefulness of this practice in better understanding and identifying the rate-limiting steps of biosynthetic pathways existing in HRs which in turn can contribute towards better productivity by utilizing metabolic engineering aspects.

Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre

Elicitation is one of the few strategies that find commercial application in the enhancement of secondary metabolite production from plants as well as cell culture systems. Due to their immense medicinal value, production of saponins in suspension cultures has been attempted by many researchers. Gymnema sylvestre is a rich source of gymnemic acids (saponins) that find application in the treatment of diabetes. The present study is an attempt to evaluate the effect of various metal salts (cadmium chloride, mercuric chloride, silver nitrate, cupric chloride, cobaltous chloride and calcium chloride) in eliciting the response from G. sylvestre suspension cultures. The maximum gymnemic acid production in the suspensions was achieved on day 12 of culture, though the maximum biomass was obtained on day 16. Among the different salts, CdCl(2) gave maximum response (59.97mg/gDCW) at 2mM concentration after a 24h time period, while, AgNO(3) gave the least response (18.35mg/gDCW) on incubation of 48h at 1mM concentration, in terms of gymnemic acid accumulation. The accumulation of gymnemic acid was found to be dependent on treatment time and concentration of the elicitor. The enhanced gymnemic acid production shown by the suspensions in response to the metal salts indicates their role in evoking the plant defense mechanisms. These elicitation studies help in providing a platform for improved commercial supply of bioactive gymnemic acids.

Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.

A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures.

Plant cell and tissue cultures can be established routinely under sterile conditions from explants, such as plant leaves, stems, roots, meristems etc for both the ways for multiplication and extraction of secondary metabolites. Strain improvement, methods for the selection of high-producing cell lines, and medium optimizations can lead to an enhancement in secondary metabolite production. However, most often trials with plant cell cultures fail to produce the desired products. In such cases, strategies to improve the production of secondary metabolites must be considered. One of the main problems encountered is the lack of basic knowledge of the biosynthetic routes, and mechanisms responsible for the production of plant metabolites. Where the productivity of the desired metabolites is limited by the lack of particular precursors, biotransformation using an exogenous supply of biosynthetic precursors, genetic manipulation and metabolic engineering may improve the accumulation of compounds. Feedback inhibition of metabolic enzymes as well as inhibition of membrane transport can be eliminated by the accumulation of synthesized products in a second phase introduced into the aqueous medium. Organ cultures and in vitro biomass production often have sites of synthesis and storage of secondary metabolites in separate compartments. Elicitors, compounds triggering the formation of secondary metabolites, can be abiotic or biotic. Natural elicitors include polysaccharides such as pectin and chitosan, which are also used in the immobilization and permeabilization of plant cells. Immobilization with suitable bioreactor system provides several advantages, such as continuous process operation, but for the development of an immobilized plant cell culture process, natural or artifically induced secretion of the accumulated product into the surrounding medium is necessary. The present review highlights the nature, applications, perspective and scale up methods for the production of valuable secondary metabolites in vitro. Key words: Plant cell cultures, secondary metabolites, biotransformation, biosynthetic pathways, elicitation, immobilization, bioreactors.

Gene-expression analysis of acidic pH shock effects on two-component systems in Streptomyces coelicolor

In this study, transcriptional analyses by using reverse transcription-polymerase chain reaction (RT-PCR), DNA chip, and quantitative real time-PCR were performed to investigate effects of acidic pH shock on two-component systems in Streptomyces coelicolor A3(2). Two-component systems of cseC/cseB and vanS/vanR known to be closely linked with self-protection against cell wall hydrolysis caused by external stimuli were upregulated by the pH shock. The chiS/chiR, afsQ2/afsQ1, ecrA2/ecrA1, bldM, ramC/ramR, and ragK/ragR known to be positively associated with the initiation of secondary metabolism were also upregulated. The cutS/cutR known to be negatively related to the secondary metabolism was, however, slightly downregulated. Upregulation or downregulation by the acidic pH shock of these two-component regulator systems might have contributed in a concerted manner to the enhancement of secondary metabolite production, at least, in this particular case of actinorhodin production in S. coelicolor A3(2).

Role of biotechnology in medicinal plants

Medicinal plants are the most important source of life saving drugs for the majority of the world's population. The biotechnological tools are important to select, multiply and conserve the critical genotypes of medicinal plants. In-vitro regeneration holds tremendous potential for the production of high-quality plant-based medicine. Cryopreservation is long-term conservation method in liquid nitrogen and provides an opportunity for conservation of endangered medicinal plants. In-vitro production of secondary metabolites in plant cell suspension cultures has been reported from various medicinal plants. Bioreactors are the key step towards commercial production of secondary metabolites by plant biotechnology. Genetic transformation may be a powerful tool for enhancing the productivity of novel secondary metabolites; especially by Agrobacterium rhizogenes induced hairy roots. This article discusses the applications of biotechnology for regeneration and genetic transformation for enhancement of secondary metabolite production in-vitro from medicinal plants. Key words: Bioreactors; genetic transformation; regeneration; secondary metabolites Abbreviations: BA: 6-Benzylaminopurine; TDZ: 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea; NAA: a-Naphthaleneacetic acid; IAA: Indole-3 acetic acid; 2iP: 6-(g-Dimethylallylamino) purine; 2,4-D: 2,4-Dichlorophenoxyacetic acid; GA3: Gibberellic acid Trop J Pharm Res , December 2003; 2(2): 243-253

Immobilisation of Solanum chrysotrichum plant cells withinCa-alginate gel beads to produce an antimycotic spirostanol saponin

Plant cell cultures of the Mexican species Solanum chrysotrichum can be established from friable calli. These cells produce an antifungal spirostanol saponin designated SC1. Plant cell immobilisation within Ca-alginate gel beads can lead to an enhancement in secondary metabolite production. Different matrices, obtained by varying calcium (0.1 to 0.8 mol·L –1) and alginate (1 to 1.5 % w/v) concentrations were used to study the influence of immobilisation parameters on the behaviour of S. chrysotrichum cells. Throughout the growth cycle and in the various immobilisation conditions, fresh and dry weight, SC1 yield and viability were measured. Important metabolic, morphological and physiological modulation seemed to be generated by the gel bead networks and by the cell-cell interactions inherent to the microenvironmental constraints. An accumulation of starch grains was observed through amyloplasts. Immobilised S. chrysotrichum cells accumulated 5 mg·g –1 dry weight of SC1. Although this was four times less than the free cells, the desired metabolite was excreted into the surrounding medium (around 40 % of the total production).

Enhancement of Secondary Metabolite Production by Immobilized Gossypium arboreum Cells

AbstractA novel reactor with simultaneous use of plant cell immobilization, permeabilization, and elicitation was designed to enhance the productivity of secondary metabolites and to overcome limitations of plant cell culture, such as slow metabolic rate, low product yield, and sensitivity to shear stresses. Production of gossypol, an antifungal agent, from Gossypium arboreum was selected as a model system. The effects of immobilization, permeabilization, and elicitation on gossypol production were investigated in free suspension culture, immobilized plant cell reactor with recycled batch operation, and bioreactor with continuous operation. G. arboreum cells were effectively immobilized on a cotton matrix by entrapment with a spirally wound configuration. Permeabilization of immobilized cells provided a tool for a continuous process increasing the release of gossypol by 30%, while the elicitation technique played a major role to enhance productivity by more than 8‐fold. In comparisons of productivity, continuous operation and combined treatment gave productivity over 20‐fold higher than batch culture. Simultaneous use of immobilization, permeabilization, and elicitation was possible in continuous operation with an immobilized plant cell reactor.