Production of specialized metabolites in plant cell and organo-cultures: the role of gamma radiation in eliciting secondary metabolism.

PURPOSE: To provide an updated summary of recent advances in the application of gamma irradiation to elicit secondary metabolism and for induction of mutations in plant cell and organ cultures for the production of industrially important specialized metabolites (SMs). CONCLUSIONS: Research on the application of gamma radiation with plants has contributed a lot to microbial decontamination of seeds, and the promotion of physiological processes such as seed germination, seedling vigor, plant growth, and development. Various studies have demonstrated the influence of gamma rays on the morphology, physiology, and biochemistry of plants. Recent research efforts have also shown that low-dose gamma (5-100 Gy) irradiation can be utilized as an expedient solution to alleviate the deleterious effect of abiotic stresses and to obtain better yields of plants. Inducing mutagenesis using gamma irradiation has also evolved as a better option for inducing genetic variability in crops, vegetables, medicinal and ornamentals for their genetic improvement. Plant SMs are gaining increasing importance as pharmaceutical, therapeutic, cosmetic, and agricultural products. Plant cell, tissue, and organ cultures represent an attractive alternative to conventional methods of procuring useful SMs. Among the varied approaches the elicitor-induced in vitro culture techniques are considered an efficient tool for studying and improving the production of SMs. This review focuses on the utilization of low-dose gamma irradiation in the production of high-value SMs such as phenolics, terpenoids, and alkaloids. Furthermore, we present varied successful examples of gamma-ray-induced mutations in the production of SMs.

Suspension culture of somatic embryos for the production of high-value secondary metabolites.

Secondary metabolites from plants are ubiquitous and have applications in medicines, food additives, scents, colorants, and natural pesticides. Biotechnological production of secondary metabolites that have economic benefits is an attractive alternative to conventional methods. Cell, adventitious, and hairy root suspension cultures are typically used to produce secondary metabolites. According to recent studies, somatic embryos in suspension culture are useful tools for the generation of secondary metabolites. Somatic embryogenesis is a mode of regeneration in several plant species. This review provides an update on the use of somatic embryogenesis in the production of valuable secondary metabolites. The factors influencing the generation of secondary metabolites using somatic embryos in suspension cultures, elicitation methods, and prospective applications are also discussed in this review.

Bioreactor configurations for adventitious root culture: recent advances toward the commercial production of specialized metabolites.

In vitro plant cell and organ cultures are appealing alternatives to traditional methods of producing valuable specialized metabolites for use as: pharmaceuticals, food additives, cosmetics, perfumes, and agricultural chemicals. Cell cultures have been adopted for the production of specialized metabolites in certain plants. However, in certain other systems, adventitious roots are superior to cell suspension cultures as they are organized structures that accumulate high levels of specialized metabolites. The cultivation of adventitious roots has been investigated in various bioreactor systems, including: mechanically agitated, pneumatically agitated, and modified bioreactors. The main relevance and importance of this work are to develop a long-lasting industrial biotechnological technology as well as to improve the synthesis of these metabolites from the plant in vitro systems. These challenges are exacerbated by: the peculiarities of plant cell metabolism, the complexity of specialized metabolite pathways, the proper selection of bioreactor systems, and bioprocess optimization. This review's major objective is to analyze several bioreactor types for the development of adventitious roots, as well as the advantages and disadvantages of each type of bioreactor, and to describe the strategies used to increase the synthesis of specialized metabolites. This review also emphasizes current advancements in the field, and successful instances of scaled-up cultures and the generation of specialized metabolites for commercial purposes are also covered.

Bioreactor systems for micropropagation of plants: present scenario and future prospects.

Plant micropropagation has been adapted in the fields of agriculture, horticulture, forestry, and other related fields for large-scale production of elite plants. The use of liquid media and adoption of bioreactors have escalated the production of healthy plants. Several liquid-phase, gas-phase, temporary immersion, and other modified bioreactors have been used for plant propagation. The design, principle, operational mode, merits, and demerits of various bioreactors used for the regeneration of propagules, such as bulblets, cormlets, rhizomes, microtubers, shoots (subsequent rooting), and somatic embryos, are discussed here. In addition, various parameters that affect plant regeneration are discussed with suitable examples.

Advances in Biotechnological Production and Metabolic Regulation of Astragalus membranaceus.

Legume medicinal plants Astragalus membranaceus are widely used in the world and have very important economic value, ecological value, medicinal value, and ornamental value. The bioengineering technology of medicinal plants is used in the protection of endangered species, the rapid propagation of important resources, detoxification, and the improvement of degraded germplasm. Using bioengineering technology can effectively increase the content of secondary metabolites in A. membranaceus and improve the probability of solving the problem of medicinal plant resource shortage. In this review, we focused on biotechnological research into A. membranaceus, such as the latest advances in tissue culture, including callus, adventitious roots, hairy roots, suspension cells, etc., the metabolic regulation of chemical compounds in A. membranaceus, and the research progress on the synthetic biology of astragalosides, including the biosynthesis pathway of astragalosides, microbial transformation of astragalosides, and metabolic engineering of astragalosides. The review also looks forward to the new development trend of medicinal plant biotechnology, hoping to provide a broader development prospect for the in-depth study of medicinal plants.

Application of Data Modeling, Instrument Engineering and Nanomaterials in Selected Medid the Scientific Recinal Plant Tissue Culture.

At present, most precious compounds are still obtained by plant cultivation such as ginsenosides, glycyrrhizic acid, and paclitaxel, which cannot be easily obtained by artificial synthesis. Plant tissue culture technology is the most commonly used biotechnology tool, which can be used for a variety of studies such as the production of natural compounds, functional gene research, plant micropropagation, plant breeding, and crop improvement. Tissue culture material is a basic and important part of this issue. The formation of different plant tissues and natural products is affected by growth conditions and endogenous substances. The accumulation of secondary metabolites are affected by plant tissue type, culture method, and environmental stress. Multi-domain technologies are developing rapidly, and they have made outstanding contributions to the application of plant tissue culture. The modes of action have their own characteristics, covering the whole process of plant tissue from the induction, culture, and production of natural secondary metabolites. This paper reviews the induction mechanism of different plant tissues and the application of multi-domain technologies such as artificial intelligence, biosensors, bioreactors, multi-omics monitoring, and nanomaterials in plant tissue culture and the production of secondary metabolites. This will help to improve the tissue culture technology of medicinal plants and increase the availability and the yield of natural metabolites.

Production of anthraquinones from cell and organ cultures of Morinda species.

Since ancient times, Morinda species, particularly Morinda citrifolia, have been used for their therapeutic benefits. Iridoids, anthraquinones, coumarins, flavonoids, lignans, phytosterols, and carotenoids are examples of natural substances with bioactivity. Anthraquinone derivatives are the most significant of these chemicals since they are utilized as natural coloring agents and have a wide range of medicinal functions. Utilizing cell and organ cultures of Morinda species, various biotechnological methods have been developed for the bioproduction of anthraquinone derivatives. The generation of anthraquinone derivatives in cell and organ cultures is summarized in this article. The methods used to produce these chemicals in bioreactor cultures have also been examined. KEY POINTS: • This review investigates the potential of cell and organ cultures for anthraquinone synthesis. • The overproduction of anthraquinones has been addressed using a variety of techniques. • The use of bioreactor technologies for anthraquinone manufacturing is highlighted.

Anthocyanin Production from Plant Cell and Organ Cultures In Vitro.

Anthocyanins are water-soluble pigments found in plants. They exist in various colors, including red, purple, and blue, and are utilized as natural colorants in the food and cosmetics industries. The pharmaceutical industry uses anthocyanins as therapeutic compounds because they have several medicinal qualities, including anti-obesity, anti-cancer, antidiabetic, neuroprotective, and cardioprotective effects. Anthocyanins are conventionally procured from colored fruits and vegetables and are utilized in the food, pharmaceutical, and cosmetic industries. However, the composition and concentration of anthocyanins from natural sources vary quantitively and qualitatively; therefore, plant cell and organ cultures have been explored for many decades to understand the production of these valuable compounds. A great deal of research has been carried out on plant cell cultures using varied methods, such as the selection of suitable cell lines, medium optimization, optimization culture conditions, precursor feeding, and elicitation for the production of anthocyanin pigments. In addition, metabolic engineering technologies have been applied for the hyperaccumulation of these compounds in varied plants, including tobacco and arabidopsis. In this review, we describe various strategies applied in plant cell and organ cultures for the production of anthocyanins.

Evaluation of growth and some unexplored bioactivities of bioreactor grown adventitious root culture of ginseng (Panax ginseng C.A. Meyer).

The purpose of the present study was to evaluate the growth potential and some rarely reported bioactivities (antioxidant, thrombolytic, anticoagulant, and anthelmintic) of Panax ginseng C.A. Meyer adventitious roots. To demonstrate the growth, shake flask and laboratory-scale bioreactor cultures have been employed. The obtained biomass was dried and extracted with water, ethanol, and methanol. The growth ratio (12.62 ± 1.03) observed in the bioreactor was significantly higher than in the shake flask culture. The presence of 10 different phytochemical classes, including carbohydrates, saponins, glycosides, and terpenoids were detected in qualitative estimation. Significant quantities of phenolics, flavonoids, proteins, and tannins were determined. Dose-dependent antioxidant activities were observed, and the IC50 values of methanolic and ethanolic extracts were very similar to the standard. The highest (29.26 ± 5.31%) thrombolytic potential was shown by the methanolic extract. The ethanolic extract significantly extended the coagulation times up to 2.5 fold. The highest anthelmintic properties in terms of paralyzing (2.21 ± 0.31 min) and killing (3.69 ± 0.41 min) of the parasitic worms were displayed by the aqueous extract. The in vitro root growth implies the commercial feasibility of ginseng production in Bangladesh and the demonstration of potential bioactivities strengthens medicinal implications and also offering new research areas.

Anthraquinone Production from Cell and Organ Cultures of Rubia Species: An Overview.

The Rubia genus includes major groups of medicinal plants such as Rubia cordifolia, Rubia tinctorum, and Rubia akane. They contain anthraquinones (AQs), particularly alizarin and purpurin, which have pharmacological effects that are anti-inflammatory, antioxidant, anticancer, hemostatic, antibacterial, and more. Alizarin and purpurin have been utilized as natural dyes for cotton, silk, and wool fabrics since the dawn of time. These substances have been used in the cosmetics and food industries to color products. The amount of AQs in different Rubia species is minimal. In order to produce these compounds, researchers have established cell and organ cultures. Investigations have been conducted into numerous chemical and physical parameters that affect the biomass and accumulation of secondary metabolites in a cell, callus, hairy root, and adventitious root suspension cultures. This article offers numerous techniques and approaches used to produce biomass and secondary metabolites from the Rubia species. Additionally, it has been emphasized that cells can be grown in bioreactor cultures to produce AQs.

A comprehensive in silico exploration of pharmacological properties, bioactivities and COX-2 inhibitory potential of eleutheroside B from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim.

Eleutherococcus senticosus (Rupr. & Maxim.) Maxim., popularly known as 'Siberian ginseng', is an important medicinal plant. Pharmacologically active compounds of this plant are called eleutherosides and among them, eleutheroside B is the most prevalent. The E. senticosus has been reported to have many medicinal properties however; very few studies are reported to understand the medicinal properties of eleutheroside B. Consequently, in the present study various computational tools have been used to predict the drug-likeness, bioactivities, and pharmacokinetic properties of eleutheroside B. Besides, the inhibitory potential of eleutheroside B has been investigated against cyclooxygenase 2 (COX-2) enzyme. This study suggests that eleutheroside B is a drug-like compound with bioactivity score (-0.08 to 0.38), having satisfactory pharmacokinetic values. Metabolism and toxicities were further studied using FAME3, GLORY, pred-hERG and Endocrine Disruptome tools. No severe toxicities (Ames, hepatotoxicity, cardiotoxicity, skin sensitization) were predicted. Rat acute toxicity, ecotoxicity and cell line cytotoxicity were evaluated based on GUSAR and CLC-pred. The compound has been predicted as non-toxic (class 5), non-hERG inhibitor and less likely to cause adverse drug interactions. Molecular docking against COX-2 enzyme revealed strong hydrogen bonds (SER530, TYR355, LEU352, SER353, VAL349, TYR385, MET522) and hydrophobic interaction (LEU352) with eleutheroside B. The docking score (-6.97 kcal/mol) suggested that this molecule can be utilized as an anti-inflammatory agent as well as a potential anticancer drug in the future. Hence, this is a comprehensive integrated in silico approach to establish the anti-inflammatory mechanism of eleutheroside B in the background of its potential in future drug development.Communicated by Ramaswamy H. Sarma.

Attributes of Polygonum multiflorum to transfigure red biotechnology.

A vast array of plant-based compounds has enriched red biotechnology to serve the human health and food. A peculiar medicinal plant which was an element of traditional Chinese medicine for centuries as a liver and kidney tonic, for life longevity and hair blackening, is Polygonum multiflorum Thunb. (PM) which is popularly known as "He shou wu" or "Fo-ti" and is rich in chemical components like stilbenes, quinones, and flavonoids which have been used as anti-aging, anti-alopecia, anti-cancer, anti-oxidative, anti-bacterial, anti-hyperlipidemia, anti-atherosclerosis, and immunomodulating and hepatoprotective agents in the modern medicine. The health benefits from PM are attained since long through commercial products such as PM root powder, extract, capsules, tincture, shampoo, and body sprays in the market. Currently, the production of these pharmaceuticals and functional foods possessing stilbenes, quinones, and flavonoids is through cell and organ cultures to meet the commercial demand. However, hepatotoxic effects of PM-based products are the stumbling blocks for its long-term usage. The current review encompasses a comprehensive account of bioactive compounds of PM roots, their biological activities as well as efficacy and toxicity issues of PM ingredients and future perspectives.

Quality, safety and efficacy profiling of ginseng adventitious roots produced in vitro.

Ginseng (Panax ginseng C. A. Meyer, Family Araliaceae) is one of the major medicinal and nutraceutical plants, which is native to oriental region. It is used worldwide as a popular herbal medicine because of its pharmacological effects like anti-oxidative, anti aging, anti-cancer, adaptogenic, and other health-improving activities. Chief components of ginseng identified till date are ginsenosides, a group of saponins with triterpenoid structure. Ginseng is cultivated under controlled conditions, and for harvesting of fully grown roots of the plant, the cultivation takes long duration of about 5-7 years and cultivated ginseng roots are inferior in quality and ginsenoside content. Wild Mountain ginseng is superior in quality and ginsenoside content but is scarce in nature. Therefore, for obtaining the useful compounds of this plant at commercial scale, cell and organ cultures especially adventitious roots have been established by using superior clones of wild mountain ginseng, ginseng biomass is produced by applying large scale bioreactors. In this paper, an effort has been made to shed light on the scientific literature and to decipher the evidences for quality, safety, and efficacy of ginseng adventitious roots produced from in vitro cultures.

Biotic elicitation of ginsenoside metabolism of mutant adventitious root culture in Panax ginseng.

Biotic elicitation is an important biotechnological strategy for triggering the accumulation of secondary metabolites in adventitious root cultures. These biotic elicitors can be obtained from safe, economically important strains of bacteria found in the rhizosphere and fermented foods. Here, we assayed the effects of filtered cultures of five nitrogen-fixing bacteria and four types of fermentation bacteria on mutant adventitious Panax ginseng root cultures induced in a previous study by colchicine treatment. The biomass, pH, and electrical conductivity (EC) of the culture medium were altered at 5 days after treatment with bacteria. The saponin content was highest in root cultures treated with Mesorhizobium amorphae (GS3037), with a concentration of 105.58 mg g-1 dry weight saponin present in these cultures versus 74.48 mg g-1 dry weight in untreated root cultures. The accumulation of the ginsenosides Rb2 and Rb3 dramatically increased (19.4- and 4.4-fold, and 18.8- and 4.8-fold) 5 days after treatment with M. amorphae (GS3037) and Mesorhizobium amorphae (GS336), respectively. Compound K production increased 1.7-fold after treatment with M. amorphae (GS3037) compared with untreated root cultures. These results suggest that treating mutant adventitious root cultures with biotic elicitors represents an effective strategy for increasing ginsenoside production in Panax ginseng.

Improvement of biosynthesis and accumulation of bioactive compounds by elicitation in adventitious root cultures of Polygonum multiflorum.

We examined the effects of abiotic (methyl jasmonate [MeJA] and salicylic acid [SA]) and biotic (yeast extract and chitosan) elicitors for improvement of bioactive compounds production on adventitious root cultures in Polygonum multiflorum. The application of yeast extract resulted in significantly (p ≤ 0.05) higher dry root biomass (9.98 g/L) and relative growth rate versus the control. Cultures treated with abiotic elicitors showed higher percentage of dry weight than the other samples. Low concentrations of all elicitors (50 µM MeJA and SA, and 50 mg/L yeast extract) improved secondary metabolite production except for chitosan, whose performance was worse than that of the control. HPLC analysis of various bioactive compounds revealed significantly higher elicitation efficiency for MeJA than for the other treatments, with an approximately 2-fold increase in root dry weight (22.08 mg/g DW) under 50 µM MeJA treatment versus the control (10.35 mg/g DW). We also investigated the feasibility of scaling up the production process by comparing shake flask cultures with 3- and 5-L balloon type bubble bioreactors (BTBB) using 50 µM MeJA as an elicitor. Growth and metabolite accumulation increased in BTBB compared with shake flask cultures. We detected a non-significant difference in biomass productivity between 3 and 5-L BTBB, but the efficiency of bioactive compound accumulation decreased with increasing volume. These findings will be useful for developing a pilot-scale P. multiflorum adventitious root cultivation process for high biomass and bioactive compound production to meet the demands for natural ingredients by the pharmaceutical and cosmetic industries without affecting the natural habitat of this plant.

Panax ginseng Adventitious Root Suspension Culture: Protocol for Biomass Production and Analysis of Ginsenosides by High Pressure Liquid Chromatography.

Panax ginseng C.A. Meyer (Korean ginseng) is a popular herbal medicine. It has been used in Chinese and Oriental medicines since thousands of years. Ginseng products are generally used as a tonic and an adaptogen to resist the adverse influence of a wide range of physical, chemical and biological factors, and to restore homeostasis. Ginsenosides or ginseng saponins are the principal active ingredients of ginseng. Since ginseng cultivation process is very slow and needs specific environment for field cultivation, cell and tissue cultures are sought as alternatives for the production of ginseng biomass and bioactive compounds. In this chapter, we focus on methods of induction of adventitious roots from ginseng roots, establishment of adventitious root suspension cultures using bioreactors, procedures for processing of adventitious roots, and analysis of ginsenosides by high pressure liquid chromatography.

Anti-inflammatory potential of saponins derived from cultured wild ginseng roots in lipopolysaccharide-stimulated RAW 264.7 macrophages.

Ginseng, namely the root of Panax ginseng Meyer, is a well-known traditional medicine that has been used in Asian countries for thousands of years. Ginseng saponins have been shown to exert a variety of prominent pharmacological effects in a number of diseases. The aim of the present study was to identify the anti-inflammatory effects of total saponins extracted from cultured wild ginseng roots (TSWG) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. An elevated production of nitric oxide (NO) was detected in the RAW 264.7 cells in response to stimulation with LPS, as shown by NO detection assay using Griess reagent. However, pre-treatment with TSWG inhibited the production of NO through the suppression of inducible NO synthase gene expression. Furthermore, the LPS-induced gene expression and production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were significantly reduced by treatment with TSWG, as shown by ELISA, and western blot analysis and RT-PCR, respectively. In the LPS-stimulated RAW 264.7 cells, nuclear factor-κB (NF-κB) was translocated from the cytosol to the nucleus, while pre-treatment with TSWG induced the sequestration of NF-κB in the cytosol through the inhibition of the inhibitor of κB degradation, as shown by immunofluorescence staining. TSWG also contributed to the downregulation of mitogen-activated protein kinases and Akt in the LPS-stimulated RAW 264.7 cells. Additionally, in the TSWG-treated RAW 264.7 cells, we observed the activation of nuclear factor (erythroid-derived 2)-like 2 and an increase in heme oxygenase-1 expression; these effects were associated with the inhibition of the generation of reactive oxygen species. The results from the present study indicate that TSWG exerts anti-inflammatory and antioxidant effects, suggesting that TSWG may be an effective therapeutic agent for inflammatory diseases and prevent cellular damage induced by oxidative stress.

The safety assessment of food ingredients derived from plant cell, tissue and organ cultures: a review.

Plant cell, tissue and organ cultures (PCTOC) have become an increasingly attractive alternative for the production of various high molecular weight molecules which are used as flavourings, fragrances, colouring agents and food additives. Although PCTOC products are cultivated in vitro in a contamination free environment, the raw material produced from PCTOC may contain many components apart from the target compound. In some cases, PCTOC raw materials may also carry toxins, which may be naturally occurring or accumulated during the culture process. Assessment of the safety of PCTOC products is, therefore, a priority of the biotech industries involved in their production. The safety assessment involves the evaluation of starting material, production process and the end product. Before commercialisation, PCTOC products should be evaluated for their chemical and biological properties, as well as for their toxicity. In this review, measures and general criteria for biosafety evaluation of PCTOC products are addressed and thoroughly discussed.

Production of biomass and bioactive compounds from adventitious roots by optimization of culturing conditions of Eurycoma longifolia in balloon-type bubble bioreactor system.

The present study aimed to optimize the conditions for the production of adventitious roots from Eurycoma longifolia Jack, an important medicinal woody plant, in bioreactor culture. The effects of the type and concentration of auxin on root growth were studied, as well as the effects of the NH4(+):NO3(-) ratio on adventitious root growth and the production of phenolics and flavonoids. Approximately 5 g L(-1) fresh weight of adventitious roots was inoculated into a 3 L balloon-type bubble bioreactor, which contained 2 L 3/4 MS medium supplemented with 30 g L(-1) sucrose and cultures were maintained in the dark for 7 weeks at 24 ± 1°C. Higher concentrations of IBA (7.0 and 9.0 mg L(-1)) and NAA (5.0 mg L(-1)) enhanced the biomass and accumulation of total phenolics and flavonoids. The adventitious roots were thin, numerous, and elongated in 3/4 MS medium supplemented with 5.0 and 7.0 mg L(-1) IBA, whereas the lateral roots were shorter and thicker with 5.0 mg L(-1) NAA compared with IBA treatment. The optimum biomasses of 50.22 g L(-1) fresh weight and 4.60 g L(-1) dry weight were obtained with an NH4(+):NO3(-) ratio of 15:30. High phenolic and flavonoid productions (38.59 and 11.27 mg L(-1) medium, respectively) were also obtained with a ratio of 15:30. Analysis of the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging activity indicated higher antioxidant activity with an NH4(+):NO3(-) ratio of 30:15. These results suggest that balloon-type bubble bioreactor cultures are suitable for the large-scale commercial production of E. longifolia adventitious roots which contain high yield of bioactive compounds.

Hypericins: biotechnological production from cell and organ cultures.

Hypericum perforatum L. (St. John's wort), a perennial flowering plant native to Europe, is widely used as a medicinal plant and has a long history of its use in the treatment of various ailments. Currently, H. perforatum is widely used as an herbal remedy for the treatment of mild to moderate depression. Hypericins are natural napthodianthrone compounds produced from H. perforatum (St. John's wort) which are having antitumor, antiviral (i.e., against human immunodeficiency and hepatitis C virus), antineoplastic, and antidepressant properties. Currently, field-grown plant materials are generally used for the commercial production of hypericins. It has been reported that hypericin accumulation in natural plants is influenced by different ecological and environmental conditions including light intensity, nitrogen availability, temperature, seasons, and growing regions. Therefore, up to 17-fold and 13-fold differences in hypericin and pseudohypericin amounts, respectively, are reported in different phytopharmaceutical preparations. Plant cell and organ cultures are effective systems for producing natural products, and attempts were made for the production of biomass and stable concentrations of hypericins through in vitro cultures of H. perforatum. Cell, callus, shoot, plantlet, and adventitious root cultures have been established and various chemical and physical factors which influence the biomass and secondary metabolite accumulation have been investigated. Large-scale plantlet and adventitious root cultures have also been attempted in H. perforatum in bioreactors, and various strategies have been applied for the production of higher biomass and secondary products. This review describes the biotechnological approaches employed for the production of hypericins and focuses upon the challenges and future prospects.