Biotechnological interventions in Withania somnifera (L.) Dunal

Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant.     

Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation

Plant secondary metabolites, including pharmaceuticals, flavorings and aromas, are often produced in response to stress. We used chemical inducers of the pathogen defense response (jasmonic acid, salicylate, killed fungi, oligosaccharides and the fungal elicitor protein, cryptogein) to increase metabolite and biomass production in transformed root cultures of the medicinal plant, Withania somnifera, and the weed, Convolvulus sepium. In an effort to genetically mimic the observed effects of cryptogein, we employed Agrobacterium rhizogenes to insert a synthetic gene encoding cryptogein into the roots of C. sepium, W. somnifera and Tylophora tanakae. This genetic transformation was associated with stimulation in both secondary metabolite production and growth in the first two species, and in growth in the third. In whole plants of Convolvulus arvensis and Arabidopsis thaliana, transformation with the cryptogein gene led, respectively, to increases in the calystegines and certain flavonoids. A similar transgenic mimicry of pathogen attack was previously employed to stimulate resistance to the pathogen and abiotic stress. In the present study of biochemical phenotype, we show that transgenic mimicry is correlated with increased secondary metabolite production in transformed root cultures and whole plants. We propose that natural transformation with genes encoding the production of microbial elicitors could influence interactions between plants and other organisms.     

Microbial secondary metabolites ameliorate growth, <Emphasis Type="Italic">in planta</Emphasis> contents and lignification in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

In the present investigation, metabolites of Streptomyces sp. MTN14 and Trichoderma harzianum ThU significantly enhanced biomass yield (3.58 and 3.48 fold respectively) in comparison to the control plants. The secondary metabolites treatments also showed significant augmentation (0.75–2.25 fold) in withanolide A, a plant secondary metabolite. Lignin deposition, total phenolic and flavonoid content in W. somnifera were maximally induced in treatment having T. harzianum metabolites. Also, Trichoderma and Streptomyces metabolites were found much better in invoking in planta contents and antioxidants compared with their live culture treatments. Therefore, identification of new molecular effectors from metabolites of efficient microbes may be used as biopesticide and biofertilizer for commercial production of W. somnifera globally.     

Strategies to enhance biologically active-secondary metabolites in cell cultures of Artemisia – current trends

The genus Artemisia has been utilized worldwide due to its immense potential for protection against various diseases, especially malaria. Artemisia absinthium, previously renowned for its utilization in the popular beverage absinthe, is gaining resurgence due to its extensive pharmacological activities. Like A. annua, this species exhibits strong biological activities like antimalarial, anticancer and antioxidant. Although artemisinin was found to be the major metabolite for its antimalarial effects, several flavonoids and terpenoids are considered to possess biological activities when used alone and also to synergistically boost the bioavailability of artemisinin. However, due to the limited quantities of these metabolites in wild plants, in vitro cultures were established and strategies have been adopted to enhance medicinally important secondary metabolites in these cultures. This review elaborates on the traditional medicinal uses of Artemisia species and explains current trends to establish cell cultures of A. annua and A. absinthium for enhanced production of medicinally important secondary metabolites.     

In silico study of binding motifs in squalene synthase enzyme of secondary metabolic pathway solanaceae family

Solanaceae is an important family with several plants of medicinal importance. These medicinal plants have distinctive pathways for secondary metabolite biosynthesis. In most of the plants, two important compounds, dimethylallyl diphosphate and isopentenyl diphosphate, synthesize isoprenoid or terpenoids. Squalene synthase (SQS) is a key enzyme of the biosynthesis of isoprenoid (farnesyl pyrophosphate (FPP) → squalene). Withania somnifera (ashwagandha), an important medicinal plant of family solanaceae produces withanolides. Withanolides are secondary metabolites synthesized through isoprenoid pathway. In this study, 13 SQS protein sequences from the plants of solanacae family and Arabidopsis thaliana were analyzed. The conserved domains in corresponding sequences were searched. The multiple sequence alignment of conserved domains revealed the important motifs and identified the residue substitution in each motif. Our result further indicated that residue substitution in motifs might not lead to functional variation, although it may affect the binding affinity of Mg⁺⁺, FPP and NAD(P)H. In addition, the homology modelling of SQS enzyme of W. somnifera was done for the prediction of three-dimensional structure. Molecular docking study of considered substrates with WsSQS was performed and the docked structure were analyzed further. The docked structures showed binding affinity for motif 2 of WsSQS. Our analysis revealed that 29 residues of motif 2 might be important for catalytic/functional activity of SQS enzyme of W. somnifera. This study may provide an understanding of metabolic pathways responsible for the production of secondary metabolites. The motifs may play a key role in regulating the pathway towards enhanced production of metabolites.     

Nitric oxide elicitation for secondary metabolite production in cultured plant cells

Nitric oxide (NO) is an important signal molecule in stress responses. Accumulation of secondary metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. NO has been reported to play important roles in elicitor-induced secondary metabolite production in tissue and cell cultures of medicinal plants. Better understanding of NO role in the biosynthesis of such metabolites is very important for optimizing the commercial production of those pharmaceutically significant secondary metabolites. This paper summarizes progress made on several aspects of NO signal leading to the production of plant secondary metabolites, including various abiotic and biotic elicitors that induce NO production, elicitor-triggered NO generation cascades, the impact of NO on growth development and programmed cell death in medicinal plants, and NO-mediated regulation of the biosynthetic pathways of such metabolites. Cross-talks among NO signaling and reactive oxygen species, salicylic acid, and jasmonic acid are discussed. Some perspectives on the application of NO donors for induction of the secondary metabolite accumulation in plant cultures are also presented.     

Exogenous application of IAA alleviates effects of supplemental ultraviolet‐B radiation in the medicinal plant Withania somnifera Dunal

• Supplemental (s)‐UV‐B radiation has adverse effects on the majority of plants. The present study was conducted to evaluate the effects of exogenous application of the growth hormone indole acetic acid (IAA) on various morphological, physiological and biochemical characteristics of Withania somnifera, an indigenous medicinal plant, subjected to s‐UV‐B.
• The s‐UV‐B‐treated plants received ambient + 3.6 kJm^?2·day^?1 biologically effective UV‐B, and IAA was applied at two doses (200 and 400 ppm) to s‐UV‐B‐exposed plants.
• The plant was forced to compromise its growth, development and photosynthetic patterns to survive under s‐UV‐B by increasing concentrations of secondary metabolites and antioxidants (thiol, proline, ascorbic acid, α‐tocopherol, ascorbate peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, superoxide dismutase) to counteract oxidative stress. Increases in secondary metabolites were evidenced as increased activity of phenylpropanoid pathway enzymes: phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, 4‐coumarate CoA ligase, chalcone isomerase and dihydroflavonol reductase. Application of different IAA doses reversed the detrimental effects of s‐UV‐B on W. somnifera by improving growth and photosynthesis and reducing concentrations of secondary metabolites and non‐enzymatic antioxidants. Antioxidant enzymes, however, had a synergistic effect on s‐UV‐B treatment and IAA application.
• The effects of s‐UV‐B on W. somnifera are ameliorated to varying degrees upon exogenous IAA application, and synergistic enhancement of antioxidant enzymes under s‐UV‐B+IAA treatment might be responsible for the partial recuperation of growth and plant protein content, as a UV‐B‐exposed plant is forced to allocate most of its photosynthate towards production of enzymes related to antioxidant defence.

     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

A 12-deoxywithastramonolide-rich somaclonal variant in Withania somnifera (L.) Dunal—molecular cytogenetic analysis and significance as a chemotypic resource

Withania somnifera, commonly known as ashwagandha or Indian ginseng, is a valuable medicinal plant, synthesizing a wide array of pharmacologically active secondary metabolites known as withanolides. In this study, we investigated variation among 54 regenerated plants attained through indirect organogenesis from leaf explants. Organogenic calli were induced on Murashige and Skoog medium containing 2?mg?l^?1 kinetin and 1?mg?l^?1 indole-3-butyric acid. High-performance liquid chromatography was used for quantitative determination of the major withanolides in the somaclones. One somaclone (WS-R-1) showed significantly higher accumulation of 12-deoxywithastramonolide (WS-12D; 0.516%) compared to the explant donor mother plant (0.002%). The incidence of somaclonal variation at the cytological level was investigated by studying mitosis and meiosis in relation to chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic chromosome count, or meiotic behavior. Fidelity at genomic level was evaluated by random amplification of polymorphic DNA (RAPD) analyses, which revealed multiple genetic polymorphisms between the WS-12D over-producing somaclone and the explant donor mother plant. This study demonstrates the capability of inducing chemotypic variability for the development of high-yielding clones due to molecular instability in W. somnifera using an in vitro approach.     

Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites

The development of plant tissue (including organ and cell) cultures for the production of secondary metabolites has been underway for more than three decades. Plant cell cultures with the production of high-value secondary metabolites are promising potential alternative sources for the production of pharmaceutical agents of industrial importance. Medicinal plant cell suspension cultures (MPCSC), which are characterized with the feature of fermentation with plant cell totipotency, could be a promising alternative “chemical factory”. However, low productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and the application to large-scale production is still limited to a few processes. This review generalizes and analyzes the recent progress of this bioproduction platform for the provision of medicinal chemicals and outlines a range of trials taken or underway to increase product yields from MPCSC. The scale-up of MPCSC, which could lead to an unlimited supply of pharmaceuticals, including strategies to overcome and solution of the associated challenges, is discussed.     

红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展

紫杉醇是一种四环二萜酰胺类化合物,是从红豆杉科红豆杉属植物中提取分离出来的次生代谢物,是世界公认广谱、活性强的天然抗癌新药。但直接从植物中提取紫杉醇的传统生产方式,不仅产量低,且会对野生红豆杉资源造成严重破坏,同时紫杉醇的化学全合成也由于其结构复杂而不具备商业价值。与之相反,细胞培养技术具有受外界影响少、生产成本低、次生代谢产物多、细胞生长周期短的优势,是目前最具前景的紫杉醇生产方式。近年来随着科研水平的不断提升,紫杉醇无论在生理代谢调控、关键基因挖掘,还是新药物制剂与剂型及其类似物的开发和运用等方面,都取得了进展,但要建立紫杉醇商业化高产体系,还必须和前人的研究经验相结合。该文对红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展进行了综述,主要包括前人对红豆杉属植物组织与细胞培养相关的外植体、培养基、激素、培养条件、褐化等问题的研究,以及从代谢调节、培养方式、基因工程等多方面提高紫杉醇含量的最新进展,最后总结了当前研究的不足,并对今后通过多种组合方式来提高紫杉醇含量的生产途径进行了展望。以期促进红豆杉组织培养技术的进步,为药用资源保护和利用提供一定的理论基础与生产指导。     

Generation of Colors and Flavors in Plant Cell and Tissue Cultures

Plant cell and tissue cultures can be used for the synthesis and production of secondary metabolites like colors, flavors, and sweeteners. Most often, plant cell cultures fail to produce the desired products. In such cases strategies to improve the production of secondary metabolites must be considered.

Plant cell culture technology has now reached the point where a variety of culture types can be critically assessed as potential sources of existing and novel flavors and pigments. This brief review gives examples where progress has been made in the development of plant tissue culture systems.     

Adventitious Roots and Secondary Metabolism

Plants are a rich source of valuable secondary metabolites and in the recent years plant cell, tissue and organ cultures have been developed as an important alternative sources for the production of these compounds. Adventitious roots have been successfully induced in many plant species and cultured for the production of high value secondary metabolites of pharmaceutical, nutraceutical and industrial importance. Adoption of elicitation methods have shown improved synthesis of secondary metabolites in adventitious root cultures. Development of large-scale culture methods using bioreactors has opened up feasibilities of production of secondary metabolites at the industrial levels. In the present review we summarize the progress made in recent past in the area of adventitious root cultures for the production of secondary metabolites.     

Adventitious Roots and Secondary Metabolism

Plants are a rich source of valuable secondary metabolites and in the recent years plant cell, tissue and organ cultures have been developed as an important alternative sources for the production of these compounds. Adventitious roots have been successfully induced in many plant species and cultured for the production of high- value secondary metabolites of pharmaceutical, nutraceutical and industrial importance. Adoption of elicitation methods have shown improved synthesis of secondary metabolites in adventitious root cultures. Development of large-scale culture methods using bioreactors has opened up feasibilities of production of secondary metabolites at the industrial levels. In the present review we summarize the progress made in recent past in the area of adventitious root cultures for the production of secondary metabolites.     

Influence of abiotic stress signals on secondary metabolites in plants

Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and industrially important biochemicals. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Secondary metabolites play a major role in the adaptation of plants to the environment and in overcoming stress conditions. Environmental factors viz. temperature, humidity, light intensity, the supply of water, minerals, and CO₂ influence the growth of a plant and secondary metabolite production. Drought, high salinity, and freezing temperatures are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plant cell culture technologies have been effective tools for both studying and producing plant secondary metabolites under in vitro conditions and for plant improvement. This brief review summarizes the influence of different abiotic factors include salt, drought, light, heavy metals, frost etc. on secondary metabolites in plants. The focus of the present review is the influence of abiotic factors on secondary metabolite production and some of important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.     

Changing trends in biotechnology of secondary metabolism in medicinal and aromatic plants

Main conclusion

Medicinal and aromatic plants are known to produce secondary metabolites that find uses as flavoring agents, fragrances, insecticides, dyes and drugs. Biotechnology offers several choices through which secondary metabolism in medicinal plants can be altered in innovative ways, to overproduce phytochemicals of interest, to reduce the content of toxic compounds or even to produce novel chemicals. Detailed investigation of chromatin organization and microRNAs affecting biosynthesis of secondary metabolites as well as exploring cryptic biosynthetic clusters and synthetic biology options, may provide additional ways to harness this resource. Plant secondary metabolites are a fascinating class of phytochemicals exhibiting immense chemical diversity. Considerable enigma regarding their natural biological functions and the vast array of pharmacological activities, amongst other uses, make secondary metabolites interesting and important candidates for research. Here, we present an update on changing trends in the biotechnological approaches that are used to understand and exploit the secondary metabolism in medicinal and aromatic plants. Bioprocessing in the form of suspension culture, organ culture or transformed hairy roots has been successful in scaling up secondary metabolite production in many cases. Pathway elucidation and metabolic engineering have been useful to get enhanced yield of the metabolite of interest; or, for producing novel metabolites. Heterologous expression of putative plant secondary metabolite biosynthesis genes in a microbe is useful to validate their functions, and in some cases, also, to produce plant metabolites in microbes. Endophytes, the microbes that normally colonize plant tissues, may also produce the phytochemicals produced by the host plant. The review also provides perspectives on future research in the field.