Withania somnifera osmotin (WsOsm) confers stress tolerance in tobacco and establishes novel interactions with the defensin protein (WsDF).

Pathogenesis-related proteins (PR), including osmotins, play a vital role in plant defense, being activated in response to diverse biotic and abiotic stresses. Despite their significance, the mechanistic insights into the role of osmotins in plant defense have not been extensively explored. The present study explores the cloning and characterization of the osmotin gene (WsOsm) from Withania somnifera, aiming to illuminate its role in plant defense mechanisms. Quantitative real-time PCR analysis revealed significant induction of WsOsm in response to various phytohormones e.g. abscisic acid, salicylic acid, methyl jasmonate, brassinosteroids, and ethrel, as well as biotic and abiotic stresses like heat, cold, salt, and drought. To further elucidate WsOsm's functional role, we overexpressed the gene in Nicotiana tabacum, resulting in heightened resistance against the Alternaria solani pathogen. Additionally, we observed enhancements in shoot length, root length, and root biomass in the transgenic tobacco plants compared to wild plants. Notably, the WsOsm- overexpressing seedlings demonstrated improved salt and drought stress tolerance, particularly at the seedling stage. Confocal histological analysis of H2O2 and biochemical studies of antioxidant enzyme activities revealed higher levels in the WsOsm overexpressing lines, indicating enhanced antioxidant defense. Furthermore, a pull-down assay and mass spectrometry analysis revealed a potential interaction between WsOsm and defensin, a known antifungal PR protein (WsDF). This suggests a novel role of WsOsm in mediating plant defense responses by interacting with other PR proteins. Overall, these findings pave the way for potential future applications of WsOsm in developing stress-tolerant crops and improving plant defense strategies against pathogens.

Functional characterization of WsPR-1 reveals its interplay with cytokinin and gibberellin signaling pathways.

Pathogenesis-related protein 1 (PR-1) is an antimicrobial protein involved in systemic acquired resistance (SAR) in plants, but its regulatory role and interactions with other pathways remain unclear. In this study, we functionally characterize WsPR-1 gene of Withania somnifera in Nicotiana tabacum to elucidate its role in plant defense, growth, and development. Interestingly, transgenic tobacco plants with increased levels of cytokinin (CK) and decreased gibberellins (GAs) exhibited stunted shoot growth, an underdeveloped root system, modified leaf morphology, reduced seed pod production, and delayed leaf senescence. Transcriptional analysis revealed that WsPR-1 overexpression downregulated the GA 20-oxidase (GA20ox) gene involved in GA biosynthesis while upregulating GA 2-oxidase (GA2ox), a GA catabolic enzyme. Moreover, transcript levels of FRUITFULL (FUL) and LEAFY (NFL2) flowering genes exhibited a decrease in WsPR-1 plants, which could explain the delayed flowering and reduced seed pod development in transgenic plants. Confocal microscopy confirmed increased lignin deposition in stem cross-sections of WsPR-1 transgenic plants, supported by gene expression analysis and lignin content quantification. Additionally, our findings also suggest the involvement of Knotted1-like homeobox (KNOX) gene in enhancing cytokinin levels. This study highlights PR-1's regulatory role in plant growth and development, with potential to boost crop yields and enhance resilience.

Retron Library Recombineering: Next Powerful Tool for Genome Editing after CRISPR/Cas.

Retron library recombineering (RLR) is a powerful tool in the field of genome editing that exceeds the scope and specificity of the CRISPR/Cas technique. In RLR, single-stranded DNA produced in vivo by harnessing the in-built potential of bacterial retrons is used for replication-dependent genome editing. RLR introduces several genomic variations at once, resulting in pooled and barcoded variant libraries, thus permitting multiplexed applications. Retron-generated RT-DNA has already shown promise for use in genome editing. Thus, this new tool will result in fresh, intriguing, and surprising developments in molecular biology and its juncture with other disciplines of research, including medicine, agriculture, and microbiology. In this review, we discuss the current state of this brand-new tool that could eventually boost genome editing research.

Plant lectins: Classical molecules with emerging roles in stress tolerance.

Biotic and abiotic stresses impose adverse effects on plant's development, growth, and production. For the past many years, researchers are trying to understand the stress induced responses in plants and decipher strategies to produce stress tolerant crops. It has been demonstrated that molecular networks encompassing an array of genes and functional proteins play a key role in generating responses to combat different stresses. Newly, there has been a resurgence of interest to explore the role of lectins in modulating various biological responses in plants. Lectins are naturally occurring proteins that form reversible linkages with their respective glycoconjugates. To date, several plant lectins have been recognized and functionally characterized. However, their involvement in stress tolerance is yet to be comprehensively analyzed in greater detail. The availability of biological resources, modern experimental tools, and assay systems has provided a fresh impetus for plant lectin research. Against this backdrop, the present review provides background information on plant lectins and recent knowledge on their crosstalks with other regulatory mechanisms, which play a remarkable role in plant stress amelioration. It also highlights their versatile role and suggests that adding more information to this under-explored area will usher in a new era of crop improvement.

Physiological and molecular insight of microbial biostimulants for sustainable agriculture.

Increased food production to cater the need of growing population is one of the major global challenges. Currently, agro-productivity is under threat due to shrinking arable land, increased anthropogenic activities and changes in the climate leading to frequent flash floods, prolonged droughts and sudden fluctuation of temperature. Further, warm climatic conditions increase disease and pest incidences, ultimately reducing crop yield. Hence, collaborated global efforts are required to adopt environmentally safe and sustainable agro practices to boost crop growth and productivity. Biostimulants appear as a promising means to improve growth of plants even under stressful conditions. Among various categories of biostimulants, microbial biostimulants are composed of microorganisms such as plant growth-promoting rhizobacteria (PGPR) and/or microbes which stimulate nutrient uptake, produce secondary metabolites, siderophores, hormones and organic acids, participate in nitrogen fixation, imparts stress tolerance, enhance crop quality and yield when applied to the plants. Though numerous studies convincingly elucidate the positive effects of PGPR-based biostimulants on plants, yet information is meagre regarding the mechanism of action and the key signaling pathways (plant hormone modulations, expression of pathogenesis-related proteins, antioxidants, osmolytes etc.) triggered by these biostimulants in plants. Hence, the present review focuses on the molecular pathways activated by PGPR based biostimulants in plants facing abiotic and biotic challenges. The review also analyses the common mechanisms modulated by these biostimulants in plants to combat abiotic and biotic stresses. Further, the review highlights the traits that have been modified through transgenic approach leading to physiological responses akin to the application of PGPR in the target plants.

Engineering medicinal plant-derived CYPs: a promising strategy for production of high-valued secondary metabolites.

MAIN CONCLUSION: Cytochorme P450s (CYPs) play a critical role in the catalysis of secondary metabolite biosynthetic pathways. For their commercial use, various strategies for metabolic pathway engineering using CYP as a potential target have been explored. Plants produce a vast diversity of secondary metabolites which are being used to treat various ailments and diseases. Some of these metabolites are difficult to obtain in large quantities limiting their industrial use. Cytochrome P450 enzymes (CYPs) are important catalysts in the biosynthesis of highly valued secondary metabolites, and are found in all domains of life. With the development of high-throughput sequencing and high-resolution mass spectrometry, new biosynthetic pathways and associated CYPs are being identified. In this review, we present CYPs identified from medicinal plants as a potential game changer in the metabolic engineering of secondary metabolic pathways. We present the achievements made so far in enhancing the production of important bioactivities through pathway engineering, giving some popular examples. At last, current challenges and possible strategies to overcome the limitations associated with CYP engineering to enhance the biosynthesis of target secondary metabolites are also highlighted.

Biotechnological interventions in banana: current knowledge and future prospects.

Banana is an important food crop responsible for ensuring food security, nutrition, and employment for a significant portion of the world population. It has fairly broad genetic diversity and is distributed widely across the globe. Due to its socio-economic importance, there has been growing demand for healthy and improved planting materials of banana. In recent years many companies and organizations are working hard to narrow down the gap between demand and supply of quality planting materials. The other challenges includes its susceptibility to adverse environmental conditions, attack of various pests/pathogens and improvement of nutritional quality of bananas. To address these issues, refinement of existing techniques and introduction of new experimental tools are required. However, the genetic improvement of bananas to a large extent is limited by using conventional methods due to polyploidy, heterozygosity, and sterility of this plant. For rapid multiplication and obtaining disease free and healthy plants, efficient in vitro propagation techniques and fine tuning of the existing protocols are being tried in many laboratories across the globe. Besides, for developing a successful protocol for propagation of different cultivars of bananas, a deeper understanding of the factors associated with various steps of its multiplication till transfer to the land is immensely critical. Similarly, developing biotic and abiotic stress tolerant banana and enhancing its commercial value through biotechnological interventions could be very useful. The key intent of this review is to highlight the research endeavor in this direction, associated challenges and future prospects.

Autophagy: a game changer for plant development and crop improvement.

MAIN CONCLUSION: Manipulation of autophagic pathway represents a tremendous opportunity for designing climate-smart crops with improved yield and better adaptability to changing environment. For exploiting autophagy to its full potential, identification and comprehensive characterization of adapters/receptor complex and elucidation of its regulatory network in crop plants is highly warranted.  Autophagy is a major intracellular trafficking pathway in eukaryotes involved in vacuolar degradation of cytoplasmic constituents, mis-folded proteins, and defective organelles. Under optimum conditions, autophagy operates at a basal level to maintain cellular homeostasis, but under stressed conditions, it is induced further to provide temporal stress relief. Our understanding of this highly dynamic process has evolved exponentially in the past few years with special reference to several plant-specific roles of autophagy. Here, we review the most recent advances in the field of autophagy in plants and discuss its potential implications in designing crops with improved stress and disease-tolerance, enhanced yield potential, and improved capabilities for producing metabolites of high economic value. We also assess the current knowledge gaps and the possible strategies to develop a robust module for biotechnological application of autophagy to enhance bioeconomy and sustainability of agriculture.

OsSalT gene cloned from rice provides evidence of its role in salinity and drought stress tolerance.

Abiotic stresses impose a huge threat to agricultural productivity and global food security. To counter this challenge, the precise identification of the right candidate gene (s) for conferring abiotic stress tolerance without compromising the growth and yield is crucial. OsSalT is identified as a salt stress responsive gene located on SalTol QTL of chromosome 1 of rice, however, there is no genetic evidence of its function and probable pathway of its regulation. To get better insights into its functioning, earlier we elucidated the structure of SALT protein at atomic scale {PDB ID (5GVY)} and solution state that provided key clues on the probable mode of its action. Herein, we report the modulation of OsSalT gene in response to various factors and its functional characterization. Results indicate that OsSalT operates through both abscisic acid and gibberellic acid-dependent pathways and is linked to the adaptive stress mechanisms of plants. Its overexpression in a model plant resulted in improved salinity and drought stress tolerance. The OsSalT transformed plants also showed vigorous root growth, early flowering, and better seed germination. The triggering of multiple responses by OsSalT suggested that modulation of such mannose-binding lectin could be a potential game-changer for the improvement of many crops in future.

Genome Editing: A Promising Approach for Achieving Abiotic Stress Tolerance in Plants.

The susceptibility of crop plants towards abiotic stresses is highly threatening to assure global food security as it results in almost 50% annual yield loss. To address this issue, several strategies like plant breeding and genetic engineering have been used by researchers from time to time. However, these approaches are not sufficient to ensure stress resilience due to the complexity associated with the inheritance of abiotic stress adaptive traits. Thus, researchers were prompted to develop novel techniques with high precision that can address the challenges connected to the previous strategies. Genome editing is the latest approach that is in the limelight for improving the stress tolerance of plants. It has revolutionized crop research due to its versatility and precision. The present review is an update on the different genome editing tools used for crop improvement so far and the various challenges associated with them. It also highlights the emerging potential of genome editing for developing abiotic stress-resilient crops.

Critical factors influencing in vitro propagation and modulation of important secondary metabolites in Withania somnifera (L.) dunal.

Withania somnifera (L.) Dunal is a valuable medicinal plant in the Solanaceae family. It is commonly known as Ashwagandha and is widely distributed around the globe. It has multiple pharmacological properties owing to the existence of diverse secondary metabolites viz., withanolide A, withanolide D, withaferin A, and withanone. It is in great demand in the herbal industry because of its extensive use. In this background, the major challenge lies in the rapid multiplication of elite cultivars of W. somnifera in order to produce genetically and phytoconstituents uniform plant material for pharmaceutical industries. Thus it is necessary to explore various biotechnological approaches for the clonal mass propagation and synthesis of pharmaceutically important constituents in W. somnifera. Though there are several studies on in vitro propagation on W. somnifera, yet many factors that critically influence the in vitro response and withanolides production need to be fine-tuned in the pretext of the existing knowledge. The current review focuses on the advancements and prospects in biotechnological interventions to meet the worldwide demands for W. somnifera and its bioactive compounds. This update on in vitro studies on W. somnifera will be useful to many researchers, entrepreneurs, and herbal industries looking for its in vitro mass multiplication and scientific utilization.

Plant Secondary Metabolite Transporters: Diversity, Functionality, and Their Modulation.

Secondary metabolites (SMs) play crucial roles in the vital functioning of plants such as growth, development, defense, and survival via their transportation and accumulation at the required site. However, unlike primary metabolites, the transport mechanisms of SMs are not yet well explored. There exists a huge gap between the abundant presence of SM transporters, their identification, and functional characterization. A better understanding of plant SM transporters will surely be a step forward to fulfill the steeply increasing demand for bioactive compounds for the formulation of herbal medicines. Thus, the engineering of transporters by modulating their expression is emerging as the most viable option to achieve the long-term goal of systemic metabolic engineering for enhanced metabolite production at minimum cost. In this review article, we are updating the understanding of recent advancements in the field of plant SM transporters, particularly those discovered in the past two decades. Herein, we provide notable insights about various types of fully or partially characterized transporters from the ABC, MATE, PUP, and NPF families including their diverse functionalities, structural information, potential approaches for their identification and characterization, several regulatory parameters, and their modulation. A novel perspective to the concept of "Transporter Engineering" has also been unveiled by highlighting its potential applications particularly in plant stress (biotic and abiotic) tolerance, SM accumulation, and removal of anti-nutritional compounds, which will be of great value for the crop improvement program. The present study creates a roadmap for easy identification and a better understanding of various transporters, which can be utilized as suitable targets for transporter engineering in future research.

Morphological Analysis, Protein Profiling and Expression Analysis of Auxin Homeostasis Genes of Roots of Two Contrasting Cultivars of Rice Provide Inputs on Mechanisms Involved in Rice Adaptation towards Salinity Stress.

Plants remodel their root architecture in response to a salinity stress stimulus. This process is regulated by an array of factors including phytohormones, particularly auxin. In the present study, in order to better understand the mechanisms involved in salinity stress adaptation in rice, we compared two contrasting rice cultivars-Luna Suvarna, a salt tolerant, and IR64, a salt sensitive cultivar. Phenotypic investigations suggested that Luna Suvarna in comparison with IR64 presented stress adaptive root traits which correlated with a higher accumulation of auxin in its roots. The expression level investigation of auxin signaling pathway genes revealed an increase in several auxin homeostasis genes transcript levels in Luna Suvarna compared with IR64 under salinity stress. Furthermore, protein profiling showed 18 proteins that were differentially regulated between the roots of two cultivars, and some of them were salinity stress responsive proteins found exclusively in the proteome of Luna Suvarna roots, revealing the critical role of these proteins in imparting salinity stress tolerance. This included proteins related to the salt overly sensitive pathway, root growth, the reactive oxygen species scavenging system, and abscisic acid activation. Taken together, our results highlight that Luna Suvarna involves a combination of morphological and molecular traits of the root system that could prime the plant to better tolerate salinity stress.

Phytohormones: Key players in the modulation of heavy metal stress tolerance in plants.

Heavy metal (HM) stress in plants has received considerable global attention as it threatens sustainable growth in agriculture worldwide. Hence, desperate efforts have been undertaken for combating the effects of this stress in plants. Interestingly, the use of phytohormones in reducing the impact of HM toxicity has gained much momentum in the recent past. Phytohormones act as chemical messengers that improve the HM stress resistance in plants, thus allowing them to retain their growth and developmental plasticity. Their exogenous application as well as manipulation of endogenous levels through precise targeting of their biosynthesis/signaling components is a promising approach for providing a protective shield against HM stress in plants. However, for the successful use of phytohormones for field plants exposed to HM toxicity, in-depth knowledge of the key pathways regulated by them is of prime importance. Hence, the present review mainly summarizes the key conceptual developments on the involvement of phytohormones in the mitigation of HM stress in plants. The role of various genes, proteins, and signaling components involved in phytohormones associated HM stress tolerance and their modulation has also been discussed. Thus, this update will pave the way for improving HM stress tolerance in plants with the advent of phytohormones for sustainable agriculture growth in the future.

Structural insights into rice SalTol QTL located SALT protein.

Salinity is one of the major stresses affecting rice production worldwide, and various strategies are being employed to increase salt tolerance. Recently, there has been resurgence of interest to characterize SalTol QTL harbouring number of critical genes involved in conferring salt stress tolerance in rice. The present study reports the structure of SALT, a SalTol QTL encoded protein by X-ray crystallography (PDB ID: 5GVY; resolution 1.66 Å). Each SALT chain was bound to one mannose via 8 hydrogen bonds. Compared to previous structure reported for similar protein, our structure showed a buried surface area of 900 Å2 compared to only 240 Å2 for previous one. Small-angle X-ray scattering (SAXS) data analysis showed that the predominant solution shape of SALT protein in solution is also dimer characterized by a radius of gyration and maximum linear dimension of 2.1 and 6.5 nm, respectively. The SAXS profiles and modelling confirmed that the dimeric association and relative positioning in solution matched better with our crystal structure instead of previously reported structure. Together, structural/biophysical data analysis uphold a tight dimeric structure for SALT protein with one mannose bound to each protein, which remains novel to date, as previous structures indicated one sugar unit sandwiched loosely between two protein chains.

Physico-chemical characterization and topological analysis of pathogenesis-related proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches.

Plants are constantly under the threat of various biotic and abiotic stress conditions and to overcome these stresses, they have evolved multiple mechanisms including systematic accumulation of different phytohormones, phytoalexins and pathogenesis related (PR) proteins. PR proteins are cluster of proteins with low molecular weight which get incited in plants under different stresses. In this paper, in-silico approaches are used to compare the physico-chemical properties of 6 PR proteins (PR1, PR2, PR5, PR9, PR10, PR12) of Arabidopsis thaliana and Oryza sativa. Topological analysis revealed the presence of transmembrane localization of PR2 and absence of transmembrane domain in PR10 of both model plants studied. Amino acid composition shows the dominance of small aliphatic amino acids i.e. alanine, glycine and serine in both plants studied. These results highlights the similarities and differences between PRs of both model plants, which provides clue towards their diversified roles in plants.

Anti-neuroinflammatory potential of Tylophora indica (Burm. f) Merrill and development of an efficient in vitro propagation system for its clinical use.

Neuroinflammation is a major risk factor associated with the pathogenesis of neurodegenerative diseases. Conventional non-steroidal anti-inflammatory drugs are prescribed but their long term use is associated with adverse effects. Thus, herbal based medicines are attracting major attraction worldwide as potential therapeutic candidates. Tylophora indica (Burm. f) Merrill is a valuable medicinal plant well known in Ayurvedic practices for its immunomodulatory, anti-oxidant, anti-asthmatic and antirheumatic activities. The present study aimed to elucidate the anti-neuroinflammatory potential of water and hydroalcoholic leaf extracts of micropropagated plants of T. indica using BV-2 microglia activated with lipopolysaccharide as an in vitro model system and development of an efficient reproducible protocol for its in vitro cloning. Non cytotoxic doses of the water and hydroalcoholic extracts (0.2µg/ml and 20µg/ml, respectively) were selected using MTT assay. α-Tubulin, Iba-1 and inflammatory cascade proteins like NFκB, AP1 expression was studied using immunostaining to ascertain the anti-neuroinflammatory potential of these extracts. Further, anti-migratory activity was also analyzed by Wound Scratch Assay. Both extracts effectively attenuated lipopolysaccharide induced microglial activation, migration and the production of nitrite via regulation of the expression of NFκB and AP1 as the possible underlying target molecules. An efficient and reproducible protocol for in vitro cloning of T. indica through multiple shoot proliferation from nodal segments was established on both solid and liquid Murashige and Skoog's (MS) media supplemented with 15µM and 10µM of Benzyl Amino Purine respectively. Regenerated shoots were rooted on both solid and liquid MS media supplemented with Indole-3-butyric acid (5-15µM) and the rooted plantlets were successfully acclimatized and transferred to open field conditions showing 90% survivability. The present study suggests that T. indica may prove to be a potential anti-neuroinflammatory agent and may be further explored as a potential therapeutic candidate for the management of neurodegenerative diseases. Further, the current study will expedite the conservation of T. indica ensuring ample supply of this threatened medicinal plant to fulfill its increasing demand in herbal industry.

In silico insights on diverse interacting partners and phosphorylation sites of respiratory burst oxidase homolog (Rbohs) gene families from Arabidopsis and rice.

BACKGROUND: NADPH oxidase (Nox) is a critical enzyme involved in the generation of apoplastic superoxide (O2-), a type of reactive oxygen species (ROS) and hence regulate a wide range of biological functions in many organisms. Plant Noxes are the homologs of the catalytic subunit from mammalian NADPH oxidases and are known as respiratory burst oxidase homologs (Rbohs). Previous studies have highlighted their versatile roles in tackling different kind of stresses and in plant growth and development. In the current study, potential interacting partners and phosphorylation sites were predicted for Rboh proteins from two model species (10 Rbohs from Arabidopsis thaliana and 9 from Oryza sativa japonica). The present work is the first step towards in silico prediction of interacting partners and phosphorylation sites for Rboh proteins from two plant species. RESULTS: In this work, an extensive range of potential partners (unique and common), leading to diverse functions were revealed from interaction networks and gene ontology classifications, where majority of AtRbohs and OsRbohs play role in stress-related activities, followed by cellular development. Further, 68 and 38 potential phosphorylation sites were identified in AtRbohs and OsRbohs, respectively. Their distribution, location and kinase specificities were also predicted and correlated with experimental data as well as verified with the other EF-hand containing proteins within both genomes. CONCLUSIONS: Analysis of regulatory mechanisms including interaction with diverse partners and post-translational modifications like phosphorylation have provided insights regarding functional multiplicity of Rbohs. The bioinformatics-based workflow in the current study can be used to get insights for interacting partners and phosphorylation sites from Rbohs of other plant species.

In silico physicochemical characterization and topology analysis of Respiratory burst oxidase homolog (Rboh) proteins from Arabidopsis and rice.

NADPH oxidase (NOX) is a key enzyme involved in the production of apoplastic superoxide (O2-), a type of reactive oxygen species (ROS). Plant Noxes are the homologs of mammalian NADPH oxidase's catalytic subunit and are documented as respiratory burst oxidase homologs (Rbohs). A number of studies have reported their diverse functions in combating various stresses and in plant growth and development. In the present study, a total of 19 Rboh proteins (10 from Arabidopsis thaliana and 9 from Oryza sativa Japonica) were analyzed. We employed in silico approaches to compute the physiochemical properties (molecular weight, isoelectric point, total number of negatively and positively charged residues, extinction coefficient, half-life, instability and aliphatic index, grand average of hydropathicity, amino acid percentage). We observed a lot of variability in these parameters among the Rbohs accounting for their functional diversification. Their topological analysis, subcellular localization and signal peptide detection are also performed. To the best of our knowledge, the present study report on in silico physiochemical characterization, topology analysis, subcellular localization and signal peptide detection of Rboh proteins within two model plants. The study elucidates the variations in the key properties among Rbohs proteins, which may be responsible for their functional multiplicity.

Organic cultivation of Ashwagandha with improved biomass and high content of active Withanolides: Use of Vermicompost.

Withania somnifera (Ashwagandha) has recently been studied extensively for its health-supplementing and therapeutic activities against a variety of ailments. Several independent studies have experimentally demonstrated pharmaceutical potential of its active Withanolides, Withaferin A (Wi-A), Withanone (Wi-N) and Withanolide A (Wil-A). However, to promote its use in herbal industry, an environmentally sustainable cultivation and high yield are warranted. In modern agriculture strategies, there has been indiscriminate use of chemical fertilizers to boost the crop-yield, however the practice largely ignored its adverse effect on the quality of soil and the environment. In view of these, we attempted to recruit Vermicompost (Vcom, 20-100%) as an organic fertilizer of choice during the sowing and growing phases of Ashwagandha plants. We report that (i) pre-soaking of seeds for 12 h in Vermicompost leachate (Vcom-L) and Vermicompost tea (Vcom-T) led to higher germination, (ii) binary combination of pre-soaking of seeds and cultivation in Vcom (up to 80%) resulted in further improvement both in germination and seedling growth, (iii) cultivated plants in the presence of Vcom+Vcom-L showed higher leaf and root mass, earlier onset of flowering and fruiting and (iv) leaves from the Vcom+Vcom-L cultivated plants showed higher level of active Withanolides, Withanone (Wi-N), Withanolide A (Wil-A) and Withaferin A (Wi-A) and showed anticancer activities in cell culture assays. Taken together, we report a simple and inexpensive method for improving the yield and pharmaceutical components of Ashwagandha leaves.