Withania somnifera

[1] [2] [3] [4] [5] [6] [7] [8]

Propagation

[9]

Germination

Recommended Protocol

1. Soak seeds in 1% potassium nitrate + 10ppm GA3 solution for 24 hours at 25-30°C in light
2. Sow seeds in deep, nutrient-rich seed starting mix at 0.5-1cm depth
3. Emergence in 5-10 days Transplant to the field in 4-6 weeks; 5-8 cm tall plants

Pretreatment with 150ppm GA3 (no specific protocol given) can increase germination rate and speed compared to higher concentrations of GA3, physical scarification, other growth regulators, and controls. The effect of GA3 treatment was even more pronounced at 25°C where the germination rate reached 98% compared to only 48% at 20°C. Though there were differences between accessions, the effect of GA3 treatment was significant.[10] Similarly, soaking seeds for 24 hours in 0.25-1 ppm resulted in a slight increase in germination rates and a reduction in mean germination time compared to no pretreatment.[11]

Heat treatment (50°C) for 5-15 min resulted in a massive decline of germination rates in petri dish seed tests. However, sowing the seeds in soil substantially mitigated the effect.[11]

Germination in the soil is roughly equivalent to germination on filter paper in Petri dishes.[11]

Somnifera seeds are commonly direct-sown in fields, sometimes mixed with sand (1:2) to ease hand distribution.[12][13][14] The practice is so common that studies sometimes use seed application rates (kg/ha) instead of planting densities (plants/m2) or row/plant spacing.[15]

Presoaking seeds for 24 hours in vermicompost and vermicompost inclusion in the starter mix significantly increases the medicinal constituents 60 days after sowing.[16] The effect may be due to the effect of dissolved salts: Presoaking seeds with various nitrate salts for 24 hours significantly increases emergence rates and seedling vigor of somnifera. The effect on vigor persists through harvest at 110 DAS. Zinc nitrate at 0.5% and sodium/potassium nitrate at 1.0% concentrate was particularly effective.[17] The effect of vermicompost tea/leachate on seed germination is less pronounced and more heterogeneous, however.[18]

Germination rates of seeds infected with fungi are lower than noninfected seeds, though they are not completely lost.[19]

[20] [10] [13] [12] [14] [21] [22] [23] [16] [15] [18] [11] [19] [17] [24] [25] [26] [27]

Vegetative

In-Vitro

[28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [9] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84]

Cultivation

[97] [98] [99] [87] [100] [91] [13] [90] [12] [101] [98] [94] [95]

Harvest

The leaves of somnifera will turn yellow to indicate the appropriate time to harvest. The pods and berries will dry out starting at the base of the plant.[102] The harvest period is mostly dictated by the weather in colder climates: harvesting is best done before the first frost.[103]

Harvesting can be done with digging implements in dry conditions[102] or by flooding and pulling [104].

[99] [105] [106] [14] [88] [107] [16]

Yield

Mechanical deflowering significantly improves withanolide yield.[121]

Root yield and quality are not linear during the growing season.[88]

Root yield is positively correlated with tillage depth. It is suggested that the change in morphology to favor deep tap roots and fewer, but thicker, lateral roots drove the increased yield in deep-tilled soil.[12]

In a comparison of yield versus planting density at 20, 40, 60, and 80 plants per square meter, maximum seed yield was obtained at 40 while maximum root yield was obtained at 60 plants per square meter.[12] Similarly, yield increased linearly from 11-33 plants per square meter without a detectable decrease in yield at the higher densities.[14]

There is a large difference in the yield of cultivated versus wild accessions.[91]

Elevated CO2 at 600ppm and 800ppm significantly increased organic carbon and biomass in leaves, stems, and roots of somnifera compared to ambient.[118]

[113] [98] [122] [108] [87] [100] [123] [114] [124] [85] [88] [125] [89] [115] [126] [13] [90] [12] [14] [101] [109] [110] [111] [98] [22] [116] [107] [16] [15] [112] [24] [17] [127] [91] [118] [25] [93] [94] [95] [96]

Soilless

[128] [114] [79] [12] [14]

Soil

[108] [129] [130] [20] [131] [85] [132] [133] [127] [81] [109] [110] [111] [22] [116] [23] [107] [16] [15] [112] [17] [25] [93] [94] [95] [96]

Fertilization

[113] [134] [135] [108] [136] [137] [129] [138] [139] [140] [141] [142] [143] [120] [144] [118] [86] [145] [130] [146] [147] [148] [49] [149] [124] [150] [132] [151] [133] [127] [89] [90] [12] [14] [101] [152] [110] [111] [21] [22] [116]² [16] [112] [24] [17] [127] [118] [25] [93] [94] [95] [96]

Temperature

Two-month-old seedlings of somnifera were unable to withstand more than 7 days of 4°C cold stress without severe necrosis.[153]

[154] [153]

Lighting

[34] [155] [156]

Pests

[157] [134] [158] [159] [160] [161] [33] [162] [163] [164] [165] [166] [167] [168] [147] [169] [170] [171] [172] [173] [174] [132] [175] [75] [176] [177]

Ecology

[178] [136] [179] [138] [141] [162] [143] [120] [163] [144] [164] [86] [146] [180] [20] [147] [131] [181] [148] [149] [170] [182] [183] [150] [132] [175] [151] [133] [176] [177] [89] [152] [184]

Morphology

Mild drought stress can improve some of the quality parameters of somnifera roots.[127]

Wild and cultivated stains differ wildly in their growth habits. Wild plants are taller and had more leaves and leaf area than cultivated varieties. Flower morphology favored self-pollination in cultivated varieties. They also differ considerably in flower production, seed weight, chemical profile, chemical yield, and, in fact, most characteristics that have been studied.[187][119][182][185][26][188]

Wild and cultivated accessions of somnifera can be easily separated by AFLP genetic variation indicating a long history of artificial selection. The genetic difference is so great that these two could be considered separate species, evidenced further by the low success rate of hybridization between wild and cultivated plants.[187]

An example of the small, easily-harvestable plants.[102]

[189] [122] [120] [149] [190] [124] [191] [187] [151] [127] [119] [192] [87] [115] [89] [12] [109] [21] [116] [23] [16] [112] [193] [117] [182] [91] [25] [26] [93] [194] [96]

Roots

[88] [125]

Stem

Leaves

Inflorescence

[26] [182] [195]

Seeds

[186] [26] [182] [196] [197] [10]

Phytochemistry

[198] [199] [200] [138] [139] [201] [37] [120] [41] [148] [202] [203] [204] [205] [206] [207] [79] [208] [91] [116] [107] [112] [25] [209] [93]

Infraspecific Variation

[210] [211] [123] [212] [213] [214] [215] [216] [217] [81] [119] [188] [194] [218]

Biosynthesis

[219] [29] [30] [157] [121] [135] [32] [220] [221] [222] [137] [34] [223] [224] [140] [225] [142] [144] [118] [226] [154] [165] [38] [145] [210] [166] [167] [168] [227] [146] [180] [181] [46] [47] [49] [50] [51] [212] [153] [155] [228] [56] [57] [58] [156] [60] [229] [230] [231] [62] [232] [64] [65] [233] [66] [234] [235] [68] [69] [236] [70] [71] [215] [72] [73] [74] [237] [76] [82] [83] [21] [22] [184]

Distribution

[199] [135] [238] [128] [156] [195] [183] [239] [240] [241] [242]

Timecourse

[189] [231] [243] [240] [127] [88]

Improvement

[9] [182] [191] [196] [187] [75] [81] [119] [194]

Identification

[97] [189] [123] [246] [212] [190] [213] [124] [214] [215] [187] [88] [247] [216] [125] [74] [185] [217] [115] [193] [248] [26] [188] [218]

Inheritance

[122] [31] [33] [87] [100] [48] [249] [63] [124] [67] [125] [115] [245]

Methods

[26] [248] [209]

History & Society

[250]

Work Log

05 Oct 2023

Harvested the bulk of the somnifera crop today with the help of two friends. Three hours of work to harvest, trim, and rinse it. Yield of 2.7 kg total wet weight. Needs to be separated, washed, and dried.

Yield data recorded: 47 plants, 16-126 g per plant.

Left two of the largest plants from each of the three varieties to see how well they survive the 4°C cold snap coming in a few days.

The shoots were layed out on a tarp to dry in the sun. I’m not sure what to do with them.

04 Oct 2023

Harvested a single plant from the garden today. Left one.

24 Mar 2023

Germination has been abysmal. Only two plants have sprouted. I don’t know what went wrong. Maybe it was the pretreatment somehow.

17 Feb 2023

EFN has a stated germination rate of 50%.

Making a 10ppm GA3 + 1% KNO3 solution. A stock solution of 10,000ppm GA3 in EtOH. Requires 1mg GA3. 0.1 ml stock GA3 solution. 1 g KNO3. 100ml tap H2O. GK1

Set each to soak in GK1 for 24 hours.

edit GK1 actually contains 8.6ppm GA3 because it ppm in 70% EtOH, not water.

19 Dec 2022

For the 2023 season: Target 1 kg of dry ashwagandha. If we assume a 500 kg/ha yield (low), that means approximately 15 square meters.

How about a row spacing of 60 cm and a plant spacing of 30 cm with a total of 10 rows and 10 plants each? Total 18 square meters; 100 plants. Sounds nice and even.

I have some seeds left over from previous years (WSS, TWF, SM; ~100), but they have an unknown germination rate. Ashwagandha seeds from SM are 4.95 per 50 seed packets. I will buy 1 each of the “African” and “Vedic” ecotypes. Two 1020 trays can hold 144 cells. That should be sufficient. At least one plant of each could be reserved for bato bucket cultivation.

https://www.weather.gov/wrh/Climate

Topeka, KS -> Climate -> NOWData -> First/last dates -> Min temp

https://mrcc.purdue.edu/freeze/freezedatetool.html

For my area, the mean day with a low of 4°C is May 1st. Ashwagandha can withstand up to 7 days at 4°C, so that’s probably late enough. 6 weeks for germination. Therefore, seed sowing time is on or about March 20th.

Bibliography

1. Pandya, Dhruv and Mankad, Archana and Pandya, Himanshu, A Review on Withania Somnifera (l.) Dunal- as an Important Ayurveda Plant, International Association of Biologicals and Computational Digest, vol. 1, no. 1, pp. 138--144, May 2022. doi: 10.56588/iabcd.v1i1.28. Abstract
   Withania somnifera (L.) Dunal is a well-known and important medicinal plant widely used in several indigenous systems of medicine for the treatment of various ailments, viz. asthma, bronchitis, inflammatory diseases, ulcer and stomach problems. Steroidal lactones have been reported as the major phytoconstituents of this species. Different pharmacological experiments in a number of in vitro and in vivo models have convincingly demonstrated the ability of W. somnifera to exhibit anti-inflammatory, anti-oxidative, antimicrobial, anti-anxiety, aphrodisiac, immunomodulation, anti- diabetic, anti-ulcer, anticancer, central nervous system depressant and hepatoprotective activities, lending support to the rationale behind several of its traditional uses. The species is also used to treat some neurological disorders like Parkinson’s and Alzheimer’s. The phytochemicals such as withaferin A, withanolide A and withanolide D isolated from this plant are potential bioactive molecules. Due to the remarkable biological activity of W. somnifera and its constituents, it will be appropriate to develop them as a medicine and make them more potent by chemical modifications and biotransformation. This review has covered botany, chemistry and pharmacology of the plant besides its traditional and folkloric uses.
2. S, Mary Princess Sulekha, A Comprehensive Review on Medicinal Herb Withania Somnifera (L.) Dunal in Women’s Health: A Rejuvenator From Siddha Medicine, Current Pharmacology Reports, vol. 8, no. 1, pp. 72--77, February 2022. doi: 10.1007/s40495-021-00274-5. Abstract
   Medicinal herbs have served mankind since several centuries and hold the rich heritage of treating dreadful diseases. One such potential herb that exists in the siddha system of medicine is Withania somnifera (WS) known by its name Amukkara. Numerous preclinical and clinical studies substantiate the biomedical application of Amukkara, some of which includes anti-cancer, antioxidant, anxiolytic, adaptogen, and anti-inflammatory properties. Despite its extensive efficacy, the herb WS is a well-known remedy for diseases and infections that are specific to women’s health. The main objective of the present review is to prime the health benefits of WS over the health and well-being of women in our society.
3. Paul, Subhabrata and Chakraborty, Shreya and Anand, Uttpal and Dey, Swarnali and Nandy, Samapika and Ghorai, Mimosa and Saha, Suchismita Chatterjee and Patil, Manoj Tukaram and Kandimalla, Ramesh and Proćków, Jarosław and Dey, Abhijit, Withania Somnifera (L.) Dunal (Ashwagandha): A Comprehensive Review on Ethnopharmacology, Pharmacotherapeutics, Biomedicinal and Toxicological Aspects, Biomedicine \& Pharmacotherapy, vol. 143, pp. 112175, November 2021. doi: 10.1016/j.biopha.2021.112175. Abstract
   Withania somnifera (L.) Dunal (Solanaceae) has been used as a traditional Rasayana herb for a long time. Traditional uses of this plant indicate its ameliorative properties against a plethora of human medical conditions, viz. hypertension, stress, diabetes, asthma, cancer etc. This review presents a comprehensive summary of the geographical distribution, traditional use, phytochemistry, and pharmacological activities of W. somnifera and its active constituents. In addition, it presents a detailed account of its presence as an active constituent in many commercial preparations with curative properties and health benefits. Clinical studies and toxicological considerations of its extracts and constituents are also elucidated. Comparative analysis of relevant in-vitro, in-vivo, and clinical investigations indicated potent bioactivity of W. somnifera extracts and phytochemicals as anti-cancer, anti-inflammatory, apoptotic, immunomodulatory, antimicrobial, anti-diabetic, hepatoprotective, hypoglycaemic, hypolipidemic, cardio-protective and spermatogenic agents. W. somnifera was found to be especially active against many neurological and psychological conditions like Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, ischemic stroke, sleep deprivation, amyotrophic lateral sclerosis, attention deficit hyperactivity disorder, bipolar disorder, anxiety, depression, schizophrenia and obsessive-compulsive disorder. The probable mechanism of action that imparts the pharmacological potential has also been explored. However, in-depth studies are needed on the clinical use of W. somnifera against human diseases. Besides, detailed toxicological analysis is also to be performed for its safe and efficacious use in preclinical and clinical studies and as a health-promoting herb.
4. Bonilla, Diego A. and Moreno, Yurany and Gho, Camila and Petro, Jorge L. and {Odriozola-Martínez}, Adrián and Kreider, Richard B., Effects of Ashwagandha (Withania Somnifera) on Physical Performance: Systematic Review and Bayesian Meta-Analysis, Journal of Functional Morphology and Kinesiology, vol. 6, no. 1, pp. 20, March 2021. doi: 10.3390/jfmk6010020. Abstract
   Ashwagandha (Withania somnifera) is considered a potent adaptogen and anti-stress agent that could have some potential to improve physical performance. This preferred reporting items for systematic reviews and meta-analyses (PRISMA)-based comprehensive systematic review and Bayesian meta-analysis aimed to evaluate clinical trials up to 2020 from PubMed, ScienceDirect, and Google Scholar databases regarding the effect of Ashwagandha supplementation on physical performance in healthy individuals. Besides implementing estimation statistics analysis, we developed Bayesian hierarchical models for a pre-specified subgroup meta-analysis on strength/power, cardiorespiratory fitness and fatigue/recovery variables. A total of 13 studies met the requirements of this systematic review, although only 12 were included in the quantitative analysis. A low-to-moderate overall risk of bias of the trials included in this study was detected. All Bayesian hierarchical models converged to a target distribution (Ȓ = 1) for both meta-analytic effect size (μ) and between-study standard deviation (τ). The meta-analytic approaches of the included studies revealed that Ashwagandha supplementation was more efficacious than placebo for improving variables related to physical performance in healthy men and female. In fact, the Bayesian models showed that future interventions might be at least in some way beneficial on the analyzed outcomes considering the 95\% credible intervals for the meta-analytic effect size. Several practical applications and future directions are discussed, although more comparable studies are needed in exercise training, and athletic populations are needed to derive a more stable estimate of the true underlying effect.
5. Mohanty, Arijita and Sahoo, Jyoti Prakash and Acharya, Akash Uditansu and Samal, Kailash Chandra, Ashwagandha (Withania Somnifera) - The Nature's Gift to Mankind, Biotica Research Today, vol. 3, no. 2, pp. 122--124, February 2021. url: https://www.biospub.com/index.php/biorestoday/article/view/756. Abstract
   Ashwagandha is a prominent herb in Indian Ayurvedic medicine and has become a popular supplement due to its health benefits. Limited evidence suggests that Ashwagandha reduces blood sugar levels through its effects on insulin secretion and sensitivity. Its supplements may help lower cortisol levels in chronically stressed individuals. It may help reduce depression. It has been shown to increase muscle mass, reduce body fat, and increase strength in men. It may help reduce the risk of heart disease by decreasing cholesterol and triglyceride levels. It supplements may improve brain function, memory and the ability to perform tasks. Although Ashwagandha is safe for most people, specific individuals should not use it unless authorized by their healthcare provider.
6. Bano, Anisha and Sharma, Navdeep and Dhaliwal, Harcharan and Sharma, Vivek, A Systematic and Comprehensive Review on Withania Somnifera (L.) Dunal- An Indian Ginseng, British Journal of Pharmaceutical Research, vol. 7, no. 2, pp. 63--75, January 2015. doi: 10.9734/BJPR/2015/17102. Abstract
   Present review article reveals the importance of species Withania somnifera (L.) Dunal, distributed in India and other parts of the world, this extensive research information on this species is highly significant for future researchers worldwide. In this article cytomorphological, phytochemical and biological activities inputs have been extensively recorded and discussed. As a part of our investigation on cytomorphological and phytochemical aspects for important medicinal plants from India, the aim of this pioneer attempt is to provide precise, truthful and detailed information of W. somnifera (L.). As per our knowledge, there is not even a single, combined, constructive review report available about this species, evaluated by using cytomorphological, phytochemical and biological activities based aspects.
7. Bhasin, Shruti and Singh, Manali and Singh, Dipti, Review on Bioactive Metabolites of Withania Somnifera. (L.) Dunal and Its Pharmacological Significance, Journal of Pharmacognosy and Phytochemistry, vol. 8, no. 3, pp. 3906--3909, 2019. url: https://www.researchgate.net/publication/344189197_Review_on_bioactive_metabolites_of_Withania_somnifera_L_Dunal_and_its_pharmacological_significance. Abstract
   Naturally active compounds from the medicinal plants are used by tribal people for many disorders. Ayurvedic medicines are most commonly used. Withania somnifera (L.) is an important medicinal plant which is widely used for the treatment of various disorders like bronchitis, ulcer, inflammatory diseases, and various stomach problems. It is a challenge to extract the bioactive plant constituent from the different parts of a plant. W. somnifera and its constituent have a remarkable biological activity which is appropriate to develop medicine and can be chemically modified and biotransform to make a more potent medicine. Numbers of in vitro and in vivo experiments have been demonstrated to find the ability of W. somnifera. This plant is a good source of bioactive constituents like withanolides and withaferin which are used for the treatment of cancer. The major phytoconstituents of this species are steroidal lactones.
8. Shinde, Ashashri and Gahunge, Pankaj and Rath, Sudipt, Conservation and Sustainability of Ashwagandha: A Medicinal Plant, Journal of Biological \& Scientific Opinion, vol. 3, pp. 94--99, April 2015. doi: 10.7897/2321-6328.03220. Abstract
   Ayurveda become a globally popular healthcare sy stem leading to constant rise in the demand for herbal medicines resulting in increased world herbal trade which stands at US\$ 120 billion and is expected to reach US\$ 7 trillion by 2050. As majority of Ayurvedic medicines are plant based, this demand has resulted in a huge pressure on the traditional sources of raw materials. Obviously, the wild sources are not able to meet the demand of raw materials. To overcome this situation alternate sources of raw materials like cultivated fields are look for supply. Dwindling forest cover, unscientific and exploitative collection leads to loss of supply source and threat to germplasm. Germplasm conservation and cultivation seems to be the best way forward for sustainable utilization of medicinal plants. Ashwagandha ( Withania somnifera Dunal) is widely used, prioritised Ayurvedic herb having annual demand 7000 tonnes /yr but its actual production is 1500 tonnes/yr. It grows naturally in subtropical region and is collected from wild and fields. Its r ampant collection has once leaded to critical pressure situation on its germplasm. This was noticed and a lot of effort was put in for its sustainable production. There i s lot of evidence we will discuss in the various research field of Ashwagandha like Agronomic research , Phy tochemical and Clinical research and Market Linkage . Ashwagndha due to such sustained and concerted multi - disciplinary effort has come near to a sustainable use mode. It is thus logical to extend the Ashwagandha model to other high demand medicinal plants and its feasibility.
9. Kaur, Kulwinder and Singh, Pritika and Guleri, Rupam and Singh, Baldev and Kaur, Kuldeep and Singh, Varinder and Pati, Pratap Kumar, Biotechnological Approaches in Propagation and Improvement of Withania Somnifera (L.) Dunal, pp. 459--478, 2017. doi: 10.1007/978-3-319-59192-6_22. Abstract
   Withania somnifera is a high value medicinal plant of the family Solanaceae. It is known for its anti-tumour, anti-stress, anti-aging, cardio-protective, neuro-protective and anti-inflammatory properties. Its pharmaceutical properties are attributed to a wide range of secondary metabolites, such as steroidal lactones, alkaloids, glycowithanolides, flavanol glycosides, phenolics and sterols. The traditional cultivation of W. somnifera is limited mainly by poor seed viability and germination, low yield and inconsistency in production of secondary metabolites. The infestation with various pests and pathogens also throw a major challenge in its cultivation. Biotechnological approaches involving organ, tissue and cell culture offer potential solution to the existing problems. In vitro propagation helps in rapid multiplication of elite cultivars and facilitate in raising quality planting materials. Genetic manipulation and secondary metabolite engineering hold great promise for enhancement of secondary metabolites and for overall crop improvement. The present chapter briefly discuss the challenges in W. somnifera and present a quick overview of biotechnological advances to address these challenges. It also highlights the futuristic approaches that would lay a foundation in the conglomeration of W. somnifera as an ideal model medicinal plant.
10. Khanna, Punit K. and Kumar, Arun and Chandra, Ratna and Verma, V., Germination Behaviour of Seeds of Withania Somnifera (L.) Dunal: A High Value Medicinal Plant, Physiology and Molecular Biology of Plants, vol. 19, no. 3, pp. 449--454, July 2013. doi: 10.1007/s12298-013-0169-3. Abstract
    In order to evolve a quick method for smooth and optimum germination for Withania somnifera- a medicinally efficacious multipurpose plant, present investigation was carried to study the effect of physico-chemical treatments, storage, temperature, photoperiod and growth regulators (GA3, IAA, IBA, 2–4 D and BA) on germinability. The most effective treatment is GA3 at 150~μg/ml concentration at 25~°C. The optimal temperature for germination is 25~°C and continuous light favored germination showing that photoperiod has a significant role. The seedlings derived from seeds performed well when grown in a glasshouse. The data have implications for conservation and cultivation of the species studied.
11. Niyaz, Afshan and Siddiqui, Enam Nabi, Seed Germination of Withania Somnifera (L.) Dunal, European Journal of Medicinal Plants, pp. 920--926, May 2014. doi: 10.9734/EJMP/2014/8916.
12. Kothari, Sushil and Singh, C. and Kumar, Y. and Singh, K., Morphology, Yield and Quality of Ashwagandha (Withania Somnifera L. Dunal) Roots and Its Cultivation Economics as Influenced by Tillage Depth and Plant Population Density, Journal of Horticultural Science, vol. 78, pp. 422--425, May 2003. doi: 10.1080/14620316.2003.11511642. Abstract
    Ashwagandha (Withania somnifera L. Dunal; Solanaceae) is being cultivated around the world mainly for its root which has rejuvenative properties. Field experiments during 1999-2001 under semi-arid tropical conditions of Hyderabad, India studied the effect of tillage depths (15 and 30 cm) and plant population densities (20, 40, 60 and 80 plants m-2) on root morphology, yield and quality and cultivation economics of ashwagandha. Preparatory tillage to 30 cm depth in combination with a density of 60 plants m-2 gave the highest root yield of 1.2 t ha-1 which was 50\% higher than that following 15 cm tillage depth and the same density. This is attributed to the favourable effect of the former treatment leading to 33, 37 and 21\% increases in plant height, number of branches per plant and shoot biomass yield, respectively. Further, this treatment produced most (68\%) best quality root pieces. Main root length, length of lateral roots and diameter of lateral roots were significantly increased, while the diameter of main root and the number of laterals decreased. Furthermore, increase in plant density from 20-80 plants m-2 increased production of best quality roots from 42\% to 59\% under shallow tillage and from 53\% to 71\% of root yield under deep tillage. The implications of change in root morphology are discussed in the light of production of different grades of roots, their market preferences and economics. Deep tillage was equally advantageous for seed production, but plant population density for maximum seed yield (211 kg ha-1) was lower 40 than at 60 plants m-2 for maximum root yield. Maximum gross and net returns and benefit-cost ratio occurred following the highest root yield.
13. Rajeswara Rao, B.R. and D.K., {\relax RAJPUT} and G, {\relax ADI}, Opportunities and Challenges in the Cultivation of Ashwagandha Withania Somnifera (L.) Dunal, Journal of Pharmacognosy, vol. 3, pp. 88--91, January 2012. Abstract
    Winter cherry or Ashwagandha (Withania somnifera) is an important medicinal plant native to India. The roots are widely used in Indian systems of medicine for more than 4000 years. The roots are credited with several medicinal properties. Recent research suggests phytochemical withaferin A present in the leaves possess antitumor activity. It is drought tolerant annual, hence is cultivated under rainfed condition in marginal soils by small and marginal farmers of Madhya Pradesh, Rajasthan, Andhra Pradesh, Karnataka and other Indian states. The ease of cultivation and high price for the roots is attracting farmers for large scale cultivation. In addition to the roots, leaves and seeds are also marketed enhancing the profits of the farmers. The future is excellent for extending its cultivation and production of numerous value added products from its roots.
14. Chavan, Mukesh, Influence of Dates of Sowing and Plant Density on Root Morphology and Root Yield of Ashwagandha (Withania Somnifera), Journal of Ecobiology, vol. 26, no. 1, pp. 285--292, 2010. url: https://www.academia.edu/12503503/INFLUENCE_OF_DATES_OF_SOWING_AND_PLANT_DENSITY_ON_ROOT_MORPHOLOGY_AND_ROOT_YIELD_OF_ASHWAGANDHA_Withania_somnifera_. Abstract
    The research study was conducted to know the influence of dates of sowing on ashwagandha (Withania somnifera Dunal) at Arabhavi during 2006 with factorial randomized block design. To study the effect of sowing dates I and II fortnights of July and
15. Wankhade, S. G. and Gholap, S. V. and Khode, P. P., Effect of Seed Rates on the Root Yield and Quality of Ashwagandha (Withania Somnifera Dunal), Agricultural Science Digest, vol. 29, no. 1, pp. 39--41, 2009. url: https://arccjournals.com/journal/agricultural-science-digest/.
16. Chaurasia, Sushmita and Singh, Rashmi, Effect of Integrated Nutrient Management System on Yield Attributes, Yield and Economics of Ashwagandha (Withania Somnifera (l, International Journal of Current Science, vol. 12, no. 2, pp. 20, 2022. Abstract
    Ashwagandha (Withania somnifera (L.) Dunal) is one of the most important medicinal crop in India. It plays an important role in medicinal industries. The demand of fleshy whitish brown roots are high but the production is low. Therefore, the present study was conducted to know the effect of integrated nutrient management system on yield attributes, yield and economics of Ashwagandha (Withania somnifera (L.) Dunal) at near the college of agriculture farm, Tikamgarh (M.P.) India during kharif season of 2017-18 and 2018-19. The experiment was laid out in randomized block design with three replications and twelve treatments viz, 100\% NPK recommended dose 50:30:30 kg/ha (T1), 100\% NPK/ha + 5 kg Zn/ha (T2), 10 tonnes FYM/ha (T3), 10 tonnes FYM/ha + 3 kg PSB/ha (T4), 10 tonnes FYM/ha + 3 kg Azotobacter/ha (T5), 10 tonnes FYM/ha + 3 kg Azotobacter/ha + 3 kg PSB/ha (T6), 10 tonnes FYM/ha + 3 kg Azotobacter/ha + 3 kg PSB/ha + 5 kg Zn/ha (T7), 50\% NPK/ha + 5 tonnes FYM/ha (T8), 50\% NPK/ha + 5 tonnes FYM/ha + 3 kg PSB/ha (T9), 50\% NPK/ha + 5 tonnes FYM/ha + 3 kg Azotobacter/ha (T10), 50\% NPK/ha + 5 tonnes FYM/ha + 3 kg Azotobacter/ha + 3 kg PSB/ha (T11) and 50\% NPK/ha + 5 tonnes FYM/ha + 3 kg Azotobacter/ha + 3 kg PSB/ha + 5 kg Zn/ha (T12). The pooled analysis of two years data indicated that an integrated nutrient management system significantly influenced different yield attributes, yield and economic parameters of Ashwagandha. The significant maximum values were recorded in number of berries per plant (121.61), number of seeds par berry (40.54), 1000 seed weight (4.46 g), root yield per plant (39.93 g), shoot yield per plant (115.80 g), seed yield per plant (12.80 g), root yield per hectare (629.69 kg/ha), gross returns (Rs. 1,20,881/ha), net returns (Rs. 81,887/ha) and benefit cost ratio (3.10) with 50\% NPK/ha + 5 tonnes FYM/ha + 3 kg Azotobacter/ha + 3 kg PSB/ha + 5 kg Zn/ha (T12) treatments. While highest seed yield per hectare (175.30 kg/ha) was recorded with the application of 100\% NPK recommended dose 50:30:30 kg/ha (T1). Based on overall experimental results, it could be concluded that 50\% NPK/ha + 5 tonnes FYM/ha + 3 kg Azotobacter/ha + 3 kg PSB/ha + 5 kg Zn/ha (T12) treatment was found to be a better integrated nutrient management system which can be used for better quality production, higher yield and maximum economic benefit of Ashwagandha (Withania somnifera (L.) Dunal) crop.
17. Kattimani, K. and Reddy, Y.N. and Rajeswara Rao, B.R., Effect of Pre-Sowing Seed Treatment on Germination, Seedling Emergence, Seedling Vigour and Root Yield of Ashwagandha (Withania Somnifera Dunal), Seed Science and Technology, vol. 27, pp. 483--488, January 1999. Abstract
    A field experiment was conducted at the College of Ag ricultule, Rajendranagar, Hyderabad to study the effect of pre-sowing seed treatments on seed vigour, root length and dry root yield of Ashwagandha under the agro-climatic conditions of Andhra Pradesh. Seedling vigour revealed that seeds soaked with nitrates of sodium and potassium at 1.0\% for 24 h produced more vigourous seedlings, higher dry matter accumulation and root length as compared to unsoaked and water soaked seeds. Unsoaked seeds recorded minimum root length throughout the different stages of crop growth except l5 days after sowing. Seeds soaked with nitrate of sodium and potassium at 1.0\% recorded maximum dry root yield of 3.93 and 3.43 g/plant respectively.
18. Kaur, Amandeep and Ohri, Puja and Kaur, Dr Arvinder, Effect of Vermicompost Extracts on In-Vitro Germination and Growth of Withania Somnifera (L.) Dunal, International Journal of Herbal Medicine, vol. 6, no. 2, pp. 28--32, 2018. url: https://www.florajournal.com/archives/2018/vol6issue2/PartA/7-1-1-510.pdf. Abstract
    Seed germination in Withania somnifera is low and erratic under natural conditions therefore in the present study we pre-soaked the seeds of W. somnifera for 12h in Vermicompost leachate (aerated and non-aerated), Vermicompost tea (aerated and non-aerated) and distilled water (DW). Treated seeds were germinated in vitro on filter papers moistened with respective pre-soaking treatments. Results indicated that seeds treated with Vcom-Ta resulted in highest germination (70\%) and biomass/seedling (0.42g). Seed germination and biomass/seedling in different treatments was Vcom-Ta {$>$} Vcom-Tna {$>$} Vcom-Lna {$>$} Vcom-La {$>$} DW. Carbon content was significantly increased in Vcom-Lna, Vcom-La and Vcom-Ta treatments as compared to control. Nitrogen content was maximum in Vcom-Ta seedlings but there was a non significant difference between control, Vcom-Ta and Vcom-Lna. We observed more root hair proliferation in Vcom-Lna and Vcom-Ta treated seedlings. Therefore, the use of such organic treatments can be extended to cultivate W. somnifera under natural conditions.
19. Subhas, Ingle and Bangale, Sachin, Comparative Study of Seed Germination and Percentage of Fungal Infection of Ashwagandha (Withania Somnifera (L.) Dunal.), Research Journal of Recent Sciences, vol. 1, pp. 2277--2502, August 2012. Abstract
    Ashwagandha (Withania somnifera (L.) Dual.), is a medicinal plant. Presently, there are five different varieties are under in cultivation. These varieties shows different rate of seed germination and fungal infection .These variation in percent of seed germination and fungal infection are investigated in the present work by blotter method.
20. Das, Amar Jyoti and Kumar, Rajesh, Bioremediation of Petroleum Contaminated Soil to Combat Toxicity on Withania Somnifera through Seed Priming with Biosurfactant Producing Plant Growth Promoting Rhizobacteria, Journal of Environmental Management, vol. 174, pp. 79--86, June 2016. doi: 10.1016/j.jenvman.2016.01.031. Abstract
    Soil contaminated by Petroleum oil cannot be utilized for agricultural purposes due to hydrocarbon toxicity. Oil contaminated soil induces toxicity affecting germination, growth and productivity. Several technologies have been proposed for bioremediation of oil contaminated sites, but remediation through biosurfactant producing plant growth promontory rhizobacteria (PGPR) is considered to be most promising methods. In the present study the efficacy of seed priming on growth and pigment of Withania somnifera under petroleum toxicity is explored. Seeds of W. somnifera were primed with biosurfactant producing Pseudomonas sp. AJ15 with plant growth promoting traits having potentiality to utilized petroleum as carbon source. Results indicates that plant arose from priming seeds under various petroleum concentration expressed high values for all the parameters studied namely germination, shoot length, root length, fresh and dry weight and pigments (chlorophyll and carotenoid) as compared to non primed seed. Hence, the present study signifies that petroleum degrarding biosurfactant producing PGPR could be further used for management and detoxification of petroleum contaminated soils for growing economically important crops.
21. Kaur, Amandeep and Pati, Pratap Kumar and Ohri, Puja and Kaur, Arvinder, Effects of Vermicompost and Vermicompost Leachate on the Biochemical and Physiological Response of Withania Somnifera (L.) Dunal, Journal of Soil Science and Plant Nutrition, vol. 22, no. 3, pp. 3228--3242, September 2022. doi: 10.1007/s42729-022-00880-2. Abstract
    Withania somnifera is a widely used herb due to its multi-health benefits. There is a growing interest to improve the growth and chemical profile of this plant because a large gap exists in the supply and demand of the quality raw material. The present study focused on using organic amendments to enhance the health and quality of this herb.
22. Pal, Shaifali and Yadav, Akhilesh Kumar and Singh, Anup Kumar and Rastogi, Shubhra and Gupta, Madan Mohan and Verma, Rajesh Kumar and Nagegowda, Dinesh A. and Pal, Anirban and Shasany, Ajit Kumar, Nitrogen Treatment Enhances Sterols and Withaferin A through Transcriptional Activation of Jasmonate Pathway, WRKY Transcription Factors, and Biosynthesis Genes in Withania Somnifera (L.) Dunal, Protoplasma, vol. 254, no. 1, pp. 389--399, January 2017. doi: 10.1007/s00709-016-0959-x. Abstract
    The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.
23. Bhosale, R S and More, A D, Response of Withania Somnifera, (L.) Dunal. to Soils from Different Locations in Satara District w.r.t Germination and Vegetative Growth, International Journal of Life Sciences, vol. 1, no. 1, pp. 76--78, 2013. Abstract
    Withania somnifera, Dunal. belongs to family Solanaceae. It is also known as Ashwagandha or Indian Genseng. Withanolides are constituents of roots of Ashwagandha. Withanolide is the major constituent with appreciable quantity of Withanone and traces of Withaferin-A. In Ayurvedic literature, is a “Rasayana” or rejuvenating drug and hence also traditionally known as Avarada which suggests the application of this plant for enhancing longevity. The seeds of Withania somnifera, Dunal. var somnifera were selected and used for following investigations. Different locations from Junnar taluka showed different response to germination and vegetative growth. From present investigation it can be concluded that soil with alkaline properties was more efficient for germination and growth of Withania somnifera, Dunal. than acidic soil.
24. Kattimani, K.N. and Reddy, Y.N. and Rajeswara Rao, B.R., Influence of Pre-Sowing Seed Treatments on Seedling Vigour, Root Length and Dry Root Yield of Ashwagandha Withania Somnifera under Semi-Arid Tropical Climate of Hyderabad., Journal of Medicinal and Aromatic Plant Sciences, vol. 22\&23, pp. 221--223, January 2001. Abstract
    A field study was undertaken on the response of pre-sowing seed treatments with nitrates of potassium, cobalt, ammonium, sodium, calcium and zinc for 24h and their impact on root length and dry root yield of ashwagandha var. Jawahar 20. Pre-soaking the seeds with nitrates of sodium, potassium and calcium showed beneficial effects on root length and dry root yield under the semi-arid tropical conditions of Hyderabad.
25. PK, • and Kumar, Arun and Ahuja, Ashok and Kaul, Maharaj, Effect of Biofertilizers on the Morphology, Root Yield and Biochemical Parameters of Withania Somnifera (L.) Dunal; Journal of Plant Biology, 33 (1\&2), Pp. 163-167, Journal of Plant Biology,, vol. 33, pp. 163--167, January 2006.
26. Mir, Bilal Ahmad and Koul, Sushma and Soodan, Amarjit Singh, Reproductive Biology of Withania Ashwagandha Sp. Novo (Solanaceae), Industrial Crops and Products, vol. 45, pp. 442--446, February 2013. doi: 10.1016/j.indcrop.2012.12.023. Abstract
    Withania ashwagandha Kaul (Solanaceae) is an annual plant species of immense medicinal importance. It is a repository of a large number of pharmacologically active secondary metabolites known as withanolides. Evidence for the delimitation of the species from W. somnifera has been provided by our group using multidisciplinary approaches. Knowledge of reproductive biology of medicinal plants is crucial for improvement, effective conservation and management plans to evolve genetically superior varieties. The present paper reports our findings on the floral biology, pollination behavior and breeding system of W. ashwagandha in natural populations grown at our experimental field under near natural conditions. Flowering (peak) takes place during April–July and anthesis occurs between 08:00 and 11:00h. The period of stigma receptivity coincides with anther dehiscence. Fruit set on pollination treatments ranged from 90.8\% (passive autogamy), 72\% (assisted autogamy), 30.30\% (xenogamy), and 56.50\% (geitonogamy) through 50.40\% (open pollination). Xenogamy brings about very low fruit set, seed-set and seed germination percentages. It is inferred that W. ashwagandha is predominantly an autogamous and self-compatible species. Self-compatibility is mainly accomplished due to close proximity of stigma and anthers. This work is the first report on the reproductive biology of W. ashwagandha and will be useful for conservation and development of improved varieties of this multipurpose herb.
27. Thapliyal, Chetna, Effect of Growth Hormones on Germination Seedling Propagation and Biomass Production of Medicinal Plants Bacopa Monnieri L Penell and Withania Somnifera Dunal, University, 2009. url: http://shodhganga.inflibnet.ac.in:8080/jspui/handle/10603/184102. Abstract
    newline
28. Vinod, Sangeetha and Natarajan, Kanimozhi and Santhanu, Krishnapriya and Selvakumar, Divya and Natesan, Senthil and Senthil, Kalaiselvi, Effect of Seaweed Elicitation and Culture Conditions on Biomass and Metabolite Production in in Vitro Shoots of Withania Somnifera, South African Journal of Botany, vol. 151, pp. 781--790, December 2022. doi: 10.1016/j.sajb.2022.10.051. Abstract
    Withania somnifera is an important medicinal plant used in various herbal preparations due the presence of its key metabolites-withanolides. The present study focuses on the effect of seaweed extract and different in vitro culture conditions on the biomass and key metabolite content in in vitro shoot cultures of Withania somnifera. The commercially available seaweed extract powder of Kappaphycus alverizii with the trade name ’Magic vita - P’ was examined for its effect on solid, suspension and bioreactor cultures of Withania somnifera. The standardisation of elicitation conditions showed the exposure of shoot cultures to 0.25\% seaweed extract for 7 days resulted in maximum biomass and withaferin A content which were 1.91 fold and 3.05 fold higher than that of untreated shoots. Further among the three culture conditions studied, the bioreactor cultures exhibited maximum growth index compared to the solid and suspension cultures thus producing maximum biomass which in turn resulted in higher accumulation of withanolides per batch of culture. Thus the seaweed treated shoots in bioreactor yielded 18.56, 8.96, and 7.29 fold increase in withaferin A, withanolide A and withanone content compared to control shoots in solid media. Further the effect of this elicitation and culture conditions on the accumulation pattern of the primary metabolites were studied using GC-MS. The Principal Component Analysis (PCA) of the GC-MS data exhibited a clear distinction between the control and seaweed treated samples. Thus our findings indicate a clear protocol for scaling up of withanolide production from in vitro shoot cultures of Withania somnifera.
29. Thorat, Sachin Ashok and Kaniyassery, Arya and Poojari, Poornima and Rangel, Melissa and Tantry, Shashikala and Kiran, Kodsara Ramachandra and Joshi, Manjunath B. and Rai, Padmalatha S. and Botha, Anna-Maria and Muthusamy, Annamalai, Differential Gene Expression and Withanolides Biosynthesis During in Vitro and Ex Vitro Growth of Withania Somnifera (L.) Dunal, Frontiers in Plant Science, vol. 13, pp. 917770, June 2022. doi: 10.3389/fpls.2022.917770. Abstract
    Ashwagandha (Withania somnifera L. Dunal) is a medicinally important plant with withanolides as its major bioactive compounds, abundant in the roots and leaves. We examined the influence of plant growth regulators (PGRs) on direct organogenesis, adventitious root development, withanolide biosynthetic pathway gene expression, withanolide contents, and metabolites during vegetative and reproductive growth phases under in vitro and ex vitro conditions. The highest shooting responses were observed with 6-benzylaminopurine (BAP) (2.0 mg L–1) + Kinetin (KIN) (1.5 mg L–1) supplementation. Furthermore, BAP (2.0 mg L–1) + KIN (1.5 mg L–1) + gibberellic acid (GA3) (0.5 mg L–1) exhibited better elongation responses with in vitro flowering. Half-strength MS medium with indole-3-butyric acid (IBA) (1.5 mg L–1) exhibited the highest rooting responses and IBA (1.0 mg L–1) with highest fruits, and overall biomass. Higher contents of withaferin A (WFA) [∼8.2 mg g–1 dry weight (DW)] were detected in the reproductive phase, whereas substantially lower WFA contents (∼1.10 mg g–1 DW) were detected in the vegetative phase. Cycloartenol synthase (CAS) (P = 0.0025), sterol methyltransferase (SMT) (P = 0.0059), and 1-deoxy-D-xylulose-5-phosphate reductase (DXR) (P = 0.0375) genes resulted in a significant fold change in expression during the reproductive phase. The liquid chromatography-mass spectrometry (LC-MS) analysis revealed metabolites that were common (177) and distinct in reproductive (218) and vegetative (167) phases. Adventitious roots cultured using varying concentrations of indole-3-acetic acid (IAA) (0.5 mg L–1) + IBA (1.0 mg L–1) + GA3 (0.2 mg L–1) exhibited the highest biomass, and IAA (0.5 mg L–1) + IBA (1.0 mg L–1) exhibited the highest withanolides content. Overall, our findings demonstrate the peculiarity of withanolide biosynthesis during distinct growth phases, which is relevant for the large-scale production of withanolides.
30. Kaur, Kuldeep and Dolker, Dechen and Behera, Shashikanta and Pati, Pratap Kumar, Critical Factors Influencing in Vitro Propagation and Modulation of Important Secondary Metabolites in Withania Somnifera (L.) Dunal, Plant Cell, Tissue and Organ Culture (PCTOC), vol. 149, no. 1, pp. 41--60, May 2022. doi: 10.1007/s11240-021-02225-w. Abstract
    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.
31. Kaur, Kuldeep and Singh, Pritika and Kaur, Kulwinder and Bhandawat, Abhishek and Nogia, Panchsheela and Pati, Pratap Kumar, Development of Robust in Vitro Culture Protocol for the Propagation of Genetically and Phytochemically Stable Plants of Withania Somnifera (L.) Dunal (Ashwagandha), Industrial Crops and Products, vol. 166, pp. 113428, August 2021. doi: 10.1016/j.indcrop.2021.113428. Abstract
    Withania somnifera (L.) Dunal is an important medicinal plant that has gained a lot of attention since ancient times due to its immense pharmaceutical potential. Scientific validations of its therapeutic potential against many diseases have immensely increased its demand in the domestic and international market. Thus, to narrow down the huge gap between its demand and supply, alternative strategies to conventional cultivation are required. In vitro propagation system provides a feasible option for the production of large-scale healthy, genetically and phytochemically uniform W. somnifera plants to boost the herbal industry and facilitate pre-clinical studies. However, the major challenge in in vitro propagation of W. somnifera is to maintain a high rate of shoot multiplication for prolonged periods in cytokinin containing MS medium. In the current investigation, for prolonged maintenance of in vitro shoots, a multi-step protocol involving the rotational subculture of Withania shoots in the auxin-cytokinin containing culture medium has been developed to minimize the adverse effects of cytokinin. In vitro shoots after prolonged subculture were checked for genetic uniformity and stable pharmaceutically important constituents. Furthermore, in vitro rooting was initiated either in an auxin-dependent or auxin-independent manner through modulation of ammonium nitrate strength. Hence, the protocol developed for in vitro propagation of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides.
32. Sivanandhan, Ganeshan and Selvaraj, Natesan and Ganapathi, Andy and Lim, Yong Pyo, Up-Regulation of Squalene Synthase in Hairy Root Culture of Withania Somnifera (L.) Dunal Yields Higher Quantities of Withanolides, Industrial Crops and Products, vol. 154, pp. 112706, October 2020. doi: 10.1016/j.indcrop.2020.112706. Abstract
    The roots of Indian ginseng, Withania somnifera (L.) Dunal (Solanaceae) are extensively used in ayurvedic and other traditional medicines. The plant produces a steroidal lactone, withanolide which has multiple health benefits. Squalene synthase plays a central role in regulating the withanolide biosynthetic pathway in W. somnifera. The present approach investigated the up-regulation of squalene synthase in withanolides biosynthesis pathway in hairy roots. The higher expression of squalene synthase was noticed in hairy root line Ws9 which exhibited withanolide A (2.82 mg/g DW), withanolide B (1.34 mg/g DW), withaferin A (1.83 mg/g DW) and withanone (1.97 mg/g DW) on the 40th day of culture with 1.08 to 1.25-times higher, respectively. Semi-quantitative and quantitative polymerase chain reaction confirmed the incorporation and expression of squalene synthase in cultured hairy roots.
33. Chakraborty, Nilanjan and Banerjee, Maitreyi and Acharya, Krishnendu, In Vitro Selection of Elite Clone of Withania Somnifera against Leaf Blight Disease Caused by Alternaria Alternata, Physiological and Molecular Plant Pathology, vol. 112, pp. 101560, December 2020. doi: 10.1016/j.pmpp.2020.101560. Abstract
    In vitro selection of Withania somnifera has been incurred against Alternaria alternata by selection pressure of fungal toxin in an innovative way. Solid green callus was maintained in Murashige and Skoog's medium supplemented with 6-Benzylaminopurine (2 mgL−1). Toxin was partially purified from A. alternata broth culture. Elite clone was generated by gradual intoxication of solid green callus in the toxin containing medium (5–400 mgL−1). Three highest dose of toxin tolerance was selected viz. 50 mgL−1, 100 mgL−1 and 200 mgL−1. Plants were successfully regenerated from those toxin selected callus. Eventually, various defense enzymes and total phenolics were measured in regenerated plants and toxin selected callus. Callus selected from 100 mgL−1 toxin dose and plants obtained from them give the best results among all and showed more than 2–5 fold increase of defense enzyme activities and total phenolics compared to control. Furthermore, those plants when challenge inoculated showed significant reduction (more than 70\%) of leaf spot incidence. Augmentation of disease resistance persists for at least two successive generations. Furthermore, the increase in defense molecules also correlated with increased nitric oxide generation (73\%). Presence of withaferin A and withanolide A has also been confirmed in the toxin regenerated plants by High-performance thin-layer chromatography (HPTLC) analysis. Over all observations suggests that fungal toxin at a concentration 100 mgL−1 and by habituation technique can be use as a selection method for the production of disease resistant medicinally important crop like W. somnifera without altering its secondary metabolite production.
34. Adil, Muhammad and Haider Abbasi, Bilal and {ul Haq}, Ihsan, Red Light Controlled Callus Morphogenetic Patterns and Secondary Metabolites Production in Withania Somnifera L., Biotechnology Reports, vol. 24, pp. e00380, December 2019. doi: 10.1016/j.btre.2019.e00380. Abstract
    Withania somnifera L. is an endangered medicinal plant of higher market value. The in vitro callus cultures were established on Murashige and Skoog (MS) media augmented with different plant growth regulators. The MS medium containing 0.5\,mg∙L−1 of each TDZ and NAA was found to be optimal for callus formation and growth. Further, callus cultures were raised in different light wavelengths to find the right wavelength carrying the photons for the ideal cell growth of W. somnifera. Among the different wavelengths, red light was best for maximum biomass accumulation in callus culture. However, violet light condition was proven to be favouring the phenols and flavonoids synthesis in the callus cultures. Compared to other wavelengths, red light grown callus extract showed significantly higher content of chlorogenic acid, and withaferin A. This study concludes that red light treatment was optimum for maximum biomass accumulation and anti-oxidant activity in calli of W. somnifera.
35. Thilip, Chandrasekaran and Mehaboob, Valiyaparambath Musfir and Varutharaju, Kandhan and Faizal, Kunnampalli and Raja, Palusamy and Aslam, Abubakker and Shajahan, Appakan, Elicitation of Withaferin-A in Hairy Root Culture of Withania Somnifera (L.) Dunal Using Natural Polysaccharides, Biologia, vol. 74, no. 8, pp. 961--968, August 2019. doi: 10.2478/s11756-019-00236-9. Abstract
    Withaferin-A (WA) is a major bioactive compound in the roots of Withania somnifera. It is reported to possess inhibitory effects against breast cancer, liver cancer, melanoma cancer and prostate cancer cells. We developed an elicitation based on the hairy root culture (HRC) protocol for the enhancement of the WA production. Agrobacterium rhizogenes strain R1000 was used for the establishment of hairy root culture. PCR analysis confirmed the integration of the root loci C (rol C) gene in transformed roots. Elicitation of the hairy roots with natural polysaccharides of sodium alginate (SA), k-carrageenan (kC) and chitosan (CH) at various concentrations in the most cases resulted in a higher amount of WA compound. Among the various elicitors, 100~mg~L−1 CH resulted 4.03-fold increase in WA production than control. This study is the first report in W. somnifera hairy roots, where the natural polysaccharides are used as elicitors for the large-scale production of WA.
36. Gaurav, Naveen and Kumar, Arun, Effect of Growth Regulators on In-Vitro Callusing of Wild Variety of Withania Somnifera L. in B5 Medium., Indian Forester, vol. 145, no. 12, pp. 1176--1181, 2019. url: https://www.cabdirect.org/cabdirect/abstract/20203122766. Abstract
    "Ashwagandha" products are one of the major herbal components of various disease tonics mentioned in Indian systems of medicine. It is an erect branching under shrub reaching about 150 cm in height, usually clothed with minutely stellate tomentum; leaves ovate up to 10 cm long, densely hairy beneath and sparsely above, flowers are greenish or yellow in axillary fascicles, bisexual, pedicel long,...
37. Johny, Leena and Conlan, Xavier A. and Adholeya, Alok and Cahill, David M., Growth Kinetics and Withanolide Production in Novel Transformed Roots of Withania Somnifera and Measurement of Their Antioxidant Potential Using Chemiluminescence, Plant Cell, Tissue and Organ Culture (PCTOC), vol. 132, no. 3, pp. 479--495, March 2018. doi: 10.1007/s11240-017-1344-y. Abstract
    Markedly increased withanolide content was found in transformed roots (TR) of Withania somnifera germplasm grown in low mineral minimal media and withanolides showed high antioxidant potential when analysed using acidic potassium permanganate chemiluminescence. Transformation frequency of explants infected with Agrobacterium rhizogenes strain A4 varied between the three germplasms tested with the highest observed as 75\,±\,0.9. Transformed root production was explant specific with leaves being the most productive among the different explants used. Withanolides, namely withaferin A, withanolide A, withanolide B and 12-deoxywithastramonolide were detected in TR cultures and differences in their content were found between germplasms. The highest concentrations of secondary metabolites were found in 4-week-old cultures and concentrations declined by the 8th and 12th week of culture. In 4-week-old cultures, the biomass of TR cultures was 4.5 fold higher than their respective non-transformed roots (NTR). Withaferin A was found in TR at levels that were 28–34 times higher than that found in NTR. A rapid method for the determination of the antioxidant potential of W. somnifera TR extracts was developed using post-column acidic potassium permanganate chemiluminescence (APPC) detection. The APPC chromatographic peaks for extract constituents showed strong alignment with those found for ultraviolet absorbance detection. The methods developed in this study for TR culture establishment and the use of a fast and sensitive way for the qualitative and quantitative determination of the antioxidant activity of their metabolites provides a new platform that will have use for similar studies in other species.
38. Saxena, Parul and Ahlawat, Seema and Ali, Athar and Khan, Shazia and Abdin, Malik Zainul, Gene Expression Analysis of the Withanolide Biosynthetic Pathway in Hairy Root Cultures of Withania Somnifera Elicited with Methyl Jasmonate and the Fungus Piriformospora Indica, Symbiosis, vol. 71, no. 2, pp. 143--154, February 2017. doi: 10.1007/s13199-016-0416-9. Abstract
    Withania somnifera, also known as Indian ginseng is known to contain valuable bioactive compounds, called withanolides that structurally resemble ginsenosides of Panax ginseng. These compounds provide the basis of pharmacological relevance in traditional systems of medicine. In the present study, 150 hairy root lines of W. somnifera were induced of which nine fast growing lines were analysed for their growth and withanolide content. Hairy root line W9 was selected due to its high specific growth rate (0.196~±~0.005 d−1) and high withanolide content. The response to different concentrations of elicitors (methyl jasmonate and P. indica cell homogenate) and various exposure durations was assessed in the W9 hairy root line. The withanolide content as well as the pattern of gene expression from MVA, MEP and sterol pathway, was evaluated using qPCR. Though gene expression and withanolide content were found to be elevated in almost all MeJ and CHP treatments, the exposure of hairy roots to 15 μM MeJ for 4 h gave the maximum withanolide yield. The results suggest that the elicitation potential of methyl jasmonate was higher than that of P. indica cell homogenate for increasing withanolide levels in hairy roots of W. somnifera.
39. Shajahan, Appakan and Thilip, Chandrasekaran and Faizal, Kunnampalli and Mehaboob, Valiyaparambath Musfir and Raja, Palusamy and Aslam, Abubakker and Kathiravan, Krishnan, An Efficient Hairy Root System for Withanolide Production in Withania Somnifera (L.) Dunal, pp. 133--143, 2017. doi: 10.1007/978-3-319-69769-7_7. Abstract
    Withania somnifera (L.) Dunal is one of the most important medicinal plant belonging to the family Solanaceae. Its root have been used as a drug since ancient times, and various pharmacological effects have been attributed to the occurrence of secondary compounds like withaferin-A and withanolide-A. Recently, huge interests are generated for production of these bioactive compounds through Agrobacterium rhizogenes-mediated hairy root culture techniques. The present review explores the culture conditions for efficient Agrobacterium-mediated hairy root culture system of W. somnifera for withanolide production. The hairy root induction is influenced by several factors like bacterial stain, type of explant, and cocultivation methods. The transformation efficiency could be enhanced by the addition of acetosyringone and SAAT treatments during cocultivation. Recent studies have also shown positive correlations of elicitors and biosynthetic pathway genes on withanolide production in hairy root culture of W. somnifera.
40. Dehdashti, Sayed Mehdi and Acharjee, Sumita and Kianamiri, Shahla and Deka, Manab, An Efficient Agrobacterium Rhizogenes-Mediated Transformation Protocol of Withania Somnifera, Plant Cell, Tissue and Organ Culture (PCTOC), vol. 128, no. 1, pp. 55--65, January 2017. doi: 10.1007/s11240-016-1081-7. Abstract
    This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75~μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120~min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5\,\% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.
41. Singh, Pritika and Guleri, Rupam and Angurala, Amrita and Kaur, Kuldeep and Kaur, Kulwinder and Kaul, Sunil C. and Wadhwa, Renu and Pati, Pratap Kumar, Addressing Challenges to Enhance the Bioactives of Withania Somnifera through Organ, Tissue, and Cell Culture Based Approaches, BioMed Research International, vol. 2017, pp. e3278494, February 2017. doi: 10.1155/2017/3278494. Abstract
    Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.
42. Singh, Pritika and Guleri, Rupam and Pati, Pratap Kumar, In Vitro Propagation of Withania Somnifera (L.) Dunal, pp. 201--213, 2016. doi: 10.1007/978-1-4939-3332-7_14. Abstract
    Withania somnifera (L.) Dunal known as Ashwagandha is commonly used in traditional Indian medicine system. It possesses immense therapeutic value against a large number of ailments such as mental diseases, asthma, inflammation, arthritis, rheumatism, tuberculosis, and a variety of other diseases including cancer. The therapeutic potential of W. somnifera is due to the presence of secondary metabolites mainly, tropane alkaloids and withanolides (steroidal lactones). The growing realization of commercial value of the plant has initiated a new demand for in vitro propagation of elite chemotypes of Withania. Micropropagation which is an important tool for rapid multiplication requires optimization of number of factors such as nutrient medium, status of medium (solid and liquid), type of explant, and plant growth regulators. Similarly, an efficient and reproducible in vitro regeneration system which is a prerequisite for the development of genetic transformation protocol requires precise manipulation of various intrinsic and extrinsic factors.
43. Mishra, Smrati and Bansal, Shilpi and Sangwan, Rajender Singh and Sangwan, Neelam S., Genotype Independent and Efficient Agrobacterium-Mediated Genetic Transformation of the Medicinal Plant Withania Somnifera Dunal, Journal of Plant Biochemistry and Biotechnology, vol. 25, no. 2, pp. 191--198, April 2016. doi: 10.1007/s13562-015-0324-8. Abstract
    Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2~days preconditioning of explants on MS basal supplemented with TDZ 1~μM, Agrobacterium density at OD600 0.4 with inclusion of 100~μM acetosyringone (As) for 20~min co-inoculation duration with 48~h of co-cultivation period at 22~°C using node explants was found optimal to improved the number of GUS foci per responding explant from 36\,±\,13.2 to 277.6\,±\,22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11~T0 transgenic plants were generated from 100 co-cultivated node explants, representing 10.6~\% transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3~months.
44. Sivanandhan, Ganeshan and Dev, Gnajothi Kapil and Theboral, Jeevaraj and Selvaraj, Natesan and Ganapathi, Andy and Manickavasagam, Markandan, Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania Somnifera (L.) Dunal, PLOS ONE, vol. 10, no. 4, pp. e0124693, April 2015. doi: 10.1371/journal.pone.0124693. Abstract
    In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84\% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90\%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10\%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.
45. Sivanesan, Iyyakkannu and Park, Se Won, Optimizing Factors Affecting Adventitious Shoot Regeneration, in Vitro Flowering and Fruiting of Withania Somnifera (L.) Dunal, Industrial Crops and Products, vol. 76, pp. 323--328, December 2015. doi: 10.1016/j.indcrop.2015.05.014. Abstract
    The effects of plant growth regulators, sucrose and temperature on adventitious shoot regeneration, in vitro flowering and fruiting of Withania somnifera were investigated. The highest frequency of shoot regeneration (96\%) with an average of 22.8 shoots per stem segment was obtained on Murashige and Skoog (MS) medium fortified with 2.0mgl−1 kinetin, 0.5mgl−1 α-naphthaleneacetic acid (NAA), 0.3mgl−1 gibberellic acid (GA3) and 40gl−1 sucrose. The greatest frequency of rooting (100\%) with an average of 14.1 roots per shoot was obtained when the regenerated shoots were cultured on the half-strength MS medium containing 2.0mgl−1 indole-3-butyric acid (IBA) and 15gl−1 sucrose. Regenerated plantlets were successfully established in the soil with 98\% survival rate. The acclimatized plants grew well, developed flowers that were morphologically similar to the donor plants. The highest frequency of flowering (88\%) with an average of 8.3 flowers per shoot and the greatest frequency of fruiting (74.9\%) with an average of 5.1 fruit per shoot were obtained when shoot tips were cultured on MS medium fortified with 0.3mgl−1 N6-benzyladenine (BA) and 60gl−1 sucrose, and maintained at 20°C. Seeds were separated from the in vitro formed mature fruits and placed on MS medium containing 0.3mgl−1 GA3 to test their viability. The percentage of seed germination was 66\%. The developed in vitro culture protocol can be useful for large scale propagation, genetic transformation and plant breeding studies.
46. Sil, Bipradut and Mukherjee, Chiranjit and Jha, Sumita and Mitra, Adinpunya, Metabolic Shift from Withasteroid Formation to Phenylpropanoid Accumulation in Cryptogein-Cotransformed Hairy Roots of Withania Somnifera (L.) Dunal, Protoplasma, vol. 252, no. 4, pp. 1097--1110, July 2015. doi: 10.1007/s00709-014-0743-8. Abstract
    Cotransformed hairy roots containing a gene that encodes a fungal elicitor protein, β-cryptogein, were established in Withania somnifera, a medicinal plant widely used in Indian systems of medicine. To find out whether β-cryptogein protein endogenously elicits the pathway of withasteroid biosynthesis, withaferin A and withanolide A contents along with transcript accumulation of farnesyl pyrophosphate (FPP) synthase, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), and sterol glycosyltransferase (SGT) were analyzed in both cryptogein-cotransformed and normal hairy roots of W. somnifera. It was observed that the withaferin A and withanolide A contents were drastically higher in normal hairy roots than cryptogein-cotransformed ones. Similar trends were also observed on the levels of transcript accumulation. Subsequently, the enzyme activity of phenylalanine ammonia lyase (PAL), one of the key enzymes of phenylpropanoid pathway, was measured in both cryptogein-cotransformed and normal hairy roots of W. somnifera along with the levels of PAL transcript accumulation. Upliftment of PAL activity was observed in cryptogein-cotransformed hairy roots as compared to the normal ones, and the PAL expression also reflected a similar trend, i.e., enhanced expression in the cryptogein-cotransformed lines. Upliftment of wall-bound ferulic acid accumulation was also observed in the cryptogein-cotransformed lines, as compared to normal hairy root lines. Thus, the outcome of the above studies suggests a metabolic shift from withanolide accumulation to phenylpropanoid biosynthesis in cryptogein-cotransformed hairy roots of W. somnifera.
47. Adil, Muhammad and Abbasi, Bilal Haider and Khan, Tariq, Interactive Effects of Melatonin and Light on Growth Parameters and Biochemical Markers in Adventitious Roots of Withania Somnifera L., Plant Cell, Tissue and Organ Culture (PCTOC), vol. 123, no. 2, pp. 405--412, November 2015. doi: 10.1007/s11240-015-0844-x. Abstract
    Light plays a pertinent role in plant photo-morphogenesis and it is believed to have an impact on the melatonin-induced physiological functions. In the current study, different light regimes were employed with varying levels of melatonin, either singly or in combination with auxins for the growth and development of adventitious roots in Withania somnifera L. It was observed that 600~µM melatonin favored maximum adventitious root induction frequency (58~\%) in cultures incubated under continuous dark conditions. However, adequate root growth (number and length of roots) was observed under 16~h light/8~h dark at 600~µM melatonin. Nevertheless, the interactive effect of light and melatonin was found stimulating for profound production of commercially important secondary metabolites. Correlation among growth parameters and biochemical markers was also observed in the current report. Data on total phenolic content and total flavonoid content were found at higher coincidence with each other and with DPPH antioxidant activity. In conclusion, exogenously applied melatonin mimics IAA activity in root growth and regulates well in 16-h light/8-h dark, thereby giving protection to plant system against light stress.
48. Fatima, Nigar and Ahmad, Naseem and Ahmad, Iqbal and Anis, Mohammad, Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania Somnifera L., Applied Biochemistry and Biotechnology, vol. 177, no. 1, pp. 118--136, September 2015. doi: 10.1007/s12010-015-1732-x. Abstract
    An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 \%) with optimum number of shoots (32\,±\,0.00)/explant were obtained on MS medium fortified with 2.5~μM 6-benzyladenine (BA) and 0.5~μM NAA and 30~g/l sucrose at pH~5.8. Among the tried TDZ concentrations, 0.5~μM resulted in maximum number of shoots (20.4\,±\,0.40)/explant after 4~weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4\,±\,0.40) with shoot length (6.80\,±\,0.12~cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100~\% with maximum roots (18.3\,±\,0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200~μM (NAA) for 15~min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.
49. Sivanandhan, Ganeshan and Arunachalam, Chinnathambi and Selvaraj, Natesan and Sulaiman, Ali Alharbi and Lim, Yong Pyo and Ganapathi, Andy, Expression of Important Pathway Genes Involved in Withanolides Biosynthesis in Hairy Root Culture of Withania Somnifera upon Treatment with Gracilaria Edulis and Sargassum Wightii, Plant Physiology and Biochemistry, vol. 91, pp. 61--64, June 2015. doi: 10.1016/j.plaphy.2015.04.007. Abstract
    The investigation of seaweeds, Gracilaria edulis and Sargassum wightii extracts was carried out for the estimation of growth characteristics and major withanolides production in hairy root culture of Withania somnifera. The extract of G.~edulis (50\%) in MS liquid basal medium enabled maximum production of dry biomass (5.46~g DW) and withanolides contents (withanolide A 5.23~mg/g DW; withaferin A 2.24~mg/g DW and withanone 4.83~mg/g DW) in hairy roots after 40 days of culture with 48~h contact time. The obtained withanolides contents were significantly higher (2.32-fold–2.66-fold) in hairy root culture when compared to the control. RT PCR analysis of important pathway genes such as SE, SS, HMGR and FPPS exhibited substantial higher expression upon the seaweed extracts treatment in hairy root culture. This experiment would paw a platform for withanolides production in hairy root culture with the influence of sea weed extracts for pharmaceutical companies in the future.
50. Sivanandhan, Ganeshan and Selvaraj, Natesan and Ganapathi, Andy and Manickavasagam, Markandan, Effect of Nitrogen and Carbon Sources on in Vitro Shoot Multiplication, Root Induction and Withanolides Content in Withania Somnifera (L.) Dunal, Acta Physiologiae Plantarum, vol. 37, no. 2, pp. 12, January 2015. doi: 10.1007/s11738-014-1758-7. Abstract
    Effect of nitrogen and carbon sources on multiple shoot regeneration and withanolides contents of Withania somnifera were evaluated. Inclusion of l-glutamine (20~mg/l) in medium fortified with the optimal levels of 1.5~mg/l 6-benzyladenine (BA) and 0.3~mg/l indole-3-acetic acid (IAA) resulted in the production of 58 shoots/explant. Sucrose at 4~\% and 20~mg/l~l-glutamine with idéal concentrations of BA and IAA improved shoot multiplication (62~shoots/explant) while 6~\% sucrose enhanced withanolides contents in regenerated multiple shoots. All the shoots were rooted (26~roots/shoot) when cultured in MS medium amended with 15~mg/l ammonium nitrate, 2~mg/l IBA, and 2~\% sucrose. The present study resulted in the production of 4.42-fold higher multiple shoots and 2.6-fold higher roots as well as enhancing the contents of all major withanolides (withaferin A, withanone, withanolide A and B) in regenerated plants when compared to previous reports on W. somnifera regeneration.
51. Rout, Jyoti Ranjan and Behera, Sadhana and Keshari, Nitin and Ram, Shidharth Sankar and Bhar, Subhajit and Chakraborty, Anindita and Sudarshan, Mathummal and Sahoo, Santi Lata, Effect of Iron Stress on Withania Somnifera L.: Antioxidant Enzyme Response and Nutrient Elemental Uptake of in Vitro Grown Plants, Ecotoxicology, vol. 24, no. 2, pp. 401--413, March 2015. doi: 10.1007/s10646-014-1389-1. Abstract
    In the present study the response of antioxidant enzyme activities and the level of expression of their corresponding genes on bioaccumulation of iron (Fe) were investigated. In vitro germinated Withania somnifera L. were grown in Murashige and Skoog’s liquid medium with increasing concentrations (0, 25, 50, 100 and 200~µM) of FeSO4 for 7 and 14~days. Root and leaf tissues analyzed for catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7), have shown an increase in content with respect to exposure time. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and observed that the isoenzymes were greatly affected by higher concentrations of Fe. Reverse transcriptase polymerase chain reaction analysis performed by taking three pairs of genes of CAT (RsCat, Catalase1, Cat1) and SOD (SodCp, TaSOD1.2, MnSOD) to find out the differential expression of antioxidant genes under Fe excess. RsCat from CAT and MnSOD from SOD have exhibited high levels of gene expression under Fe stress, which was consistent with the changes of the activity assayed in solution after 7~days of treatment. Analysis by proton induced X-ray emission exhibited an increasing uptake of Fe in plants by suppressing and expressing of other nutrient elements. The results of the present study suggest that higher concentration of Fe causes disturbance in nutrient balance and induces oxidative stress in plant.
52. Patel, Neha and Patel, Parth and Kumari, Uma and Kendurkar, Shuchishweta V and Khan, Bashir M, Microprojectile Bombardment Assisted Agroinfection Increases Transformation Efficiency of Withania Somnifera (L.), Research in Biotechnology, pp. 12, 2014. Abstract
    Withania somnifera (L.) Dunal, an evergreen shrub distributed throughout the drier parts of India, is well known for its medicinal properties. Three different approaches were employed towards development of an efficient transformation system in Withania somnifera, namely Agrobacterium tumefaciens-mediated transformation, microprojectile bombardment and microprojectile bombardment assisted agroinfection. A. tumefaciens strain GV2260 containing a binary vector pCAMBIA 1301 was used carrying β-Glucuronidase (gus) reporter gene and hygromycin phosphotransferase (hptII) selection gene under the transcriptional control of Cauliflower Mosaic Virus 35S promoter. The apical and nodal segments of about one cm length excised from in vitro grown shoots were chosen as explants for transformation studies. The selection of transgenic plants was attained in the presence of 10 mg L-1 hygromycin B. Analysis of transgenic plants was done by histochemical Gus assay followed by polymerase chain reaction for the presence of gus and hptII genes. The overall transformation efficiencies obtained were 3.86\%, 3.62\% and 8.71\% for A. tumefaciensmediated, microprojectile bombardment and a combination of both the techniques, respectively.
53. Udayakumar, Rajangam and Ganapathi, Andy and SeiChang, Kim and ChangWon, Choi, Indirect Regeneration of Withania Somnifera from Nodal Explants., British Biotechnology Journal, vol. 4, no. 4, pp. 366--378, 2014. doi: 10.9734/BBJ/2014/7511. Abstract
    Withania somnifera is an important medicinal plant and used to cure many diseases. Indirect regeneration protocol for multiple shoots development was established using nodal explants of W. somnifera from 50-60 days old seedlings. The callus induction was observed from nodal explants, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of...
54. Rani, Archana and Kumar, M. and Kumar, Sanjeev, In Vitro Propagation of Withania Somnifera (L.) Dunal. from Shoot Apex Explants, Journal of Applied and Natural Science, vol. 6, no. 1, pp. 159--163, June 2014. doi: 10.31018/jans.v6i1.393. Abstract
    In vitro rapid micropropagation of Withania somnifera; has been achieved using shoot apex explants, when cultured on MS medium with different auxins: Indole Butyric Acid (IBA) 0.5-4 mg/l, Indole Acetic Acid (IAA) 2 mg/l and Naphthalene Acetic Acid (NAA) 0.5-3 mg/l and cytokinin: Benzyl Amino Purine (BAP) 0.5-4 mg/l. NAA and BAP stimulated direct as well as callus mediated multiple shoot. IBA alone and in combination with IAA stimulated rhizogenesis, while IBA with NAA proliferated callus-mediated rhizogenesis. The rooted plantlets through sequential hardening process could be established in the field.
55. Sathees Kannan, T. M. and Sownthariya, S. and Anbazhakan, S., In Vitro Mass Propagation of Withania Somnifera Dunal Using Seaweed Extract, International Letters of Natural Sciences, vol. 19, 2014. url: http://agro.icm.edu.pl/agro/element/bwmeta1.element.agro-f45ddf96-3b8b-45b5-808a-74d5753b1e27.
56. Sivanandhan, Ganeshan and Selvaraj, Natesan and Ganapathi, Andy and Manickavasagam, Markandan, Improved Production of Withanolides in Shoot Suspension Culture of Withania Somnifera (L.) Dunal by Seaweed Extracts, Plant Cell, Tissue and Organ Culture (PCTOC), vol. 119, no. 1, pp. 221--225, October 2014. doi: 10.1007/s11240-014-0521-5. Abstract
    The influence of Gracilaria edulis and Sargassum wightii extracts was investigated for the production of biomass and withanolides in the multiple shoot suspension culture of Withania somnifera. Supplementation of 40~\% G. edulis extract in MS liquid medium for 24~h exposure time in the culture recorded the highest biomass accumulation [62.4~g fresh weight and 17.82~g dry weight (DW)] and withanolides production (withanolide A 0.76~mg/g DW; withanolide B 1.66~mg/g DW; withaferin A 2.80~mg/g DW and withanone 2.42~mg/g DW) after 5~weeks of culture, which were 1.45–1.58-fold higher than control culture. This naturally available G. edulis extract-treated multiple shoot suspension culture protocol offers a potential alternative for the optimum production of biomass and withanolides utilizing shake-flasks.
57. Varghese, Smini and Keshavachandran, R. and Baby, Bincy and Nazeem, P. A., Genetic Transformation in Ashwagandha ( Withania Somnifera (L.) Dunal) for Hairy Root Induction and Enhancement of Secondary Metabolites, Journal of Tropical Agriculture, vol. 52, no. 1, pp. 47--53, October 2014. url: http://jtropag.kau.in/index.php/ojs2/article/view/302. Abstract
    Genetic transformation was carried out in ashwagandha (Withania somnifera (L.) Dunal) using three different Agrobacterium rhizogenes strains viz., A4, ATCC 15834 and MTCC 2364, for inducing hairy roots. The explants such as hypocotyls, cotyledonary segments, leaf segments, shoot tips and nodal segments were used for genetic transformation. A4 and ATCC 15834 strains produced successful transformation and hairy (transformed) roots were induced from leaf segments and shoot tips. A4 strain produced transformation by direct inoculation of bacteria from single cell colonies as well as in the suspension form, but ATCC 15834 produced transformation only in the suspension form. Among the liquid media tested, half MS was found to be superior in promoting hairy root growth. The transformation was confirmed by PCR and dot blot analysis. A Thin Layer Chromatographic method was employed for withanolide estimation. The spot corresponding to withaferin A was observed under UV at 254 nm. Field root possessed more withaferin A followed by hairy roots and in vitro roots contained the least. Enhancement of secondary metabolite production was attempted through addition of osmoregulator, precursor feeding and elicitation. Withaferin A content in the hairy root biomass and the culture medium were estimated. The biotic elicitor Aspergillus homogenate (250 and 500 μl /125 ml) had a positive influence in the enhancement of secondary metabolites.
58. Mir, Bilal Ahmad and Khazir, Jabeena and Hakeem, Khalid R. and Koul, Sushma and Cowan, Don A., Enhanced Production of Withaferin-A in Shoot Cultures of Withania Somnifera (L) Dunal, Journal of Plant Biochemistry and Biotechnology, vol. 23, no. 4, pp. 430--434, October 2014. doi: 10.1007/s13562-014-0264-8. Abstract
    Withania somnifera (L) Dunal, commonly known as ashwagandha or Indian ginseng, is the source of large number of pharmacologically active withanolides. Withaferin-A (WS-3), a major withanolide of W. somnifera, has been proven to be an effective anti-cancer molecule. In this study, a liquid culture system for shoot proliferation, biomass accumulation and withaferin-A production of an elite accession (AGB002) of W. somnifera was investigated. The nodal explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with various concentrations of 6-benzyl adenine (BA) and Kinetin (Kn) elicited varied responses. The highest number of regenerated shoots per ex-plant (35\,±\,3.25) and the maximum average shoot length (5.0\,±\,0.25~cm) were recorded on MS medium supplemented with BA (5.0~μM). The shoots were further proliferated in half and full strength MS liquid medium supplemented with the same concentration BA. It was interesting to note that shoots cultured on MS half strength liquid medium fortified with 4 gL-1 FW (fresh weight) shoot inoculum mass derived from 5~week old nodal explants of W. somnifera showed highest accumulation of biomass and withaferin A content in 5~weeks. Withaferin A was produced in relatively high amounts (1.30~\% and 1.10~\% DW) in shoots cultured in half and full strength MS liquid media respectively as compared to natural field grown plants (0.85~\% DW). A considerable amount of the withaferin A was also excreted in the culture medium. Successful proliferation of shoots in liquid medium and the synthesis of withaferin A in vitro opens new avenues for bioreactor scale-up and the large-scale production of the compound.
59. Mishra, Bhawana and Sangwan, Rajender S. and Mishra, Smrati and Jadaun, Jyoti S. and Sabir, Farzana and Sangwan, Neelam S., Effect of Cadmium Stress on Inductive Enzymatic and Nonenzymatic Responses of ROS and Sugar Metabolism in Multiple Shoot Cultures of Ashwagandha (Withania Somnifera Dunal), Protoplasma, vol. 251, no. 5, pp. 1031--1045, September 2014. doi: 10.1007/s00709-014-0613-4. Abstract
    Withania somnifera is one of the most important medicinal plant and is credited with various pharmacological activities. In this study, in vitro multiple~shoot cultures were exposed to different concentrations (5–300~μM) of cadmium (Cd) as cadmium sulphate to explore its ability to accumulate the heavy metal ion and its impact on the metabolic status and adaptive responses. The results showed that supplemental exposure to Cd interfered with N, P, and K uptake creating N, P, and K deficiency at higher doses of Cd that also caused stunting of growth, chlorosis, and necrosis. The study showed that in vitro shoots could markedly accumulate Cd in a concentration-dependent manner. Enzymatic activities and isozymic pattern of catalase, ascorbate peroxidase, guaiacol peroxidase, peroxidase, glutathione-S-transferase, glutathione peroxidase, monodehydroascorbate reductase, and dehydroascorbate reductase were altered substantially under Cd exposure. Sugar metabolism was also markedly modulated under Cd stress. Various other parameters including contents of photosynthetic pigments, phenolics, tocopherol, flavonoids, reduced glutathione, nonprotein thiol, ascorbate, and proline displayed major inductive responses reflecting their protective role. The results showed that interplay of enzymatic as well as nonenzymatic responses constituted a system endeavor of tolerance of Cd accumulation and an efficient scavenging strategy of its stress implications.
60. Dhar, Niha and Rana, Satiander and Razdan, Sumeer and Bhat, Wajid Waheed and Hussain, Aashiq and Dhar, Rekha S. and Vaishnavi, Samantha and Hamid, Abid and Vishwakarma, Ram and Lattoo, Surrinder K., Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases from Withania Somnifera (L.) Dunal *, Journal of Biological Chemistry, vol. 289, no. 24, pp. 17249--17267, June 2014. doi: 10.1074/jbc.M114.571919. Abstract
    {$<$}p{$>$}Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from \emph{Withania somnifera} (\emph{Ws}), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, β-amyrin synthase (\emph{Ws}OSC/BS), lupeol synthase (\emph{Ws}OSC/LS), and cycloartenol synthase (\emph{Ws}OSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in \emph{Schizosaccharomyces pombe}, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three \emph{Ws}OSCs were found to be spatially regulated at transcriptional level with \emph{Ws}OSC/CS being maximally expressed in leaf tissue. Promoter analysis of three \emph{Ws}OSCs genes resulted in identification of distinct \emph{cis}-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like \emph{Ws}OSC/BS and \emph{Ws}OSC/LS possibly leads to diversion of substrate pool toward \emph{Ws}OSC/CS for increased withanolide production.{$<$}/p{$>$}
61. Sivanandhan, G. and Selvaraj, N. and Ganapathi, A. and Manickavasagam, M., An Efficient Hairy Root Culture System for Withania Somnifera (L.) Dunal, African Journal of Biotechnology, vol. 13, no. 43, 2014. doi: 10.4314/ajb.v13i43. Abstract
    Withania somnifera is an important aromatic medicinal plant and possesses wide array of pharmacological properties. In the present investigation, an improved version of hairy root culture system was developed by optimizing various transformation parameters such as type of explant, concentration of acetosyringone, Agrobacterium types and co-cultivation period. Between the leaf and cotyledon explants and two Agrobacterium rhizgenes strains (R1000 and A4) tested, leaf explants infected with R1000 and cocultured for five days on MS basal half strength medium in the presence of acetosyringone (100 µM) attained a higher frequency (88\%) of hairy root induction. By adopting this protocol, we could utilize the hairy root culture for industrial scale production of withanolides.Keywords: Leaf explant, Agrobacterium rhizogenes, Withania somnifera, co-cultivation period, acetosyringone.African Journal of Biotechnology, Vol 13(43) 4141-4147
62. Sivanandhan, Ganeshan and Kapil Dev, Gnanajothi and Jeyaraj, Murugaraj and Rajesh, Manoharan and Arjunan, Annavi and Muthuselvam, Manickam and Manickavasagam, Markandan and Selvaraj, Natesan and Ganapathi, Andy, Increased Production of Withanolide A, Withanone, and Withaferin A in Hairy Root Cultures of Withania Somnifera (L.) Dunal Elicited with Methyl Jasmonate and Salicylic Acid, Plant Cell, Tissue and Organ Culture (PCTOC), vol. 114, no. 1, pp. 121--129, July 2013. doi: 10.1007/s11240-013-0297-z. Abstract
    Withania somnifera, an important medicinal plant that possesses a variety of bioactive secondary metabolites collectively known as withanolides. Hairy roots with an initial inoculum mass of 5~g FW were elicited separately with methyl jasmonate (MeJ) and salicylic acid (SA) at various concentrations for different exposure times after 30~days of culture. Enhanced production of biomass (32.68~g FW and 5.54~g DW; 1.23-fold higher), withanolide A (132.44~mg/g DW; 58-fold higher), withanone (84.35~mg/g DW; 46-fold higher), and withaferin A (70.72~mg/g DW; 42-fold higher) were achieved from 40~day-old harvested hairy roots elicited with 150~μM SA for 4~h exposure time. The present study reports a higher production of withanolide A, withanone and withaferin A from the elicited-hairy roots of W. somnifera under optimal inoculum mass, harvest time, elicitor exposure time and its concentration. These results will be useful for biochemical and bioprocess engineering for the viable production of withanolides in hairy root culture.
63. Nayak, S. A. and Kumar, S. and Satapathy, K. and Moharana, A. and Behera, B. and Barik, D. P. and Acharya, L. and Mohapatra, P. K. and Jena, P. K. and Naik, S. K., In Vitro Plant Regeneration from Cotyledonary Nodes of Withania Somnifera (L.) Dunal and Assessment of Clonal Fidelity Using RAPD and ISSR Markers, Acta Physiologiae Plantarum, vol. 35, no. 1, pp. 195--203, January 2013. doi: 10.1007/s11738-012-1063-2. Abstract
    An efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings. Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) (MS) medium supplemented with 1.0~mg~l−1N6-benzyladenine (BA) was found to be optimum for production of multiple shoots (100~\% shoot proliferation frequency and 16.93 shoots per explant). Successive shoot cultures were established by repeatedly sub-culturing the original cotyledonary node on a fresh medium after each harvest of newly formed shoots. Multiple shoot proliferation was also achieved from nodal segments derived from in vitro raised shoots on MS medium augmented with 1.0~mg~l−1 BA. Regenerated shoots were best rooted (95.2~\%, 38.7 roots per shoot) in half-strength MS medium supplemented with 1.0~mg~l−1 indole-3-butyric acid. The plantlets were successfully acclimated and established in soil. Random amplified polymorphic DNA and inter-simple sequence repeats analysis revealed a homogeneous amplification profile for all micropropagated plants analyzed validating the genetic fidelity of the in vitro regenerated plants.
64. Sivanandhan, Ganeshan and Rajesh, Manoharan and Arun, Muthukrishnan and Jeyaraj, Murugaraj and Kapil Dev, Gnanajothi and Arjunan, Annavi and Manickavasagam, Markandan and Muthuselvam, Manickam and Selvaraj, Natesan and Ganapathi, Andy, Effect of Culture Conditions, Cytokinins, Methyl Jasmonate and Salicylic Acid on the Biomass Accumulation and Production of Withanolides in Multiple Shoot Culture of Withania Somnifera (L.) Dunal Using Liquid Culture, Acta Physiologiae Plantarum, vol. 35, no. 3, pp. 715--728, March 2013. doi: 10.1007/s11738-012-1112-x. Abstract
    The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera. Elicitation of shoot inoculum mass (2~g~l−l FW) with SA at 100~μM in the presence of 0.6~mg~l−l BA and 20~mg~l−l spermidine for 4~h exposure time at the 4th week in 20~ml liquid medium recorded higher withanolides production (withanolides A [8.48~mg~g−l DW], withanolides B [15.47~mg~g−l DW], withaferin A [29.55~mg~g−l DW] and withanone [23.44~mg~g−l DW]), which were 1.14 to 1.18-fold higher than elicitation with MeJ at 100~μM after 5~weeks of culture. SA-elicited cultures did not exhibit much variation in biomass accumulation when compared to control. This cytokinin induces and SA-elicited multiple shoot culture protocol provides a potential alternative for the optimal production of biomass and withanolides utilizing liquid culture.
65. Rout, Jyoti R. and Ram, Shidharth S. and Das, Ritarani and Chakraborty, Anindita and Sudarshan, Mathummal and Sahoo, Santi L., Copper-Stress Induced Alterations in Protein Profile and Antioxidant Enzymes Activities in the in Vitro Grown Withania Somnifera L., Physiology and Molecular Biology of Plants, vol. 19, no. 3, pp. 353--361, July 2013. doi: 10.1007/s12298-013-0167-5. Abstract
    Withania somnifera L. seedlings were grown in half-strength MS (Murashige and Skoog) basal medium for 4~weeks and then transferred to full-strength MS liquid medium for 3~weeks. The sustainable plants were subcultured in the same medium but with different concentrations (0, 25, 50, 100 and 200~μM) of Cu for 7 and 14~days. The growth parameters (root length, shoot length, leaf length and total number of leaves per plant) showed a declining trend in the treated plants in a concentration dependant manner. Roots and leaves were analyzed for protein profiling and antioxidant enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7)]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude protein extracts showed the appearance of some new proteins due to Cu treatment. In plant samples grown with 25 and 50~μM of Cu, a rapid increase in antioxidant activities were noticed but at higher concentration (100 and 200~μM) the activities declined. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and concentration specific new isoforms were noticed during the study. Isoforms of the antioxidant enzymes synthesized due to Cu stress may be used as biomarkers for other species grown under metal stress.
66. Murthy, H. N. and Praveen, N., Carbon Sources and Medium pH Affects the Growth of Withania Somnifera (L.) Dunal Adventitious Roots and Withanolide A Production, Natural Product Research, vol. 27, no. 2, pp. 185--189, January 2013. doi: 10.1080/14786419.2012.660691. Abstract
    This work deals with optimisation of adventitious root suspension culture of Withania somnifera (L.) Dunal (Solanaceae) for the production of biomass and withanolide A and we investigated the effect of carbon source and the initial medium pH on growth and production of withanolide A in adventitious root cultures of Withania somnifera. A 2\% sucrose concentration was found to be best for both biomass (113.58 g L−1 fresh weight (FW) and 11.33 g L−1 dry weight (DW)) and secondary metabolite accumulation (8.93 mg g−1 DW) in the tested range of concentration (1–8\%). The biomass of adventitious roots was optimal when the initial medium pH was 5.8 (113.26 g L−1 FW and 11.33 g L−1 DW) but the withanolide A production was highest at the medium pH level of 5.5 (9.09 mg g−1 DW).
67. Fatima, Nigar and Ahmad, Naseem and Anis, Mohammad and Ahmad, Iqbal, An Improved in Vitro Encapsulation Protocol, Biochemical Analysis and Genetic Integrity Using DNA Based Molecular Markers in Regenerated Plants of Withania Somnifera L, Industrial Crops and Products, vol. 50, pp. 468--477, October 2013. doi: 10.1016/j.indcrop.2013.08.011. Abstract
    Non-embrogenic, synthetic seeds were produced by encapsulating nodal segments (containing axillary buds) of Withania somnifera L. in calcium alginate hydrogel containing Murashige and Skoog (MS) medium. A 3\% sodium-alginate with 100mM calcium cloride found to be the optimum concentration for the production of uniform synthetic seeds. Effect of different treatments (M1–M5), i.e. MS medium containing different concentrations of cytokinins (0.5, 1.0, 2.5, 5.0 \& 10.0μM) along with optimal level of auxins NAA (0.5μM) on in vitro morphogenic response of synthetic seeds was evaluated. The maximum frequency (86.2\%) of conversion of encapsulated beads into plantlets was achieved on MS (M3) medium containing 6-benzyladenine, BA (2.5μM) and α-naphthalene acetic acid, NAA (0.5μM) after 4 weeks of culture. Rooting in plantlets was achieved on 1/2MS+NAA (0.5μM). Plantlets obtained from stored synthetic seeds were hardened, acclimatized and established in field, where they grew well without any detectable malformation. Significant enhancement in the pigment contents (chlorophyll, carotenoids and net photosynthetic rates) with an increase in acclimatization days may be attributed to chlorophyll biosynthesis. Activities of antioxidant enzymes i.e. superoxide dismutase, catalase and peroxidase activity) were significantly increased which suggests their preventive role in membrane oxidation and damage to biological molecules. Also, an enhanced level of lipid peroxidation, as indicated by MDA content, a sensitive diagnostic index of oxidative injury clearly indicating its positive determining role in combating oxidative stress during acclimatization of plantlets. The generated RAPD and ISSR profiles from regenerated plantlets with mother plant were monomorphic which confirms the genetic stability among the clones. This synthetic seed technology could possibly paves the way for the conservation, short-term storage, germplasm exchange with potential storability and limited quarantine restrictions.
68. Sabir, Farzana and Sangwan, Rajender S. and Kumar, Ritesh and Sangwan, Neelam S., Salt Stress-Induced Responses in Growth and Metabolism in Callus Cultures and Differentiating In Vitro Shoots of Indian Ginseng (Withania Somnifera Dunal), Journal of Plant Growth Regulation, vol. 31, no. 4, pp. 537--548, December 2012. doi: 10.1007/s00344-012-9264-x. Abstract
    In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO3, NaNO3, and CaCl2. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO3 compared to other salts and the in vitro shoots appeared healthy at 50~mM concentration of NaCl and KNO3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl2 produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress.
69. Sivanandhan, Ganeshan and Arun, Muthukrishnan and Mayavan, Subramanian and Rajesh, Manoharan and Jeyaraj, Murugaraj and Dev, Gnanajothi Kapil and Manickavasagam, Markandan and Selvaraj, Natesan and Ganapathi, Andy, Optimization of Elicitation Conditions with Methyl Jasmonate and Salicylic Acid to Improve the Productivity of Withanolides in the Adventitious Root Culture of Withania Somnifera (L.) Dunal, Applied Biochemistry and Biotechnology, vol. 168, no. 3, pp. 681--696, October 2012. doi: 10.1007/s12010-012-9809-2. Abstract
    Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70~g FW) on 30-day-old adventitious root cultures with 150~μM SA for 4~h elicitor exposure period resulted in the production of 64.65~mg~g−l dry weight (DW) withanolide A (48-fold), 33.74~mg~g−l DW withanolide B (29-fold), 17.47~mg~g−l DW withaferin A (20-fold), 42.88~mg~g−l DW withanone (37-fold), 5.34~mg~g−l DW 12-deoxy withastramonolide (nine fold), 7.23~mg~g−l DW withanoside V (seven fold), and 9.45~mg~g−l DW withanoside IV (nine fold) after 10~days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.
70. Murthy, H.N. and Praveen, N., Influence of Macro Elements and Nitrogen Source on Adventitious Root Growth and Withanolide-A Production in Withania Somnifera (L.) Dunal, Natural Product Research, vol. 26, no. 5, pp. 466--473, March 2012. doi: 10.1080/14786419.2010.490914. Abstract
    Withania somnifera (L.) Dunal. (Indian ginseng) is an important medicinal plant which yields pharmaceutically active compounds, namely withanolides. This study deals with the optimisation of the adventitious root suspension culture of W. somnifera for the production of biomass and withanolide-A. We investigated the effects of macro elements (NH4NO3, KNO3, CaCl2, MgSO4 and KH2PO4) and nitrogen source [ ] of Murashige and Skoog (MS) medium on the accumulation of biomass and withanolide-A content. The highest accumulation of fresh and dry biomass (127.52 and 12.45 g L−1) was recorded in the medium with 0.5× concentration of NH4NO3 and the highest production of withanolide-A was recorded in the medium with 2.0× KNO3 (14.00 mg g−1 DW). The adventitious root growth was greater when the concentration was higher than that of and the withanolide-A production was highest in the absence of . Maximum biomass growth was achieved at ratio of 14.38 : 37.60, while withanolide-A production was greatest (11.76 mg g−1 DW) when the ratio was 0.00 : 18.80 mM. The results of this study are useful for scale-up processes.
71. Saravanakumar, Ananthapadmanaban and Aslam, Abubakker and Shajahan, Appakan, Development and Optimization of Hairy Root Culture Systems in Withania Somnifera (L.) Dunal for Withaferin-A Production, African Journal of Biotechnology, vol. 11, no. 98, 2012. doi: 10.4314/ajb.v11i98. Abstract
    Transformation of Withania somnifera was carried out by using three Agrobacterium rhizogenes strains (ATCC 15834, R1000 and K599) for hairy root induction. Induction of hairy root was carried out in leaf, petiole and internodal explants. Hairy root induction was successful only in ATCC 15834 and R1000. The highest frequency of hairy root was obtained in petiole explants (64\%) infected with R1000 and it resulted in five distinct morpho-types (callus (fragile), callus (hard), callus + hairy roots, hairy roots and callusing roots). The frequency of R1000 transformation was increased up to 93.2\% by the addition of acetosyringone during various steps of infection. Molecular identification through PCR analysis of rolC confirmed the presence of Ri T-DNA. The half strength Murashige and Skoog (MS) liquid medium was found to be the best medium that supports the high root biomass accumulation than the other tested medium types (MS full strength, Gamborg B5 medium (B5) full strength and B5 half strength). High performance liquid chromatography (HPLC) analysis of hairy roots revealed the accumulation of withaferin-A (72.3 mg/g dw). This study reports the influence of Agrobacterium strains, explant types and acetosyringone in the hairy root induction of W. somnifera.Key words: Withania somnifera, hairy root, Agrobacterium rhizogenes, hairy root morpho-types.
72. Sivanandhan, Ganeshan and Mariashibu, Thankaraj Salammal and Arun, Muthukrishnan and Rajesh, Manoharan and Kasthurirengan, Sampath and Selvaraj, Natesan and Ganapathi, Andy, The Effect of Polyamines on the Efficiency of Multiplication and Rooting of Withania Somnifera (L.) Dunal and Content of Some Withanolides in Obtained Plants, Acta Physiologiae Plantarum, vol. 33, no. 6, pp. 2279--2288, November 2011. doi: 10.1007/s11738-011-0768-y. Abstract
    An efficient mass multiplication protocol was developed for Withania somnifera (L.) Dunal from nodal explants of field-grown plants on Murashige and Skoog medium (MS) supplemented with 6-benzyladenine (BA) [1.5~mg~L−l], indole-3-acetic acid (IAA) [0.3~mg~L−l] and with the addition of polyamine, spermidine (20~mg~L−l) (shoot multiplication medium). A total of 46.4 shoots were obtained from nodal explants and they were elongated in the same medium in a culture duration of 6~weeks. The elongated shoots produced roots in MS medium fortified with putrescine (20~mg~L−l) after 4~weeks, and all the rooted plants were successfully hardened and acclimatized with a survival rate of 100\%. An average of 276 shoots (46~×~6) was produced when at least six nodal explants obtained from each of the 46 in vitro grown shoots were cultured by microcutting method in the same shoot multiplication medium. On an average, 12,696 plants could be produced from all the shoots (276~×~46) by microcuttings in a period of 7~months. HPLC revealed a significant increase in the quantities of withanolide A, withanolide B, withaferin A and withanone in the leaves, stems, and roots of in vitro regenerated plants compared to the field-grown parent plants. Ploidy analysis using flow cytometry revealed genetic stability of in vitro regenerated plants. This protocol will be useful for scale-up production of withanolides on commercial scale.
73. Fatima, Nigar and Ahmad, Naseem and Anis, Mohammad, Enhanced in~Vitro Regeneration and Change in Photosynthetic Pigments, Biomass and Proline Content in Withania Somnifera L. (Dunal) Induced by Copper and Zinc Ions, Plant Physiology and Biochemistry, vol. 49, no. 12, pp. 1465--1471, December 2011. doi: 10.1016/j.plaphy.2011.08.011. Abstract
    In the present study the effect of inorganic nutrients (CuSO4 \& ZnSO4) on morphogenic and biochemical responses from nodal explants in Withania somnifera L. was investigated. Incorporation of either Copper sulphate (25–200~μM) or Zinc sulphate (50–500~μM) in the optimized Murashige and Skoog (MS) medium highly influenced the shoot bud formation and subsequent elongation, which induced maximum percentage (95\%) regeneration, number (61.7~±~0.25) of shoots with shoot length (5.46~±~0.16~cm) on CuSO4 (100~μM) and maximum percentage regeneration (100\%), number of shoots (66.1~±~0.96) with shoot length (6.24~±~0.21~cm) on ZnSO4 (300~μM) after 12 weeks of culture. Healthy growing in~vitro microshoots rooted efficiently on ½ MS medium supplemented with NAA (0.5~μM), which induced (16.2~±~0.12) roots with root length (3.30~±~0.12~cm) after 4 weeks. Pigment content increased with increasing concentration of Cu and Zn and the maximum Chl. a (0.47), (0.41); Chl. b (0.52), (0.42); total Chl. (0.99), (0.83) and Carotenoid (0.16), (0.16) mg/g FW contents in regenerants were found on CuSO4 (100~μM) and ZnSO4 (300~μM), respectively. Maximum proline content (0.17), (0.16)~μg/g FW was observed on high concentrations of CuSO4 (200~μM) and ZnSO4 (500~μM) respectively, in the basal medium. Regenerated plantlets were acclimatized successfully in soilrite with a survival rate of 95\%. No morphological variations were detected among the micropropagated plants when compared with seedling raised plants of the same age.
74. Gupta, Parul and Akhtar, Nehal and Tewari, Shri Krishna and Sangwan, Rajender Singh and Trivedi, Prabodh Kumar, Differential Expression of Farnesyl Diphosphate Synthase Gene from Withania Somnifera in Different Chemotypes and in Response to Elicitors, Plant Growth Regulation, vol. 65, no. 1, pp. 93--100, September 2011. doi: 10.1007/s10725-011-9578-x. Abstract
    Withania somnifera (L.) Dunal (Family, Solanaceae), commonly known as Ashwagandha is one of the most valuable medicinal plants synthesizing large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized a gene encoding farnesyl diphosphate synthase (FPPS; EC 2.5.1.10), a key enzyme in the pathway of biosynthesis of isoprenoids, from W. somnifera. The full-length cDNA of Withania somnifera FPPS (WsFPPS) of 1,253~bps encodes a polypeptide of 343 amino acids. The amino acid sequence homology and phylogenetic analysis suggest that WsFPPS has close similarity to its counterparts from tomato (SlFPPS) and capsicum (CaaFPPS). Using semi quantitative RT–PCR, the expression pattern of the WsFPPS gene was analyzed in different tissues of Withania chemotypes (NMITLI-101, NMITLI-108, NMITLI-118 and NMITLI-135) as well as in response to elicitors (salicylic acid and methyl jasmonate) and mechanical wounding. The expression analysis suggests that WsFPPS expression varies in different tissues (with maximal expression in flower and young leaf) and chemotypes (with highest level in NMITLI-101) and was significantly elevated in response to salicylic acid, methyl jasmonate and mechanical injury. This is the first report on characterization of an isoprenoid pathway gene involved in withanolide biosynthesis.
75. Jhankare, A. and Tiwari, G. and Tripathi, M. K. and Pandey, G. N. and Patel, R. P. and Tiwari, S. and Baghel, B. S., Development of Resistant Lines against Leaf Blight Disease of Withania Somnifera (L.) Dunal. Caused by Alternaria Alternata through in Vitro Selection., Plant Cell Biotechnology and Molecular Biology, vol. 12, no. 1/4, pp. 21--30, 2011. url: https://www.cabdirect.org/cabdirect/abstract/20193133638. Abstract
    Disease tolerant/resistant Withania (Withania somnifera (L.) cv JA-20 and MWS-100) cell lines were selected against leaf blight disease caused by Alternaria alternata. For this purpose callus and cell suspension cultures derived from mature embryos and hypocotyl explants were exposed to purified toxic culture filtrate produced by the fungus supplemented with MS culture medium. Two selection...
76. Singh, Satyapal and Tanwer, Babeet Singh and Khan, Moinuddin, Callus Induction and in Vivo and in Vitro Comparative Study of Primary Metabolites of Withania Somnifera, pp. 6, 2011. Abstract
    In present study callus was raised from the leaf explant of Withania somnifera. Maximum callus was obtained on MS medium supplemented with 2, 4- D 1.0 mg/liter and IBA 0.5 mg/liter. The callus and different plant parts were used for primary metabolite quantification and antioxidant activity. Maximum soluble sugars found in callus, maximum amount of starch, protein and phenolic contents were found in stem and maximum lipid found in leaf.
77. Joshi, Aruna Girish and Padhya, Mainavati A., Shoot Regeneration from Leaf Explants of Withania Somnifera (L.) Dunal, Notulae Scientia Biologicae, vol. 2, no. 1, pp. 63--65, March 2010. doi: 10.15835/nsb213609. Abstract
    Regeneration from leaf explants of Withania somnifera (L.) for mass propagation was studied on Murashige and Skoog’s medium supplemented with Kinetin (Kn) and 6-benzylaminopurine (BAP) alone or in combination. Shoot buds were induced from the midrib on the abaxial side in presence of Kn and BAP (4 µM). These shoot buds developed into shoots on the same medium. Rooting of these shoots was achieved in 0.5 µM of IBA.
78. Sinha, Arunima and Jain, Rohit and Kachhwaha, Sumita and Kothari, S. L., Optimization of the Level of Micronutrient Copper in the Culture Medium Improves Shoot Bud Regeneration in Indian Ginseng [Withania Somnifera (L.) Dunal]., National Academy Science Letters, vol. 33, no. 1/2, pp. 11--16, 2010. url: https://www.cabdirect.org/cabdirect/abstract/20113218518. Abstract
    The effect of copper sulphate (CuSO4) on the formation of shoot buds and roots from nodal segments of Withania somnifera was investigated. Nodal segments formed multiple shoots through bud break on MS medium with BAP (22 µM) and IAA (5.7 µM). Formation of shoot buds was positively influenced by the presence of higher levels of copper ions in the medium. Upto 1.9 fold increase in the number of...
79. Dewir, Y. H. and Chakrabarty, D. and Lee, S. -H. and Hahn, E. -J. and Paek, K. -Y., Indirect Regeneration of Withania Somnifera and Comparative Analysis of Withanolides in in Vitro and Greenhouse Grown Plants, Biologia Plantarum, vol. 54, no. 2, pp. 357--360, June 2010. doi: 10.1007/s10535-010-0063-6. Abstract
    The present study reports an efficient protocol for indirect shoot organogenesis and plantlets regeneration of Withania somnifera (L.) Dunal. Leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). The highest callus induction rate (89.5 \%) and shoot regeneration rate (92 \%) were obtained when 2 mg dm−3 BAP was combined with 0.5 mg dm−3 IAA. Three major withanolides (withaferine A, 12-deoxywithastramonolide and withanolide A) were investigated in different plant organs from in vitro and greenhouse grown plants. Leaves contained higher contents of withanolides and phenolics than roots or stems, whereas roots contained the highest contents of flavonoids and polysacharides. In vitro grown plants contained greater contents of phenolics, flavonoids and polysaccharides while lower contents of withanolides than greenhouse grown plants.
80. Shukla, Durga Dutt and Bhattarai, Nabin and Pant, Bijaya, {\emph{In-Vitro}} Mass Propagation of {\emph{Withania Somnifera}} (L.) Dunal, Nepal Journal of Science and Technology, vol. 11, pp. 101--106, 2010. doi: 10.3126/njst.v11i0.4131. Abstract
    Ashwagandha (Withania somnifera L.) Dunal] is an important medicinal plant and a major source of alkaloids and steroids (withanolids), which is regularly used in pharmaceutical industries. Various vegetative parts were studied for its mass propagation through tissue culture technique. Seeds were pretreated with GA3 (50 and 100 mgl-1) for 24 h and 80\% germination was achieved. All the explants were taken from in-vitro germinated plant. Among the different explants tested, multiple shoot formation was achieved from shoot-tip and nodal explants in MS medium + 0.25, 0.5, and 1.0 mgl-1 kinetin. Nodal explants were selected for mass propagation protocol because it formed maximum number of shoots (16.25 shoots per explant) on MS medium + 1mgl-1 kinetin after eight weeks of culture. Increase in concentration of kinetin was most effective for callus formation. For further multiplication these shoots were sub-cultured on MS +0.5 mgl-1 kinetin. Presence of IAA at 0.5 mgl-1 was most effective medium for rooting of in-vitro propagated shoots. However, hardening was not achieved for these propagated plants. Key words: IAA; IBA; NAA; kinetin; in-vitro multiplication DOI: 10.3126/njst.v11i0.4131Nepal Journal of Science and Technology 11 (2010) 101-106
81. Mallubhotla, S. and Ahuja, A. and Kumar, A. and Sharma, R.D. and Verma, V. and Suri, K.A. and Vij, S.P., Field Performance, Rapd Analysis and Withanolide Profiling of Micropropagated Plants of Wsr - an Elite Cultivar of Indian Ginseng (Withania Somnifera L. Dunal), Acta Horticulturae, no. 865, pp. 199--207, June 2010. doi: 10.17660/ActaHortic.2010.865.25.
82. Chitturi, D. and Venisetty, R. K. and Molmoori, R. K. and Kokate, C. K. and Apte, S. S., Enhanced Bioproduction of Withaferin A from Suspension Cultures of Withania Somnifera., Annals of Biological Research, vol. 1, no. 2, pp. 77--86, 2010. url: https://www.cabdirect.org/cabdirect/abstract/20103256568. Abstract
    Callus cultures of Withania somnifera from leaves were established on MS (Murashige and Skoog) media supplemented with Dicamba (2 mg/l), Kinetin (0.1 mg/l) and Sucrose (3\% w/v). Suspension cultures were established and the growth and production kinetics were studied. For growth kinetics MS media supplemented with Dicamba (2 mg/l), Kinetin (0.1 mg/l) and Sucrose (3\% w/v) without agar was used...
83. Sabir, Farzana and Kumar, Anil and Tiwari, Pragya and Pathak, Neelam and Sangwan, Rajender S. and Bhakuni, Rajendra S. and Sangwan, Neelam S., Bioconversion of Artemisinin to Its Nonperoxidic Derivative Deoxyartemisinin through Suspension Cultures of Withania Somnifera Dunal, Zeitschrift für Naturforschung C, vol. 65, no. 9-10, pp. 607--612, October 2010. doi: 10.1515/znc-2010-9-1013. Abstract
    Biotransformation of artemisinin was investigated with two different cell lines of suspension cultures of Withania somnifera. Both cell lines exhibited potential to transform artemisinin into its nonperoxidic analogue, deoxyartemisinin, by eliminating the peroxo bridge of artemisinin. The enzyme involved in the reaction is assumed to be artemisinin peroxidase, and its activity in extracts of W. somnifera leaves was detected. Thus, the non-native cell-free extract of W. somnifera and suspension culture-mediated bioconversion can be a promising tool for further manipulation of pharmaceutical compounds.
84. Pandey, Vibha and Ansari, Waquar Akhter and Misra, Pratibha and Atri, Neelam, Withania Somnifera: Advances and Implementation of Molecular and Tissue Culture Techniques to Enhance Its Application, Frontiers in Plant Science, vol. 8, 2017. doi: 10.3389/fpls.2017.01390. Abstract
    Withania somnifera, commonly known as Ashwagandha an important medicinal plant largely used in Ayurvedic and indigenous medicine for over 3,000 years. Being a medicinal plant, dried powder, crude extract as well as purified metabolies of the plant has shown promising therapeutic properties. Withanolides are the principal metabolites, responsible for the medicinal properties of the plant. Availability and amount of particular withanolides differ with tissue type and chemotype and its importance leads to identification characterization of several genes/ enzymes related to withanolide biosynthetic pathway. The modulation in withanolides can be achieved by controlling the environmental conditions like, different tissue culture techniques, altered media compositions, use of elicitors, etc. Among all the in vitro techniques, hairy root culture proved its importance at industrial scale, which also gets benefits due to more accumulation (amount and number) of withanolides in roots tissues of W. somnifera. Use of media compostion and elicitors further enhances the amount of withanolides in hairy roots. Another important modern day technique used for accumulation of desired secondary metabolites is modulating the gene expression by altering environmental conditions (use of different media composition, elicitors, etc.) or through genetic enginnering. Knowing the significance of the gene and the key enzymatic step of the pathway, modulation in withanolide contents can be achieved upto required amount in therapeutic industry. To accomplish maximum productivity through genetic enginnering different means of Withania transformation methods have been developed to obtain maximum transformation efficiency. These standardized transformation procedues have been used to overexpress/silence desired gene in W. somnifera to understand the outcome and succeed with enhanced metabolic production for the ultimate benefit of human race.
85. Shrivastava, Atul Kumar and Sahu, Pankaj K., Economics of Yield and Production of Alkaloid of Withania Somnifera (L.) Dunal, American Journal of Plant Sciences, vol. 04, no. 10, pp. 2023, September 2013. doi: 10.4236/ajps.2013.410253. Abstract
    The yield parameters and cost economics of Withania somnifera were studied using Integrated Nutrient Management (INM) treatments. Withania somnifera is cultivated in around 10,780 ha with a production of 8429 tones in India. The annual demand of this herb increased from 7028 tones (2001-2002) to 9127 tones (2004-2005). The field experiment was conducted at the research farm of Department of Plant Physiology, College of Agriculture, Jabalpur during Kharif season of 2008-2009 (1st year) and 2009-2010 (2nd year), whereas the laboratory work was carried out in Quality Laboratory, College of Agriculture, Jabalpur. The soil of experimental field was sandy loam in texture with pH 7.5, EC 0.18 dsm-1 having good drainage. Soil analysis revealed that available nitrogen was low (202.0 kg·ha-1) whereas available phosphorus (16.25 kg·ha-1) and potassium (236.0 kg·ha-1) were in the medium range. The present paper shows how to determine the economics of varying INM treatments. Cultivation of W. somnifera in India is gaining popularity among farmers; however, due to poor soil fertility, and costly chemical fertilizers and pesticides its production is not economical or profitable.
86. Anuroopa, N., Inoculation with Selected Microbial Consortia Not Only Enhances Growth and Yield of Withania Somnifera but Also Reduces Fertilizer Application by 25\% under Field Conditions, Proceedings of the Indian National Science Academy, vol. 93, no. INVALID\_SCITE\_VALUE, August 2017. doi: 10.16943/ptinsa/2017/49127.
87. KUMAR, {\relax MITHLESH} and PATEL, {\relax MANUBHAI} and CHAUHAN, {\relax RAVINDRASINGH} and TANK, {\relax CHANDRESH} and SOLANKI, {\relax SATYANARAYAN} and PATEL, {\relax PRAVINBHAI} and BHADAURIA, {\relax HITENDRA} and GAMI, {\relax RAMAN} and PACHCHIGAR, {\relax KAREN} and SONI, {\relax NISHIT} and PATEL, {\relax PRANAY} and SINGH, {\relax ANUJ} and PATEL, {\relax NITIN} and PATEL, {\relax RAMESH}, Elucidation of Genotype–Environment Interactions and Genetic Stability Parameters for Yield, Quality and Agromorphological Traits in Ashwagandha (Withania Somnifera (L.) Dunal), Journal of Genetics, vol. 99, no. 1, pp. 59, July 2020. doi: 10.1007/s12041-020-01207-9. Abstract
    The present study was undertaken to delineate genotype–environment interactions and stability status of 16 genotypes of ashwagandha (Withania somnifera (L.) Dunal) in context to the 12 characters, namely plant height, number of primary branches, number of secondary branches, days to flowering, days to maturity, number of berries, number of seeds/berry, root length, root diameter, root branches, dry root yield and total alkaloid content (\%). Experiment was carried out in a randomized complete block design with three replications over three different locations (S. K. Nagar, Jagudan and Bhiloda) in north Gujarat for three years (2016–17, 2017–18 and 2018–19). Pooled analysis of variance revealed that the mean squares due to genotypes and genotype\,×\,environment interaction along with linear and nonlinear components were highly significant (P{$<$}0.01) for most of the traits under study. Stability parameters for component traits through Eberhart and Russell model showed that genotypes that can be used directly in breeding programme are SKA-4 for early flowering, SKA-21 for early maturity and SKA-1, SKA-4, SKA-6 and SKA-17 for shorter plant height. Further, SKA-21 could be used for improving number of primary branches per plant, SKA-11 and SKA-17 for number of secondary branches per plant, SKA-19 for number of berries per plant, SKA-6, SKA-21, SKA-27 and AWS-1 for root branches and SKA-17 for root length as these genotypes were found to be most stable across the environments for mentioned traits. The result revealed that some reliable predictions about genotype\,×\,environment interaction and its unpredictable components were involved significantly in determining the stability of genotypes. Hence, the present investigation can be exploited for the identification of more productive genotypes in specific environments, leading to significant increase in root productivity of ashwagandha.
88. Ramesh Kumar, R. and Prasanna Anjaneya Reddy, L. and Niranjana Kumar, A. and Komaraiah, K. and Purnanand, S. and Sastry, K. P., Root Textural Quality in Ashwagandha (Withania Somnifera) as Influenced by Crop Growth Periods and Morphotypes, Industrial Crops and Products, vol. 34, no. 1, pp. 1231--1234, July 2011. doi: 10.1016/j.indcrop.2011.03.016. Abstract
    Ashwagandha (Withania somnifera) is a commercially important medicinal crop and roots are the main portion of the plant used therapeutically. The root textural quality determined by high starch–fiber ratio fetches premium price for the produce. Influence of morphological variants and crop growth intervals on accumulation of starch and crude fiber in roots was studied to assess morphotypical differences and to determine the right harvest stage for high root yield with better physical quality. The morphotypes showed similar trend for dry root yield, whereas differences were observed for starch and crude fiber accumulation. The pattern of starch and fiber accumulation varied with different crop growth periods. In general, crude fiber content increased with the crop duration, whereas starch content and starch–fiber ratio followed a trend of decrease–increase–decrease as influenced by different phenophases. Discussions on suitable harvest stage based on dry root yield and starch–fiber ratio are made.
89. KUMAR, {\relax VIVEK} and SOLANKI, {\relax AMAR} and SHARMA, {\relax SHIVESH}, Yield and Economics of Withania Somnifera Influenced by Dual Inoculation of Azotobacter Chroococcum and Pseudomonas Putida, Turkish Journal of Biology, vol. 33, no. 3, pp. 219--223, January 2009. doi: 10.3906/biy-0711-7. Abstract
    The yield parameters and cost economics of Withania somnifera were studied using dual inoculation of Azotobacter chroococcum and Pseudomonas putida. The experiment was conducted in a randomized block design with 3 replications for 2 consecutive years, along with 2 levels of organic manure, 10 t ha−1 and 20 t ha−1. Inoculation with the inoculants generated encouraging results; root yield (1185.6 kg ha−1), seed yield (208.13 kg ha−1), number of offshoots per plant (6.07), and plant height (108.4 cm) were maximum with treatment T6 (organic manure [OM] 20 t ha−1 + both bacteria), followed by T9 (OM 10 t ha−1 + both bacteria), T4 (OM 20 t ha−1 + A. chroococcum), and T5 (OM 20 t ha−1 + P. putida), as compared to T1 (control), T2 (OM 10 t ha−1), and T3 (OM 20 t ha−1). The survival count of inoculated bacteria was highest 70 days after inoculation and declined thereafter. Economic analysis revealed that the net profit from treatment T6 was the highest with dual inoculation of bacteria and OM 20 t ha−1, followed by OM 10 t ha\_1 and dual inoculation of bacteria.
90. Thakur, N S and Verma, Kartar and Rana, Rajinder, Growth and Yield Performance of Ashwagandha (Withania Somnifera) under Agroforestry, Indian Journal of Agricultural Sciences, vol. 84, pp. 937--941, August 2014. Abstract
    Investigations on effect of tree-crop combinations and nitrogen levels on growth, yield and withanolides content and yield of Withania somnifera L. Dunal were carried out during 2005-2006 and 2006-2007, in mid hills of Himachal Pradesh (India). W. somnifera was grown in association with Prunus persica (fruit), Grewia optiva, Morus alba (fodder) and Setaria sphacelata (grass). The distinctive tree-crop combinations (agroforestry systems) formed were Peach + Grewia + Setaria + W. somnifera, Peach + Morus + Setaria + W. somnifera, Peach + Setaria + W. somnifera, Grewia + Setaria + W. somnifera, Morus + Setaria + W. somnifera and W. somnifera as sole crop. Three nitrogen doses, viz. 40, 80 and 120 kg/ha were applied to W. somnifera. The plant height and leaf area of ashwagandha plants was not affected by tree-crop combinations. The nitrogen dose of 120 kg/ha resulted in maximum height (44.55 cm) and leaf area (17.76 cm 2/leaf). Leaf area index was maximum (0.97) for plants grown in association with Peach+Grewia+Setaria. Belowground, aboveground and total biomass yield was not affected significantly by various tree-crop combinations. It ranged from 3.23 to 3.91, 2.30 to 2.63 and 5.53 to 6.46 q/ha, respectively. The N dose 120 kg/ha gave significantly higher below, aboveground and total biomass yield to the tune of 5.23, 3.37 and 8.60 q/ha, respectively. Withanolide content in roots varied from 0.73 to o.79 per cent but was not affected due to different treecrop combinations and nitrogen doses.
91. Kaul, M.K. and Kumar, Arun and Ahuja, Ashok and Mir, B.A. and Suri, K.A. and Qazi, G.N., Production Dynamics of Withaferin A in Withania Somnifera (L.) Dunal Complex, Natural Product Research, vol. 23, no. 14, pp. 1304--1311, September 2009. doi: 10.1080/14786410802547440. Abstract
    Withaferin A (WA) is an important withanolide holding promise in cancer treatment and as a relatively safe radiosensitive/chemotherapeutic agent, which is present in traces in all parts of Withania somnifera except the leaves, where as it is reported to be present in only two non-Indian chemotypes (South African chemotype/Israel chemotype 1). The present studies have marked its presence in all Indian populations (wild/cultivated), as well as two identified Indian chemotypes (AGB002 and AGB025). The quantitative dynamics of WA production in Indian populations and interchemotypic hybrids developed at our institute have been studied, and the results were compared with five previously reported chemotypes from Israel, South Africa and India. An analysis on inheritance characteristics based on presence/absence of WA in hybrid plants and their respective parents is given for future studies on the chemogenetics of this complex species in greater detail. Further, the production potential of WA in vitro propagated plants of elite varieties developed at our institute is discussed, in view of maintaining chemotypic fidelity and stability from a production point of view. Also, evidence-based clues suggesting the leaves as the site of the synthesis of WA is provided.
92. Kubsad, V S and Palled, Y B and Mansur, C P and Channal, H T and Basavaraj, N and Koti, R V, Performance of Ashwagandha (Withania Somnifera Dunal) as Influenced by Dates of Sowing and Stages of Harvesting, pp. 5, 2004. Abstract
    A field experiment was conducted during late kharif seasons of 2004-05 and 2005-06 to find out an optimum date of sowing and stage of harvesting to maximize productivity and enhance the quality of ashwagandha in vertisols under rainfed conditions at Agricultural Research Station, Annigeri (Karnataka). There were twelve treatments consisting of four date of sowing ( Aug. 15th , Aug. 30th , Sep. 15th and Sep. 30th ) and three stage of harvesting (120, 150 and 180 DAS). Pooled results over two years indicated that crop sown on Sep 15th gave more dry root yield by 11.9, 9.1 and 6.3\% and net returns by 14.5, 11.2 and 7.8\% than Aug 15th , Aug 30th and Sep 30th sowings respectively. While the late harvested crop at 180 DAS gave significantly higher dry root yield by 28.2 and 18.8\% over early harvested crop at 120 and 150 DAS respectively. This treatment also provided the highest net returns (Rs. 48,430/ha) compared to others. The total withanolide content and total withanolide yield were maximum with Sep 15th sowing (0.537\% and 7.233 kg/ha respectively) and also with late harvested crop (0.560\% and 8.181 kg/ha respectively) compared to other sowing dates and harvesting stages. Similar trend was observed during both the years.
93. Manohar, S. and Choudhary, M.R. and Yadav, B. L. and Dadheech, S. and Singh, S. P., Analyzing the Efficacy of Organic and Inorganic Sources of Nitrogen and Phosphorus on Growth of Ashwagandha (Withania Somnifera Dunal.), Journal of Horticultural Science, vol. 7, no. 2, pp. 161--165, January 2020. url: https://jhs.iihr.res.in/index.php/jhs/article/view/369. Abstract
    Ashwagandha (Withania somnifera~Dunal.) is an important medicinal plant whose roots are prescribed as medicine for several disorders of females, bronchitis, dropsy, stomach problems, lung inflammation, tuberculosis, arthritis, skin diseases and male impotency. The present experiment was designed to work out a suitable dose of organic manures and fertilizers for ashwagandha. Treatments consisted of nitrogenous (N) and phosphatic (P) fertilizers at 20 kg ha-1~and 40 kg ha-1~each, and two levels of farm yard manure (FYM) and vermicompost and combinations thereof, along with control. The treatments were replicated thrice in Randomized Block Design. Results revealed that application of 40 kg ha-1~of N and P each as urea and SSP + 2.5 t ha-1~vermicompost registered significant values for plant height, number of branches per plant, leaf area, yield attributing traits, root (8.60 q ha-1) and seed yield (85.6 kg ha-1) as well as soil physical properties like organic carbon, hydraulic conductivity and water retention at 33 and 1500kpa besides the highest B:C ratio (2.57).
94. Shrivastava, Atul and Upadhyay, V. and Gautam, Damodar and Sarvade, S. and Sahu, R., Effect of Integrated Nutrient Management on Growth and Productivity of Withania Somnifera (L.) Dunal in Kymore Plateau and Satpura Hills of Madhya Pradesh, India, Archives of Agriculture and Environmental Science, vol. 3, pp. 202--208, June 2018. doi: 10.26832/24566632.2018.0302015. Abstract
    The field experiment was carried out at the Dusty Acres Farm, Jawaharlal Nehru Krishi Vishwa Vidyalaya (JNKVV), Jabalpur, Madhya Pradesh to study the effect of integrated nutrient man�agement for growth and yield of Withania somnifera (L.) Dunal (Variety: Jawahar Ashwagandha- 134). Tallest plants (49.35 cm) were observed at harvest stage, whereas higher number of branches per plant (5.78) of W. somnifera was observed at 90 DAS with T11, respectively. The mean number of leaves per plant (125.40) and LAI (10.345) of W. somnifera was higher at 90 DAS with T11. Mean CGR was maximum (2.536 g m-2 week-1 ) and mean RGR was highest (0.098 gg-1 day-1 ) of W. somnifera at 90 DAS and thereafter decline at 120 DAS and harvest stage with T11. Whereas, mean dry matter (1392.60 kg/ha) production of W. somnifera was higher at harvesting stage of crop with T11. Significantly higher mean root length (16.30 cm), root girth (2.26 cm) and mean dry root yield (612.8 kg/ha) of W. somnifera was recorded under T11 than the rest of treatments. Mean number of berries per plant (30.78) and mean number of seeds per berry (194.17) of W. somnifera were significantly higher under T11. Higher mean seed yield (62.6 kg/ha) and harvest index (43.61\%) of W. somnifera was observed under T11 as compared to other treatments. Therefore, 100\% recommended dose of NPK through fertiliz�ers along with vermicompost (2.5 t/ha), FYM (5.0 t/ha) and ZnSO4 20kg/ha (T11) followed by 50\% recommended dose of NPK through fertilizers along with vermicompost (2.5 t/ha), FYM (5.0 t/ha) and ZnSO4 20kg/ha (T14) and 100\% recommended dose of NPK through fertilizers along with vermicompost (2.5 t/ha) and ZnSO4 20kg/ha (T10) were found to be better integrated nutrient management for the cultivation of W. somnifera. �
95. Pramodkumar, T. and Shiragur, Mukund and Chandrakant, B., Effect of Bio-Inoculants on Growth, Dry Root Yield and Quality in Ashwagandha (Withania Somnifera L. Dunal.), International Journal of Current Microbiology and Applied Sciences, vol. 7, no. 09, pp. 2203--2212, September 2018. doi: 10.20546/ijcmas.2018.709.272.
96. Kumar, Mayank and Kumar, Kaushal and Kumar, Neeraj and {patel}, Arjun, Impact of Different Manures on Ashwagandha (Withania Somnifera Dunal.) Production under Rainfed Condition, International Journal of Current Microbiology and Applied Sciences, vol. 6, no. 10, pp. 50439--5059, October 2017. doi: 10.20546/ijcmas.2017.610.479.
97. Kumar, A. and Jnanesha, A. C. and Kumar M, V. and Lal, R. K., Quantification of Genotype × Environment Interaction of Agronomic Traits in Ashwagandha (Withania Somnifera (L.) Dunal), Acta Ecologica Sinica, July 2022. doi: 10.1016/j.chnaes.2022.07.002. Abstract
    Ashwagandha is a popular industrially medicinal plant used in Ayurveda and other traditional medical systems. We looked at the stability of the sixteen economic traits in the five ashwagandha genotypes in four locations over two years in south India. The pooled ANOVA showed significant differences among different lines of ashwagandha. The five genotypes (G), genotypes × locations interaction (G~×~L), and treatments were highly significant for all sixteen traits. The character's associations directly impact the genotype's ability to sustain stability. The traits X10-VS-X13 and X3-VS-X4 were positively and significantly related across the locations over the three years, according to correlations among the selected traits, indicating that choosing one trait had a beneficial impact on its associated attributes. Except for location I year two, the traits, specifically X11-VS-X13, were highly significant across all locations. The characteristics, specifically X12-VS-X13, were also highly significant, except for location I year I. The genotypes nearest the ideal variety are more desirable than the other. The genotypes W.S. 3 and W.S. 5 for the traits X11 and X16, W.S. 2, and W.S. 5 for the trait X15 were more desirable for selection. In a large amount, both predictable and unanticipated components contributed to the stability. The PCA distribution also showed a significant amount of morphologically based genetic variability. Different genotypes of ashwagandha showed a substantial variation in X15~=~Withaferin-A (\%) and X16~=~Withanolide-A (\%). The percentages of X15~=~Withaferin-A (W.S.2~{$>~$}W.S.5~{$>~$}W.S.~{$>~$}4~W.S.3~{$>~$}W.S.1) and X16~=~Withanolide-A (W.S.3~{$>~$}W.S.5~{$>~$}W.S.4~{$>~$}W.S.2~{$>~$}W.S.1) were shown to be 0.07 to 1.24\% and 0.088 to 1.88\%, respectively. Based on the mean performance of the most economically important traits and stability parameters, the two superior genotypes, W.S. 3 and W.S. 5 were highly stable for dry root yield with high-quality alkaloids. In south India, these genotypes are recommended for further cultivation.
98. Saiyem, {\relax Md}. A. and Sabur, Shaikh A. and Khan, {\relax Md}. A. and Hossain, Mohammad I., Profitability Options of Medicinal Plants Production under Risk: Understanding from Aloe Vera, Bombax Ceiba Root and Withania Somnifera Cultivation in North-West Region, Bangladesh, Journal of Applied Research on Medicinal and Aromatic Plants, vol. 31, pp. 100416, December 2022. doi: 10.1016/j.jarmap.2022.100416. Abstract
    Medicinal plants (MPs) cultivation are an integral part of agricultural diversification, sustainable livelihoods, and profitable options for farmers in Bangladesh. The study is conducted to examine the profitability scenarios of MPs under different risks, i.e. price, yield and market absorption. Primary data were collected from 196 MPs growers of Aloe vera, Bombax ceiba root and Withania somnifera from Natore and Bogura districts of Bangladesh. Monte-Carlo simulation method is applied with considering the embedded risks. Results show that MPs production is profitable alternative to traditional crops in the study area. A. vera production is more profitable compared with B. ceiba root and W. somnifera. The lion share of cost is for human labor followed by seedlings for the selected MPs. Results from Monte-Carlo simulation indicate that there is a significant negative impact on growers’ profits taking into consideration of price, yield and market absorption risks, however, there is a greater possibility of making a profit from A. vera, B. ceiba and W. somnifera production by addressing these risks. The policy should be triggered on taking risk reduction strategies emphasizing more prices and market absorption of MPs by the farmers through establishing contract farming. Farmers should exploit more efficient marketing channels by contracting upstream market actors and avoid traditional long channels that they solely rely on.
99. Jnanesha, A. C. and Kumar, Ashish, Improved Production and Postharvest Technologies in Ashwagandha (Indian Ginseng), 2022. doi: 10.1201/9781003277408-8. Abstract
    Withania somnifera is a miracle medicinal crop used traditionally in Ayurveda and Siddha for treating various diseases in South Asia. The root is the principal productive part of the Ashwagandha plant employed widely in the Indian system of medicine. The root is having potential medicinal properties and contains several alkaloids. One of the major alkaloids, Withanoloides, helps improving reproductive system in men and women, antistress, antioxidant, antiaging, diuretic, hypothyroid, immunomodulatory, antidementia, antihyperglycemic, anti-hypercholesterolemic, and cardiovascular activity. Leaves contain Withaferin A and B used for the treatment of thyroid and insomnia problems. It is drought tolerant cultivated mainly in Andhra Pradesh, Telangana, Karnataka, Rajasthan, Madhya Pradesh, and other states of India. Simple cultivation practices and higher root price attracting the farmer to grow Withania somnifera crop in large scale. The main challenges posed by farmers are interference of middlemen, lack of organized market, fluctuation in root price, demand-supply of roots, climatic variation, lower root yield, lack of availability of improved varieties, high fiber content in some location, lack of knowledge about improved post-harvest technology and problem occurrence with long term storage of roots.
100. Kumar, Mithlesh and Patel, M. P. and Chauhan, R. M. and Tank, C. J. and Solanki, S. D. and Gami, R. A. and Soni, N. V. and Patel, P. C. and Patel, P. T. and Bhadauria, H. S. and Patel, N. B. and Patel, R. M. and Rani, Kirti, Delineating G × E Interactions by AMMI Method for Root Attributes in Ashwagandha [Withania Somnifera (L.) Dunal], INDIAN JOURNAL OF GENETICS AND PLANT BREEDING, vol. 80, no. 04, pp. 441--449, December 2020. doi: 10.31742/IJGPB.80.4.10. Abstract
     In the present study, additive main effects and multiplicative interactions (AMMI) biplot analyses was used to dissect genotype x environment interaction (GEI) and to identify location specific and widely adapted genotypes for root branches, diameter and length in ashwagandha [Withania somnifera (L.) Dunal]. Trials were conducted in randomized complete block design (RCBD) with two replications over three consecutive years at three different locations. ANOVA analysis revealed environment, G×E interaction and genotype effects to contribute significantly (p less than 0.001) towards total sum of squares for root branches (61.00\%, 22.18\% and 14.00\%); root diameter (51.06\%, 24.26\% and 15.34\%) and root length (65.67\%, 20.82\% and 11.39\%). Further, the GEI for these traits was mostly explained by the first, second and third principal component axis (IPCA1, IPCA2 and IPCA3). AMMI1 and AMMI2 biplot analyses showed differential stability of genotypes for root branches, diameter and length with few exceptions. Environmental contribution towards the genotypic performance from AMMI1 and AMMI2 analysis for root traits except environment Bhi16 contribution for root diameter and root length. AMMI1 biplots and simultaneous selection index (SSI) statistics identified SKA-11 as the most desirable genotype for root branches and length while SKA-26 and SKA-27 for root diameter. The ashwagandha genotypes identified for root attributes could be advocated either for varietal recommendation or in varietal development program.
101. Gk, Ningaraju and R, Praveenakumar and G, Gopinath, Integrated Nutrient Management in Ashwagandha (\<Em\>Withania Somnifera\</Em\>): A Review, Journal of Pharmacognosy and Phytochemistry, vol. 7, no. 3S, pp. 118--121, 2018. url: https://www.phytojournal.com/special-issue/2018.v7.i3S.4737/integrated-nutrient-management-in-ashwagandha-ltemgtwithania-somniferaltemgt-a-review. Abstract
     \<em\>Withania somnifera\</em\> commonly called as Ashwagandha is a widely used medicinal herb in Ayurveda. It is considered to be a Rasayana herb, an adaptogen, and is commonly referred to as \‘Indian ginseng\’. It is one of the most important medicinal plant coined with two words \<em\>viz\</em\>., Ashwini and Gandha. Ashwini means horse and Gandha means power. Although an important medicinal plant, it is still seen growing on waste and marginal lands, with little or no manures and fertilizers. Use of organic manures, biofertilizers and inorganic fertilizers has assumed great importance for sustainable production and for maintaining soil health. These not only supply macro- and micro-nutrients to the crop but also improve the physical, chemical and biological properties of the soil, leading to good crop production. The advantage of combining inorganic and organic sources of nutrients generally results in better use of each component which is responsible for crop growth and development. In this paper, the literatures pertaining to the different aspects of integrated nutrient management are reviewed.
102. {HerbalGram - American Botanical Council}, Medicine Hunter Presents: Ashwagandha Harvest, September 2017. url: https://www.youtube.com/watch?v=SnSWLvddqxM. Abstract
     Our mission is to provide education using science-based and traditional information to promote responsible use of herbal medicine — serving the public, researchers, educators, healthcare professionals, industry and media. Disclaimer: This video was produced independently by Chris Kilham. Information provided is for educational purposes. The views expressed by all contributors belong to them and do not necessarily reflect the views of the American Botanical Council.
103. {The Homestead Adventure}, How to Harvest and Dry Ashwagandha, November 2021. url: https://www.youtube.com/watch?v=br20BDL36SY. Abstract
     Ashwagandha is an Ayurvedic herb that has amazing health benefits for your body and mind. It can reduce cortisol levels, increase energy, improve blood sugar levels, balance hormones, reduce inflammation, reduce stress and anxiety, help with thyroid function, improve sleep, support adrenal function, improve brain function, increase muscle mass, boost libido, and so much more!
104. {GuidanceGD}, Ashwagandha Harvest and Processing. Indian Ginseng Root and Seed Collection., April 2021. url: https://www.youtube.com/watch?v=F4fkf0QkhhM. Abstract
     \#Ashwagandha \#ginsengRoot Ashwagandha harvest and processing. Root and Seed collection. ginseng root. Indian ginseng root. Ginseng root cultivation. Ginseng refers to eleven different varieties of a short, slow-growing plant with fleshy roots. Ginseng is believed to restore and enhance wellbeing. It is one of the most popular herbal remedies. Ginseng has traditionally been taken to aid a range of medical conditions. \#Ashwagandha \#Medicinal\_herb In rural areas of India , farmer are cultivating medicinal herbs and getting high profit than other crops. Here in this video you will see how the farmers of West Bengal are cultivating indian ginseng i.e ashwagandha and becoming economically stable. Ashwagandha or indian ginseng ( Withaniasomnifera) is a highly effective ayurvedic medicine. There are many ayurvedic medicines which are prepared by Ashwagandha. The demand and supply of ashwagandha is not equal in the market that's why the price is always high. The avarage price of dried ashwagandha root in the market is Rs. 25000 /- to Rs. 40000/-. So those farmer who want to earn high profit from cultivation may take a chance in ashwagandha. More research is needed to confirm its benefit as a supplement. However, it is claimed that ginsenosides, chemical components found in ginseng, are responsible for the clinical effect of the herb.
105. Tsaltaki, C. and Katsouli, M. and Kekes, T. and Chanioti, S. and Tzia, C., Comparison Study for the Recovery of Bioactive Compounds from Tribulus Terrestris, Panax Ginseng, Gingko Biloba, Lepidium Meyenii, Turnera Diffusa and Withania Somnifera by Using Microwave-Assisted, Ultrasound-Assisted and Conventional Extraction Methods, Industrial Crops and Products, vol. 142, pp. 111875, December 2019. doi: 10.1016/j.indcrop.2019.111875. Abstract
     Heat reflux (CONV), Soxhlet (SOX), microwave-assisted (MAE) and ultrasound-assisted (UAE) extraction methods, using ethanol, water and their mixtures, were studied for the recovery of bioactive compounds from Tribulus terrestris, Panax ginseng, Gingko biloba, Lepidium meyenii, Turnera diffusa and Withania somnifera. The study revealed that SOX method achieved extracts with the highest yieldTPC in terms of the total phenolic content (TPC) (66.65\,±\,1.50\%); MAE and UAE resulted also in extracts with high TPC and shorter treatment times compared to SOX. More specifically, the TPC values for MAE and UAE were 91.783\,±\,0.026\,mg GA/g dw and 81.113\,±\,0.103\,mg GA/g dw, respectively, the antioxidant activity (DPPH) was 63.577\,±\,0.057\,mg Trolox/g dw and 64.923\,mg Trolox/g dw, and the yieldTPC was 60.82\% and 61.89\%, respectively. All were higher compared to those obtained by the CONV method. The use of pure ethanol as the extraction solvent, provided maximum TPC and similar results for DPPH and yieldTPC (\%). Moreover, maximum TPC (183.70\,mg GA/g dw) and DPPH (377.21\,mg Trolox/g dw) values were achieved for T. diffusa. The HPLC analysis of T. terrestris, P. ginseng and G. biloba extracts showed that G. biloba contained its characteristic bioactive compound in the highest concentration (13.512\,mg gingkolide/g herb).
106. Assessment, US EPA National Center for Environmental, Novel Method to Isolate Withaferin A from Withania Somnifera Roots and Its Bioactivity, March 2009. url: https://hero.epa.gov/hero/index.cfm/reference/details/reference_id/7738795.
107. {Al-Doghachi}, Essam H A and Hassan, Abdul-Razzak O and Faris, Nada N, Response of Ashwagandha (Withania Somnifera L.) to Sowing Dates and Organic Liquid Fertilizer (Vit-ORG), Asian Journal of Plant Science and Research, vol. 6, no. 1, pp. 1--7, 2016. Abstract
     The experiment was conducted to study the response of Ashwagandha to sowing dates and organic liquid fertilizer ( Vit-ORG) on chemical components of Ashwagandha sown at Medicinal and Aromatic Plants fields of the College of Agriculture , Basra University ,Iraq, during the growing season of 2013/2014 and 2014/2015. The experiment was laid out in Randomized Complete Block Design in a factorial experiment having three replications. The experiment involved two factors: sowing dates at three levels of 1 or 20 September and 10 October and organic liquid fertilizer ( Vit-ORG) (0 and 2) ml.l-1.Theresults showed that the nutritional contents were significantly affected by sowing dates and organic liquid fertilizer ( Vit-ORG).Percentage of fixed oil and total alkaloid were not significantly affected by sowing dates during both growing seasons. The organic liquid fertilizer ( Vit-ORG) caused increase the percentage of fixed oil in leaves and roots. The interactions between sowing dates and organic liquid fertilizer ( VitORG)gave a significant increase in most studied parameters during both seasons.
108. Basak, B. B. and Saha, Ajoy and Gajbhiye, N. A. and Manivel, P., Potential of Organic Nutrient Sources for Improving Yield and Bioactive Principle of Ashwagandha (Withania Somnifera) through Enhanced Soil Fertility and Biological Functions, Communications in Soil Science and Plant Analysis, vol. 51, no. 6, pp. 779--793, March 2020. doi: 10.1080/00103624.2020.1729368. Abstract
     Ashwagandha (Withania somnifera L. Dunal) is an industrially important medicinal herb and root and is a rich source of bioactive withanolides. To investigate the impacts of different organic nutrient management practices on ashwagandha yield and quality, a field experiment was conducted for 2 consecutive years. The experiment was carried out in randomized bock designed (RBD) with treatments comprised of different organic manures (farmyard manure, vermicompost, and castor cake) and microbial consortium along with recommended doses of fertilizer and the control. The root yield parameters (root girth, length, and fresh and dry root weight) of ashwagandha were influenced significantly by the treatments containing organic manures and microbial consortium alone or in combination. The highest fresh (1505 kg ha−1) and dry (767 kg ha−1) root yields as well as total withanolide contents (0.947 mg g−1) were recorded under the treatment receiving castor cake + microbial consortium (T7) followed by application of vermicompost + microbial consortium (T8). Significant improvements in soil fertility status (mineral N, available P and S) and soil biochemical parameters (microbial biomass and enzymes) were observed under the treatments containing castor cake and vermicompost. A positive correlation coefficient ({$<$} 0.01) was found between dry root yield, withanolide content and soil properties (r = 0.34–0.64). Thus, the results indicate that castor cake and vermicompost could be promising nutrient source for production of quality medicinal herb by improving soil properties.
109. Ahirwar, S.K. and Agrawal, K.K. and Kushwaha, H.S., Growth and Yield of Ashwagandha [Withania Somnifera (L.)] as Influenced by Different Intercropping System in Kymore Plateau of Madhya Pradesh, International Journal of Current Microbiology and Applied Sciences, vol. 8, no. 04, pp. 513--524, April 2019. doi: 10.20546/ijcmas.2019.804.056.
110. Vajantha, B. and Uma Devi, Makam and Chandini Patnaik, Manthri and Rajkumar, M., Growth and Yield of Ashwagandha (Withania Somnifera L.) as Effected by INM and Panchagavya, International Journal of Agricultural Sciences, vol. 8, no. 1, pp. 128--134, January 2012. url: http://researchjournal.co.in/upload/assignments/8_128-134.pdf. Abstract
     Investigations were carried out for two years to study the effect of different levels of NPK (0, 50, 100 and 150\%), organic manures (castor cake @ 2.5 t ha-1 and vermi-compost @ 1 t ha-1), panchagavya and bio fertilizers (Azospirillum and phosphorus solubilising bacteria each of 5 kg ha-1) on growth and root yield of medicinal crop Ashwagandha at College Farm, College of Agriculture, Hyderabad during Rabi 2007-08 and Kharif 2008 by using split plot design. During both the years at flowering and harvest, the highest dry matter production (3314 and 3083 kg ha-1 and 6204 and 5101 kg ha-1) and dry root yield (127 and 125 kg ha-1 and 348 and 333 kg ha-1, respectively) was recorded with conjunctive use of 150 per cent NPK with castor cake @ 2.5 t ha-1.
111. Saranya, S. and Santhi*, R. and Appavu, K. and Rajamani, K., Soil Test Based Integrated Plant Nutrition System for Ashwagandha on Inceptisols, Indian Journal of Agricultural Research, vol. 46, no. 1, pp. 88--90, 2012. url: https://arccjournals.com/journal/indian-journal-of-agricultural-research/.
112. Chaudhary, S.R. and Kumar, Jitendra and Chaudhary, A. P. and Basak, Biraj, Organic Nutrient Sources Influence Yield and Quality of Ashwagandha (Withania Somnifera), and Soil Biochemical Properties, Agrochimica, vol. 61, no. 3, pp. 221--236, July 2017. doi: 10.12871/00021857201735. Abstract
     A field experiment was conducted to study the effect of organic nutrient management on yield and quality parameters in ashwagandha (Withania somnifera) as well as soil biochemical properties. The experiment was laid out in a split plot design with three replications. The organic manures (farmyard manure, vermicompost and castor cake) were applied as main plot treatments, and biofertilizers and jivamrut were applied in different combination as subplot treatments. The results indicate that the application of organic fertilizers invariably improved root growth, yield and total withanolide content in ashwagandha. The highest dry root yield (714.6 kg ha⁻¹) and total withanolide content (0.91 mg g⁻¹) were recorded with application of castor cake, which was at par with vermicompost. Soil available nutrients and biochemical properties were significantly improved under vermicompost and castor cake applications as compared to other treatments. Thus, organic manures, particularly castor cake and vermicompost, could be a promising source of nutrients in organic cultivation of ashwagandha.
113. Mazeed, Abdul and Maurya, Pooja and Kumar, Dipender and Suryavanshi, Priyanka, The Enhancement of Root Yield and Quality of Ashwagandha [Withania Somnifera (L.) Dunal] by Weeds Leaves Extracts, Indian Journal of Weed Science, vol. 54, no. 1, pp. 81--86, 2022. doi: 10.5958/0974-8164.2022.00014.4. Abstract
     Ashwagandha, having multiple therapeutic uses, is a highly valuable medicinal plant for pharmaceutical industry. In order to meet the industrial demand, both quality as well as yield of ashwagandha needs to be improved agronomically. In this study, effect of weed leaves extracts (WLE) as bio-stimulants to improve yield and quality of ashwagandha roots were studied in pot experiments during 2020-21. The treatments consisted of combinations of four commercial preparations with microorganisms (Pusa zinc solubilizing biofertilizer, Pusa Azotobacter liquid biofertilizer, Pusa PSB liquid biofertilizer, Pusa Potash solubilizing liquid biofertilizer) with four weeds [Cyperus rotundus L., Amaranthus viridis L., Echinochloa colona (L.) Link, Digera arvensis Forsk.] leaves extracts. The treated plants exhibited stimulatory responses in growth and physiology, leading to enhanced dry root yield of ashwagandha compared to control. Yield enhancing effects of different treatments, when used separately, without combination was the highest in case of Amaranthus viridis WLE, followed by Digera arvensis WLE and PSB solubilizing biofertilizer, however co-application resulted in synergistic effect. Among different combination of treatments, Pusa PSB liquid biofertilizer + Amaranthus viridis WLE recorded the highest whole ashwagandha plant dry matter production (157.3 g/plant), root fresh weight per plant (65.0 g) and root dry weight (23.0 g). Different bioactive compounds in ashwagandha roots (withanoloides A, withanosides IV and withanone) were also enhanced with this treatment indicating the potentiality of weed leaves extracts as biostimulants, with a possibility to use as a novel eco-friendly approach for enhancing root yield and quality of ashwagandha.
114. von Bieberstein, Philipp and Xu, Ya-ming and Gunatilaka, A. A. Leslie and Gruener, Raphael, Biomass Production and Withaferin A Synthesis by Withania Somnifera Grown in Aeroponics and Hydroponics, HortScience, vol. 49, no. 12, pp. 1506--1509, December 2014. doi: 10.21273/HORTSCI.49.12.1506. Abstract
     The medicinal herb Withania somnifera (L.) Dunal (Solanaceae) was grown in two soilless systems to determine optimal conditions for production of biomass and withaferin A, the major secondary metabolite responsible for its claimed medicinal properties. Withaferin A content was analyzed using high-performance liquid chromatography (HPLC). The results show that there was no statistically significant difference (P {$>$} 0.05; t test) in biomass production between the plants grown aeroponically and hydroponically. Aeroponically grown plants produced an average of 49.8 g dried aerial plant material (DW) (sd 20.7) per plant, whereas hydroponically grown plants produced an average of 57.6 g DW (sd 16.0). In contrast, withaferin A content was statistically higher in plants grown hydroponically. These plants contained an average of 7.8 mg·g−1 DW (sd 0.3), whereas the aeroponically grown plants contained an average of 5.9 mg·g−1 DW (sd 0.6). These results demonstrate that hydroponic techniques are optimal in reproducibly and efficiently generating withaferin A. These findings may be of importance to the natural products industry in seeking to maximize production of biologically active compounds from medicinal plants.
115. Singh, Govind and Dodiya, N and Joshi, Arunabh and Khatik, Champa, Variability, Character Associations and Path Analysis in Ashwagandha (Withania Somnifera (L). Dunal) with Respect to Root Yield and Biochemical Aspects, January 2017. Abstract
     ABSTRACTBackground: The experimental study was carried out to know the genetic variability, characters association,interrelationship and cause and effect of various characters in ashwagandha with respect to dry root yield andbiochemical aspects.Methods: Twenty genotypes (including 3 checks) were evaluated for 13 traits (10 quantitative and 3 qualitative)and analysis of variance, correlation and path analysis were performed for the mean data.Results: All characters were found to be differing significantly among genotypes. Estimates of variability parametersrevealed that a high genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) were foundfor total antioxidants content in root, dry root weight per plant, dry plant weight per plant and harvest index(\%). High heritability (h2) was found for total crude fiber content in root, total antioxidants content in root, plantheight and number of secondary branches per plant. Total antioxidants content in root, dry plant weight and totalcrude fiber content in root were recorded with high genetic advance (GA). A high heritability coupled with highgenetic advance was found for total antioxidants and total crude fiber content in root. Root diameter, plant height,dry plant weight and days to 75\% maturity were shown significant positive correlation with dry root yield perplant. Path coefficient analysis revealed that root diameter, days to75\% maturity and plant height had shown highpositive and direct effect on dry root yield per plant.Conclusion: The heritable variability and estimates of variability can be used for crop improvement. Rootdiameter, days to 75\% maturity, plant height and dry plant weight could be used to select high dry root yieldinggenotypes. Biochemical data (alkaloid and antioxidants content) will be useful to select genotypes of high medicinalvalue.Keywords: Variability, Correlation, Path analysis, Heritability (h2), Genetic advance (GA), Biochemical aspects Variability, Character Associations and Path Analysis in Ashwagandha (Withania somnifera (L). Dunal) with Respect to Root Yield and Biochemical Aspects.
116. Vishal, Goel and B., S. Duhan, Ashwagandha (Withania Somnifera L. Dunal) Crop as Affected by the Application of Farm Yard Manure (FYM) and Inorganic Phosphorus in Typic Torripsamment of Hisar, African Journal of Biotechnology, vol. 13, no. 6, pp. 743--748, February 2014. doi: 10.5897/AJB2013.13538. Abstract
     The vegetative parameters of ashwagandha (viz. plant height, number of primary branches, plant spread and dry weight of shoot) were enhanced significantly with the application of 12.5 mg P2O5 kg-1 soil, whereas; dry weight of roots was enhanced up to the application level of 25 mg P2O5 kg-1 soil. FYM at the rate of 12.5 t ha-1 in combination with 12.5 mg P2O5 kg-1 soil significantly improved all the vegetative parameters, whereas, FYM at the rate of 12.5 t ha-1 in combination with 25 mg P2O5 kg-1 soil significantly enhanced the dry weight of the roots. The alkaloids yield (mg pot-1) in ashwagandha roots increased significantly with the application of 25 mg P2O5 kg-1 soil. The application of FYM at the rate of 12.5 t ha-1 improved the alkaloids yield (mg pot-1) but the significantly highest yield of total alkaloids (mg pot-1) was found in the treatment combination of 12.5 t FYM ha-1 + inorganic-P at the rate of 25 mg P2O5 kg-1 soil as compared with other eleven treatments. Nutrients (NPK) uptake by ashwagandha shoot increased significantly with the application level of 12.5 mg P2O5 kg-1 soil over control whereas in case of ashwagandha roots, the increase in nutrients uptake at the level of 25 mg P2O5 kg-1 soil over control. Application of FYM at the rate of 12.5 t ha-1 + 12.5 mg P2O5 kg-1 soil was the best treatment combination for ashwagandha roots in terms of nutrients uptake. Application of fertilizer-P significantly improved the status of organic carbon, Available-P in post harvest soil but decreased the Available-N, Available-K and DTPA extractable micronutrients (Fe, Cu, Zn and Mn) up to the level of 25 mg P2O5 kg-1 soil. FYM at the rate of 12.5 t ha-1 helped in maintaining the soil fertility status after harvest of the crop alone or in combination with fertilizer-P.
117. Goel, Vaibhav and Duhan, Balraj and Madan, V.K. and Raj, D.E.V. and Prakash, Ram, Effect of Nitrogen and FYM on Yield, Growth Parameters and Quality of Ashwagandha (Withania Somnifera), vol. 16, pp. 101--106, December 2011. Abstract
     Application of 25 mg N/kg soil and FYM 12.5 t/ha significantly increased the mean height of ashwagandha to the tune of41.6 and 8.1\%, respectively, over control. Combined use of 25 mg N/kg soil and FYM @ 12.5 t/ha produced significantly taller plants (47.7 cm) in comparison to other treatments over control (31.7 cm). Application of 25 mg N/kg soil produced average number of primary branches (2.7) over control (1.5). Application of FYM @ 12.5 and 25 t/ha increased the average number of primary branches significantly from 2.0 to 2.3 and 2.6 over control. Application of 25 mg N/kg soil significantly increased the average plant spread from 134.7 to 287.6 cm2over control. The average plant spread also increased from 232.0 to 246.6 and 260.0 cm2 with the application of FYM @ 12.5 and 25 t/ha, respectively, over control. Average fresh and dry weight of ashwagandha roots increased at the tune of 17.4 and 8.9\%, respectively, with the application of 37.5 mg N/kg soil. Similarly, FYM @ 25 t/ha increased the fresh and dry weight of roots to the tune-of 10.2 and 4.3\%, respectively, over control. Application of 25 mg N/kg soil significantly increased mean fresh and dry weight of ashwagandha shoot to the extent of 31.6 and 31.7\%, respectively, over control. Sole application of FYM @ 25 t/ha resulted in significant increase in fresh and dry weight of ashwagandha shoots to the extent of 22.2 and 20.0\% over control. Application of 25 mg N/kg soil increased mean total alkaloids (\%) of ashwagandha roots to the extent of 19.6\% over control. Application of FYM @ 25 t/ha also increased the total alkaloid content significantly upto the extent of 14.8\% over control. Similarly, application of nitrogen @ 25 mg N/kg soil increased the mean total alkaloids (\%) of ashwagandha roots to the extent of 0.61\% over control (0.51\%) and FYM @ 25 t/ha also increased the mean alkaloids yield of ashwagandha roots from 0.54 to 0.62\% over control.
118. Sharma, Rupali and Singh, Hukum and Kaushik, Monica and Nautiyal, Raman and Singh, Ombir, Adaptive Physiological Response, Carbon Partitioning, and Biomass Production of Withania Somnifera (L.) Dunal Grown under Elevated CO2 Regimes, 3 Biotech, vol. 8, no. 6, pp. 267, May 2018. doi: 10.1007/s13205-018-1292-1. Abstract
     Winter cherry or Ashwagandha (Withania somnifera) is an important medicinal plant used in traditional and herbal medicine system. Yet, there is no information available on response of this plant to changing climatic conditions particularly elevated atmospheric CO2 concentrations. Therefore, we conducted an experiment to examine the effect of elevated CO2 concentrations (ECs) on Withania somnifera. The variations in traits of physiological adaptation, net primary productivity, carbon partitioning, morphology, and biomass in response to elevated CO2 concentrations (ambient, 600 and 800~µmol mol−1) during one growth cycle were investigated within the open top chamber (OTC) facility in the foothill of the Himalayas, Dehardun, India. ECs significantly increased photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, soil respiration, net primary productivity and the carbon content of plant tissues (leaf, stem, and root), and soil carbon. Furthermore, ECs significantly enhanced biomass production (root and shoot), although declined night leaf respiration. Overall, it was summarized that photosynthesis, stomatal conductance, water use efficiency, leaf, and soil carbon and biomass increased under ECs rendering the physiological adaptation to the plant. Increased net primary productivity might facilitate mitigation effects by sequestering elevated levels of carbon dioxide. We advocate further studies to investigate the effects of ECs on the accumulation of secondary metabolites and health-promoting substances of this as well as other medicinal plants.
119. Joshi, Chandrika and Gajbhiye, Narendra and Phurailatpam, Arunkumar and Geetha, K. A. and Maiti, Satyabrata, Comparative Morphometric, Physiological and Chemical Studies of Wild and Cultivated Plant Types of Withania Somnifera (Solanaceae), Current Science, vol. 99, no. 5, pp. 644--650, 2010. url: https://www.jstor.org/stable/24108318. Abstract
     Ashwagandha (Withania somnifera Dunal.) is an important medicinal plant cultivated in about 4000 ha in India. It is mainly distributed in the northwestern region of Madhya Pradesh and also in limited areas of different states. However, wild collection of the species continues to be a source of raw drug in Ayurvedic preparations. In the present communication, results of a comparative study of the wild type distributed locally in Anand, Gujarat and the superior variety, Jawahar Ashwagandha (JA-20), are presented. Results revealed conspicuous differences between the cultivated and wild-type plants in most of the characters studied. One of the major differences between cultivated and wild-type plants is that the former are annual, whereas the latter are perennial. Photosynthetic rate was higher in the wild type, which was reflected in its higher biomass production. Another distinguishing character was the floral structure which favours self-pollination in the cultivated plants because of short stigma covered with anther lobes, which is in contrast to the wild type having long, projected stigma inviting cross-pollination. The cultivated plants were in full bloom during December–February; however, in the wild type flowering was a continuous process throughout its lifespan. Flow cytometer study revealed the same ploidy level for both the plant types. However, chemical profile showed variation between the two plant types, even though targeted chemical constituents tested in the study were common to both. However, HPLC quantification of these constituents showed superiority of the wild type compared to JA-20.
120. Parihar, Pratibha and Bora, Madhumati, Effect of Mycorrhiza (Glomus Mosseae) on Morphological and Biochemical Properties of Ashwagandha (Withania Somnifera) (L.) Dunal, Journal of Applied and Natural Science, vol. 10, no. 4, pp. 1115--1123, December 2018. doi: 10.31018/jans.v10i4.1797. Abstract
     Mycorrhizal inoculation in the plant causing increase in growth and production of phytochemicals is well reported, however little information is available related to the effect of mycorrhiza on morphological and biochemical properties of the medicinal plants like Ashwagandha. The present study is an attempt on diversity analysis in Withania somnifera with an aim to ascertain the nature and extent of genetic diversity present among different accessions in presence of mycorrhiza. The major biochemical constituents of Ashwaganda roots are with nolides which are well known for its medicinal properties. Mycorrhizal associations confer benefits like better nutrition acquisition, enhanced growth, defense enhancement and improved abiotic and biotic stress tolerance in plants. The present investigation was undertaken to assess genetic diversity among five different accessions of W. somnifera using morphological and biochemical markers and the effect of mycorrrhizal inoculation on these marker. The present study concluded that presence of mycorrhiza was effective on plant growth and phytochemical constituents more than non-treated plants. Amongst five selected germplasms IC 283662, JA 134, RAS 23, MPAS 6 and MWS 205 of W. somnifera, JA 134 showed best response in pretext of the selected morphological and biochemical features in presence of mycorrhiza.
121. Choyal, Prince and Tomar, Maharishi and Rana, Virendra Singh and Suthar, Manish Kumar and Tripathi, Kailashpati and Kalariya, Kuldeepsingh A. and Manivel, Ponnuchamy and Singh, Bhupinder, Chemical Manipulation of Source and Sink Dynamics Improves Significantly the Root Biomass and the Withanolides Yield in Withania Somnifera, Industrial Crops and Products, vol. 188, pp. 115577, November 2022. doi: 10.1016/j.indcrop.2022.115577. Abstract
     Roots are the main economic part of Withania somnifera having high commercial value in the pharmaceutical industry. Since W. somnifera has the indeterminant type of growth habit, it exhibits a continuous berry formation which ultimately reduces the root yield. The present study was undertaken to determine the effect of altered source - sink relationship by regulating the sink availability to increase the root yields. Maleic hydrazide (MH @ 200, 500 and 1000 μmol mol−1) and Gibberellic acid (GA @ 500 and 1000 μmol mol−1) were used as chemical gametocide for inducing male sterility while mechanical deflowering was used to prevent the berry formation. In all three approaches, the root biomass and withanolides content, in general, were significantly increased. The dry matter partitioning pattern showed that only 6\% of total biomass was accumulated in the roots whereas 37\% accululated in the shoot and a significant amount i.e., 57\% was partitioned to the berries under control condition. An application of MH and GA and the mechanical deflowering treatment, however, changed this pattern in favour of the roots. As much as 26\% of the total biomass was partitioned to the roots under the MH treatments. Whereas, GA (500 μmol mol−1) and mechanical deflowering treatment increased the root biomass by 75 and more than 300\% respectively. More than 200\% increase in withaferin A and ∼100\% increase in withanolide A content was observed in leaves under the GA (500 μmol mol−1) treatment. Mechanical deflowering treatment was also equally efficient as GA in increasing the withanolides both in the leaves and the roots. The photosynthetic traits, total sugars and starch content, and sugar metabolizing enzyme analysis showed that modification in source-sink relationship through inhibited berry formation triggers a higher partitioning of carbon assimilates from source to the roots and thus improves significantly the root biomass and the withanolide yields in W. somnifera. The same was validated through the radiotracer studies involving 14C.
122. Sardarkrushinagar, Gujarat and Balakrishnan, Adithya P and Patel, N. B. and {Dept. of Genetics and Plant Breeding, C. P. College of Agriculture, S. D. Agricultural University, Sardarkrushinagar, Gujarat (385 506), India} and Patel, M. P. and {Pulses Research Station, S. D. Agricultural University, Sardarkrushinagar, Gujarat (385 506), India}, Assessment of Heterosis for Root Yield and Attributing Traits in Ashwagandha [Withania Somnifera (L.) Dunal], International Journal of Bio-resource and Stress Management, vol. 13, no. 1, pp. 93--98, January 2022. doi: 20220209025844892. Abstract
     Astudy was conducted was conducted to estimate the extent of heterosis for root yield and attributing traits in ashwagandha at the Botanical Garden, Department of Genetics and Plant Breeding, C. P. C. A, S. D. Agricultural University, Sardarkrushinagar, Gujarat, India. Thirty six hybrids were generated from the diallel mating design of 9 diverse parents during October 2018 – March 2019 (rabi season). Fourty five entries were evaluated in RBD with 3 replications during October 2019–March 2020 (rabi season) for the yield and attribuiting traits. The root traits are given more emphasis because of its economic value.All the forty-five genotypes exhibited moderate to good mean performance along with an adequate amount of variability among and between the parents and hybrids. Most of the hybrids showed highly significant better parent and standard heterosis in desirable direction for the traits considered. The hybrids like SKA 10×JA 20, JA 134×AWS 1, SKA 24×SKA 26, SKA 24×AWS 1, SKA 11×AWS 1, SKA 11×SKA 26 and JA 20×AWS 1 were with a markedly significant heterotic response for fresh root yield trait. The results also suggest that the cross JA 134×AWS 1is appropriate to exploit heterosis in root length, fresh root yield and dry root yield. Further evaluation and generation advancement of these successful crosses can derive transgressive segregants and hybrids with high yield potential for commercialization.
123. Lal, R. K., Quantification of Adaptability and Stability among Genotypes/Cultivars for Root Yield in Ashwagandha (Withania Somnifera L.), Industrial Crops and Products, vol. 77, pp. 648--657, December 2015. doi: 10.1016/j.indcrop.2015.09.035. Abstract
     Ashwagandha (Withania somnifera L.), is one of the very important medicinal plant in Ayurveda since ancient times. It is a native of Mediterranean region grows wildly in arid and semi arid parts of India. Under the germplasm enhancement programme, thirty genetic stocks/cultivars of ashwagandha originated from selective divergence belongs to eight states of India (U.P., M.P., Rajasthan, Maharashtra, Kerala, J and K., A.P. and W.B.) were evaluated in the four consecutive years for the estimation of stability/adaptability. Using the AMMI model, ashwagandha genetic stocks namely, W 20, W 1 (cv. Pratap), W 2, W 3 (cv. Chetak), W 4 and W 6 (cv.Poshita) were expressed the high adaptability and stability for root yield over years. Therefore, above six selected genetic stocks/varieties recommended for commercial cultivation in India on large scale.
124. Sangwan, O. and Avtar, Ram and Singh, Amit, Genetic Variability, Character Association and Path Analysis in Ashwagandha [Withania Somnifera (L.) Dunal] under Rainfed Conditions., Research in Plant Biology, vol. 3, no. 2, pp. 32--36, 2013. url: https://www.cabdirect.org/cabdirect/abstract/20133182722. Abstract
     Twenty six diverse genotypes of ashwagandha [Withania somnifera (L.) Dunal] were evaluated for fourteen quantitative traits under rainfed conditions. Both phenotypic and genotypic coefficients of variation were higher for important traits like number of berries/plant, fresh weight of berries/plant, biomass yield at maturity and ratio of fresh above ground biomass: fresh root biomass. High...
125. Kumar, Ramesh R. and Reddy, Anjaneya Prasanna L. and Subbaiah, Chinna J. and Kumar, Niranjana A. and Prasad, Nagendra H. N. and Bhukya, Balakishan, Genetic Association among Root Morphology, Root Quality and Root Yield in Ashwagandha (Withania Somnifera), Genetika, vol. 43, no. 3, pp. 617--624, 2011. doi: 10.2298/GENSR1103617K. Abstract
     Ashwagandha (Withania somnifera) is a dryland medicinal crop and roots are used as valuable drug in traditional systems of medicine. Morphological variants (morphotypes) and the parental populations were evaluated for root - morphometric, quality and yield traits to study genetic association among them. Root morphometric traits (root length, root diameter, number of secondary roots/ plant) and crude fiber content exhibited strong association among them and showed significant positive genotypic correlation with yield. Starch-fiber ratio (SFR), determinant of brittle root texture showed strong negative association with root yield. The total alkaloid content had positive genotypic correlation with root yield. So genetic upgradation should aim at optimum balance between two divergent groups of traits i.e. root yield traits (root morphometric traits and crude fiber content) and root textural quality traits (starch content and SFR) to develop superior genotypes with better yield and quality.
126. Venugopal, S. and Padma, M. and Rajkumar, M. and Seenivasan, N. and Saidaiah, P. and Sathish, G. and Jnanesha, A. C. and Kumar, R. S. and Kumar, A. and Srivastava, J. and Kumari, M. and Lal, R. K., Comparative Investigation of the Genetic Components of Withania Somnifera L. Yield and Quality Traits Using Multivariate and Associations Analysis in the Deccan Plateau Region, Industrial Crops and Products, vol. 188, pp. 115696, November 2022. doi: 10.1016/j.indcrop.2022.115696. Abstract
     The vital herb ashwagandha was farmed for commercial purposes in India. This study aimed to estimate the nature and amount of genetic variation in the 20 attributes in the 29 genotypes of ashwagandha in order to understand the correlations and character contributions to dry root weight/plant and alkaloid content with the selection of potential parents for ashwagandha crop improvement. Twenty-nine ashwagandha genotypes were assessed using a range of genetic metrics, including analysis of variance (ANOVA), genetic variability components, correlation, principal component analysis (PCA), and Mahalanobis D2. The ANOVA confirms a large amount of variability in the genotypes/varieties under study. Out of twenty the four traits, namely fresh leaf weight per plant (X8), dry leaf weight per plant (X9), seed yield per plant (X13), and dry root weight per plant (X16)—indicates the presence of additive gene action in the current study were highly heritable and had a high genetic advance mean value. Starch estimation, main root length, and root diameter were found to be positively and significantly correlated with dry root production per plant and total alkaloid content. The principle component analysis (PCA) results showed that the first four PCA accounted for the bulk (79.56\%) of the differences. All genotypes are divided into four clusters based on the distances between and within clusters. Based on the findings mentioned above, it is recommended that genotypes from clusters IV (Poshita, CIMAP-Chetak), cluster II (AKAS-10, AKAS-11, NMITLI-101, and NMITLI-118), and cluster I (AKAS-02, IC-286632, and CIMAP Pratap) be chosen for a different hybridization program. Based on the multidisciplinary investigation, some of the genotypes with desired qualities have been found. They can be employed in upcoming breeding programs to increase alkaloid content and dry root yield.
127. Shah, Sonal and Saravanan, Raju and Gajbhiye, Narendra Atmaram, Phytochemical and Physiological Changes in Ashwagandha (Withania Somnifera Dunal) under Soil Moisture Stress, Brazilian Journal of Plant Physiology, vol. 22, pp. 255--261, 2010. doi: 10.1590/S1677-04202010000400005. Abstract
     Ashwagandha, commonly known as the Indian Ginseng (Withania somnifera) is an ingredient in many herbal preparations used for healing various ailments. It is cultivated in semi-arid regions of India as rain-fed crop. The response of this crop (cv. JA-134) to progressive soil moisture deficit on growth, physiology and content of phytochemicals was studied. At the end of treatment period, soil water potential (Ψw) decreased to -10.93, -1.15 MPa at 30 cm depth, predawn leaf water potentials were reduced to -0.62 and -0.51 MPa in the severe and moderate stress. Leaf area was reduced by 27.4\% and 34.4\% in moderate and severe water stress treatment compared to control. Chlorophyll content was reduced by 60\% in the severe water stress. Changes in the steroidal alkaloids and lactones that are the major medicinal active principles derived from root extracts of this plant species was also analyzed. A reduction of 52.6\% in net photosynthesis was noticed under severe stress. At the end of the treatment period, moderate and severe stress treatments had 0.751 and 0.714 mg g-1 12-deoxywithastramonolide (12-DWS) content in the roots respectively. The content of 12-DWS and withanolide were at 77\% and 93\% of the control in the moderate and severe stress, respectively, while yield of these compounds were at 65\% and 78\% of control in mild and severe stress. Thus, preferential partitioning of biomass in roots leads to higher root yield in moderate stress. Higher root yield partially offset the loss of bioactive compounds under soil moisture stress.
128. Kaul, Sunil and Ishida, Yoshiyuki and Tamura, Kazuya and Wada, Teruo and Iitsuka, Tomoko and Garg, Sukant and Kim, Mijung and Gao, Ran and Nakai, Shoichi and Okamoto, Youji and Terao, Keiji and Wadhwa, Renu, Novel Methods to Generate Active Ingredients-Enriched Ashwagandha Leaves and Extracts, PLOS ONE, vol. 11, pp. e0166945, December 2016. doi: 10.1371/journal.pone.0166945. Abstract
     Ashwagandha (Withania somnifera) is an Ayurvedic herb commonly used in world-renowned traditional Indian home medicine system. Roots of Ashwagandha have been traditionally known to possess a variety of therapeutic and health promoting potentials that have not been sufficiently supported by laboratory studies. Nevertheless, most, if not all, of the preventive and therapeutic potentials have been assigned to its bioactive components, steroidal alkaloids and lactones. In contrast to the traditional use of roots, we have been exploring bioactivities in leaves of Ashwagandha. Here, we report that the leaves possess higher content of active Withanolides, Withaferin-A (Wi-A) and Withanone (Wi-N), as compared to the roots. We also established, for the first time, hydroponic cultivation of Ashwagandha and investigated the effect of various cultivation conditions on the content of Wi-A and Wi-N by chemical analysis and bioassays. We report that the Withanone/Withaferin A-rich leaves could be obtained by manipulating light condition during hydroponic cultivation. Furthermore, we recruited cyclodextrins to prepare extracts with desired ratio of Wi-N and Wi-A. Hydroponically grown Ashwagandha and its extracts with high ratio of withanolides are valuable for cancer treatment.
129. Nigam, Nidhi and Yadav, Vineet and Khare, Puja and Singh, Raghuvendra Pratap and Das, Paurabi and Shanker, Karuna and Sharma, Ram Suresh, Exploring the Benefits of Biochar over Other Organic Amendments for Reducing of Metal Toxicity in Withania Somnifera, Biochar, vol. 1, no. 3, pp. 293--307, September 2019. doi: 10.1007/s42773-019-00023-4. Abstract
     Biochar is considered as a good metal sequester and ameliorates the metal toxicity and uptake in plants. However, its effectiveness over other organic amendments is not well discussed. The aim of this study is to compare the biochar amendments alleviating the levels of Pb and Cd in Withania somnifera L. Dunal with other organic manure. Farmyard manure, vermicompost, and biochar were applied in the field spiked with highly available Pb and Cd. Metal bioavailability and accumulation, plant growth and plant physiology, antioxidant enzymes and metabolite content of W. somnifera along with soil properties were evaluated in all treatments. Study indicates that the application of organic manures significantly alleviated the metal levels (33–72\%) in the plant in comparison with control. The herbage yield was significantly higher (33–69\%) under different organic manures as compared to the metal treatments. Secondary metabolite contents and antioxidant enzymes were higher in metal treatments in comparison with control. Organic manures were not only able to restrain the Pb and Cd in soil but also enhanced the soil microbial activities. Results indicate that biochar amendments were more promising than farmyard manure and vermicompost due to the presence of more stable carbon in biochar and more alleviation in metal uptake. In addition, net profit in the cultivation of W. somnifera was higher for biochar amendments (50\% higher) compared to control. The study recommended that biochar could be a better option for commercial and safer production of W. somnifera.
130. Gupta, Manjul and Srivastava, Pankaj Kumar and {Shikha} and Niranjan, Abhishek and Tewari, Shri Krishna, Use of a Bioaugmented Organic Soil Amendment in Combination with Gypsum for Withania Somnifera Growth on Sodic Soil, Pedosphere, vol. 26, no. 3, pp. 299--309, June 2016. doi: 10.1016/S1002-0160(15)60044-3. Abstract
     Limited availability of organic matter is a problem to sustain crop growth on sodic soil. Organic soil amendments are a cost-effective source of nutrients to enhance crop growth. A field study was conducted to evaluate the effect of an organic soil amendment bioaugmented with plant growth-promoting fungi (SFOA) in combination with gypsum on soil properties and growth and yield attributes of Withania somnifera, one of the most valuable crops of the traditional medicinal system in the world, on a sodic soil at the Aurawan Research Farm of CSIR-National Botanical Research Institute, Lucknow, India. The SFOA used was prepared by pre-enriching farm waste vermicompost with plant growth-promoting fungi before mixing with pressmud and Azadirachta indica seed cake. The application of SFOA at 10 Mg ha−1 after gypsum (25.0 Mg ha−1) treatment significantly (P {$<$} 0.05) increased root length (by 96\%) and biomass (by 125\%) of Withania plants compared to the control without SFOA and gypsum. Similarly, the highest withanolide contents were observed in leaves and roots of Withania plants under 10 Mg ha−1 SFOA and gypsum. Combined application of SFOA and gypsum also improved physical, chemical and enzymatic properties of the soil, with the soil bulk density decreasing by 25\%, water-holding capacity increasing by 121\%, total organic C increasing by 90\%, pH decreasing by 17\% and alkaline phosphatase, β-glucosidase, dehydrogenase and cellulase activities increasing by 54\%, 128\%, 81\% and 96\%, respectively, compared to those of the control. These showed that application of the SFOA tested in this study might reclaim sodic soil and further support Withania cultivation and results were better when the SFOA was applied after gypsum treatment.
131. Kumar, Rajesh and Das, Amar Jyoti and Juwarkar, Asha A., Reclamation of Petrol Oil Contaminated Soil by Rhamnolipids Producing PGPR Strains for Growing Withania Somnifera a Medicinal Shrub, World Journal of Microbiology and Biotechnology, vol. 31, no. 2, pp. 307--313, February 2015. doi: 10.1007/s11274-014-1782-1. Abstract
     Soil contaminated by hydrocarbons, cannot be used for agricultural intents due to their toxic effect to the plants. Surfactants producing by plant growth promotory rhizobacteria (PGPR) can effectively rig the problem of petroleum hydrocarbon contamination and growth promotion on such contaminated soils. In the present study three Pseudomonas strains isolated from contaminated soil identified by 16S rRNA analysis were ascertained for PGPR as well as biosurfactants property. Biosurfactants produced by the strains were further characterized and essayed for rhamnolipids. Inoculation of the strains in petrol hydrocarbon contaminated soil and its interaction with Withania somnifera in presence of petrol oil hydrocarbons depict that the strains helped in growth promotion of Withania somnifera in petrol oil contaminated soil while rhamnolipids helped in lowering the toxicity of petrol oil. The study was found to be beneficial as the growth and antioxidant activity of Withania sominfera was enhanced. Hence the present study signifies that rhamnolipids producing PGPR strains could be a better measure for reclamation of petrol contaminated sites for growing medicinal plants.
132. Pandey, Rakesh and Mishra, A.K. and Tiwari, S. and Kalra, A., Nematode Inhibiting Organic Materials and a Strain of Trichoderma Harzianum Effectively Manages Meloidogyne Incognita in Withania Somnifera Fields, Biocontrol Science and Technology, vol. 21, no. 12, pp. 1495--1499, December 2011. doi: 10.1080/09583157.2011.625396. Abstract
     The usefulness of Trichoderma harzianum was tested along with farmyard manure, cow urine, neem oil seed cake, and vermicompost separately and in combination to manage Meloidogyne incognita in Withania somnifera. A treatment combination of nematode inhibitory vermicompost and T. harzianum was found to be most effective against M. incognita.
133. Abhilash, P. C. and Singh, Nandita, Withania Somnifera Dunal-Mediated Dissipation of Lindane from Simulated Soil: Implications for Rhizoremediation of Contaminated Soil, Journal of Soils and Sediments, vol. 10, no. 2, pp. 272--282, March 2010. doi: 10.1007/s11368-009-0085-x. Abstract
     Lindane is an organochlorine chemical that has been used both as an agricultural insecticide and as a treatment for head lice and scabies. It is a neurotoxin that interferes with GABA neurotransmitter function. In humans, lindane primarily affects the nervous system, liver, and kidneys and may be a carcinogen and/or endocrine disruptor. Currently, India is the largest consumer and producer of lindane in the world. Due to its continuous use and indiscriminate industrial production, lindane-contaminated soils are widespread in the country. Apart from India, historical lindane production sites were found in Austria, France, Spain, Bulgaria and in China, Turkey, and the former USSR. Before 1984, lindane was also manufactured in the German Democratic Republic, Poland, Yugoslavia, Romania, and Hungary; since then, all production has been stopped in Germany, Japan, The Netherlands, the UK, and the USA. Because of its worldwide use for more than 50~years, lindane-contaminated soils can be found in most countries of the world. Although many countries have restricted or eliminated its usage, obsolete stock piles continue to pose a threat to various ecosystems and human health. Physical, chemical, and biological methods can all be used for the remediation of contaminated sites, but phytoremediation is now recognized as a cost-effective method for the decontamination of soil sites. The present study examines the potential of Withania somnifera Dunal (previously shown to accumulate lindane from contaminated industrial area; Abhilash et al., Chemosphere 72:79–86, 2008) to take up lindane (γ-HCH) and the subsequent plant-mediated dissipation of lindane from an artificially contaminated soil.
134. Kaur, Amandeep and Kaur, Arvinder and Ohri, Puja, Combined Effects of Vermicompost and Vermicompost Leachate on the Early Growth of Meloidogyne Incognita~Stressed Withania Somnifera (L.) Dunal, Environmental Science and Pollution Research, vol. 29, no. 34, pp. 51686--51702, July 2022. doi: 10.1007/s11356-022-19264-1. Abstract
     Roots of Withania somnifera, an important medicinal herb, are prone to the infection of Meloidogyne incognita (a root parasitic nematode). The infection lowers the quality and quantity of plant material and poses a challenge in crop cultivation and obtaining desirable yield. In the present study, in vitro inhibitory activity of vermicompost leachate (Vcom-L) was assessed against \% hatch and survival of M. incognita in a 96~h assay. Then, Vcom-L was used as soil supplement in combination with vermicompost (Vcom) to evaluate their nematode inhibitory and stress alleviating effect in W. somnifera, in a pot experiment. Root galling intensity and growth performance of nematode-stressed W. somnifera raised from seeds pre-soaked in distilled water (DW), Vcom-L, vermicompost tea (Vcom-T) and different dilutions of Vcom-L were assessed. We observed 79\% suppression of egg hatching and 89\% juvenile (J2) mortality after 96~h compared to control, at 100\% concentration of Vcom-L. Significant reduction in gall formation with increase in growth parameters of seedlings was observed after combined application of Vcom (60\% or 100\%)\,+\,Vcom-L and was evident as enhancement in seedling biomass and contents of chlorophyll and protein. However, proline, total phenolics and malondialdehyde (MDA) content declined significantly in these combinations compared to the control (0\% Vcom). Activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidise (APX), guaiacol peroxidise (GPX) and glutathione reductase (GR) declined with Vcom as well as Vcom\,+\,Vcom-L and corresponded with decline in the accumulation of reactive oxygen species in leaves. Further, 1:5 and 1:10 dilutions of Vcom-L in combination with Vcom (60\%) proved beneficial in mitigating the nematode-induced stress in W. somnifera. Present results showed the potential of Vcom and Vcom-L in standardised combination as an effective strategy in controlling the pathogenicity of M. incognita in medicinal plants such as W. somnifera.
135. Qadir, Sami Ullah and Raja, Vaseem and Siddiqui, Weqar A. and Alyemeni, Mohammed Nasser and Ahmad, Parvaiz, Foliar Concentrations of Selected Elements, Assessment of Oxidative Stress Markers and Role of Antioxidant Defense System Is Associated with Fly Ash Stress Tolerance in Withania Somnifera, Journal of Plant Growth Regulation, vol. 40, no. 4, pp. 1450--1465, August 2021. doi: 10.1007/s00344-020-10200-6. Abstract
     Increased dependence on thermal power has resulted in a significant increase in the generation of fly ash~(FA), which exacerbates environmental pollution. In order to mitigate this source of pollution, we propose covering FA dumps with a layer of planted vegetation. Due to varying degrees of tolerance and their sessile nature, plants are themselves susceptible to stress from pollution. This suggests that an investigation to assess the role of wild growing plants for management of FA dumps, where selected wild plants could be grown to mitigate consequences of FA. The present study assesses oxidative damage and the foliar concentration of metals in Withania somnifera growing wild at the Badarpur Thermal Power Plant~(BTPP) compared to those growing at a control site. Plants growing at the BTPP showed significantly higher foliar concentrations of Pb, Mn, and Fe, and low concentrations of Ni and Cd. The plants at the BTPP site showed signs of oxidative stress as indicated by enhanced levels of malondialdehyde and electrolyte leakage from cells. The CO2 assimilation rate, net photosynthetic rate, rate of transpiration, stomatal conductance decrease, while water use efficiency, and air pollution tolerance index increase. Among air pollution tolerance index parameters, relative water content showed a significant increase with FA pollution stress at the BTPP. A significant decrease was observed in leaf morphology single leaf area, leaf length, and leaf width and biochemical parameters (Chlorophyll a, Chlorophyll b, total chlorophyll, and carotenoids. Moreover, FA pollution stress induces oxidative stress in W. somnifera through a significant and enhanced production of reactive oxygen species~(ROS). According to our observations, the ability of W. somnifera to effectively coordinate superoxide dismutase, ascorbate peroxidase, and glutathione reductase activities involved in the scavenging of ROS along with the enhanced increment of nonenzyme activities (total ascorbic acid, proline, and oxidized glutathione) could be related to FA stress tolerance in W. somnifera.
136. Sahu, Sharda and Prakash, Anil and Shende, Kishor, Talaromyces Trachyspermus, an Endophyte from Withania Somnifera~with Plant Growth Promoting Attributes, Environmental Sustainability, vol. 2, no. 1, pp. 13--21, March 2019. doi: 10.1007/s42398-019-00045-5. Abstract
     The medicinal plant, Withania somnifera is attributed by valuable medicinal properties and is widely cultivated. It is a need to take care of this plant from synthetic agrochemicals that may be hazardous for health and environment. The aim of the present study was to isolate and screen the endophytic fungi of W. somnifera that ~have potential of plant growth promotion and antagonism against plant pathogens. In this study, 22 potential fungal endophytes comprising of species of Alternaria, Aspergillus, Fusarium, Nigrospora, Colletotrichum and Talaromyces identified at National Fungal Culture Collection of India (NFCCI), Pune~were isolated. The potential isolate, Talaromyces trachyspermus was confirmed by BLAST and phylogenetic analysis of sequences of rDNA ITS, LSU (D1 D2) and β-tubulin genes. Among all the isolates, T. trachyspermus exhibited comparatively higher activity for hydrolytic enzymes, protease, chitinase, amylase, cellulase and pectinase that are required for antagonistic property. It was~observed to be a~promising biocontrol agent against plant pathogen, Sclerotinia sclerotiorum. This strain is also characterized with high level of indole acetic acid (IAA), siderophore synthesis, and phosphate solubilization activities~that are important for plant growth promotion. This is the first report on endophyte, T. trachyspermus from W. somnifera having potential plant growth promoting traits and biocontrol, which can be further exploited to enhance the medicinal value of the plant.
137. Mishra, Bhawana and Chand, Sukhmal and Singh Sangwan, Neelam, ROS Management Is Mediated by Ascorbate-Glutathione-α-Tocopherol Triad in Co-Ordination with Secondary Metabolic Pathway under Cadmium Stress in Withania Somnifera, Plant Physiology and Biochemistry, vol. 139, pp. 620--629, June 2019. doi: 10.1016/j.plaphy.2019.03.040. Abstract
     Being static, plants are frequently exposed to various essential and non-essential heavy metals from the surroundings. This exposure results in considerable ROS generation leading to oxidative stress, the primary response of the plants under heavy metal stress. Withania somnifera is a reputed Indian medicinal plant in Ayurveda, having various pharmacological activities due to the presence of withanolides. The present study deals with the understanding endurance of oxidative stress caused by heavy metal exposure and its management through antioxidant partners in synchronization with secondary metabolites in W. somnifera. The quantitative assessment of enzymatic/non-enzymatic antioxidants revealed significant participation of ascorbate-glutathione-α-tocopherol triad in ROS management. Higher activities of glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) resulted in glutathione and ascorbate accumulation. In addition, superoxide dismutase (SOD), glutathione peroxidase (GPX) and peroxidase (POD) were contributed considerably in ROS homeostasis maintenance. In-situ localization and assays related to ROS generation/scavenging revealed key management of ROS status under Cd stress. Higher antioxidative and reducing power activity attributed to the tolerance capability to the plant. Increased expression of withanolide biosynthetic pathway genes such as WsHMGR, WsDXS, WsDXR and WsCAS correlated with enhanced withanolides. The present study indicated the crucial role of the ascorbate-glutathione-α-tocopherol triad in co-ordination with withanolide biosynthesis in affording the oxidative stress, possibly through a cross-talk between the antioxidant machinery and secondary metabolite biosynthesis. The knowledge may be useful in providing the guidelines for developing abiotic stress resistance in plants using conventional and molecular approaches.
138. Kushwaha, Ramesh Kumar and Singh, Sucheta and Pandey, Shiv Shanker and Rao, D. K. Venkata and Nagegowda, Dinesh A. and Kalra, Alok and Vivek Babu, Chikkarasanahalli Shivegowda, Compatibility of Inherent Fungal Endophytes of Withania Somnifera with Trichoderma Viride and Its Impact on Plant Growth and Withanolide Content, Journal of Plant Growth Regulation, vol. 38, no. 4, pp. 1228--1242, December 2019. doi: 10.1007/s00344-019-09928-7. Abstract
     Withania somnifera (Ashwagandha), also known as Indian ginseng, is an important ancient medicinal plant, used in the Indian traditional systems of medicine. In view of increasing demand for roots of Ashwagandha, the present study was undertaken to investigate the compatibility of inherent fungal endophytes along with the biocontrol agent, Trichoderma viride, for enhancing W. somnifera plant growth and root secondary metabolites (withaferin A). It has frequently been emphasized by the World Health Organization the use of healthy roots of Ashwagandha for therapeutic applications. To maintain quality of W. somnifera roots, an option could be eco-friendly management of root-knot diseases and co-inoculation of native endophytes along with T. viride. The in vitro antagonistic activity of T. viride (TV) against the W. somnifera pathogens, Alternaria alternata and Sclerotium rolfsii, showed 64.3\% and 69.5\% growth inhibition, respectively. Here, we investigated the compatibility of TV along with the native endophytic fungi Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) for the cultivation of W. somnifera. The co-inoculation of TV and native endophytic fungi resulted in increased shoot, root weight, and plant height to 65–150\%, 35–74.5\%, and 15–35\%, respectively, compared to untreated plants. Withanolide A content in leaves of TV-treated plants increased significantly by 260\%, whereas in co-inoculation treatments, it was enhanced up to 109–242\%. However, no considerable change was noticed with withaferin A content in leaves, except the 2aWF\,+\,TV treatment significantly increased by 27\%. In contrast, withanolide A content in roots was not affected by TV alone but co-inoculation with endophyte treatments significantly increased its content (19–73\%). TV alone had increased chlorophyll a by 23\%; however, in combination treatments, it increased up to 115–164\% compared to control. Besides secondary metabolites in roots and leaves, co-inoculation of TV and native endophytes modulated the expression of the withanolide biosynthetic pathway genes HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1, and CYP710A1 compared to control treatments. Apart from withanolide biosynthetic pathway genes, co-inoculation of TV also ameliorated the host-resistant-related gene NPR1 which was upregulated by ninefold in the TV treatment and 3- to 7-fold in the combination treatment. Overall, our results show that co-inoculation of TV along with inherent endophytes of W. somnifera enhanced plant growth and withanolides accumulation.
139. Rout, Jyoti Ranjan and Kerry, Rout George and Panigrahi, Debasna and Sahoo, Santi Lata and Pradhan, Chinmay and Ram, Shidharth Sankar and Chakraborty, Anindita and Sudarshan, Mathummal, Biochemical, Molecular, and Elemental Profiling of Withania Somnifera L. with Response to Zinc Stress, Environmental Science and Pollution Research, vol. 26, no. 4, pp. 4116--4129, February 2019. doi: 10.1007/s11356-018-3926-6. Abstract
     Zn stress seriously induces various toxic responses in Withania somnifera L., when accumulated above the threshold level which was confirmed by investigating the responses of protein, expression of antioxidant enzymes, and elemental profiling on accumulation of Zn. Zn was supplemented in the form of ZnSO4 (0, 25, 50, 100, and 200 μM) through MS liquid medium and allowed to grow the in vitro germinated plants for 7 and 14 days. The study revealed that when the application of Zn increased, a significant reduction of growth characteristics was noticed with alterations of proteins (both disappearance and de novo synthesis). The activity of CAT, SOD, and GPX were increased up to certain concentrations and then declined, which confirmed through in-gel activity under different treatments. RT-PCR was conducted by taking three sets of genes from CAT (RsCat, Catalase1, Cat1) and SOD (SodCp, TaSOD1.2, MnSOD) and found that gene RsCat from CAT and MnSOD from SOD have shown maximum expression of desired genes under Zn stress, which indicate plant’s stress tolerance mechanisms. The proton-induced X-ray emission study confirmed an increasing order of uptake of Zn in plants by suppressing and expressing other elemental constituents which cause metal homeostasis. This study provides insights into molecular mechanisms associated with Zn causing toxicity to plants; however, cellular and subcellular studies are essential to explore molecule-molecule interaction during Zn stress in plants.
140. Mishra, Bhawana and Singh Sangwan, Neelam, Amelioration of Cadmium Stress in Withania Somnifera by ROS Management: Active Participation of Primary and Secondary Metabolism, Plant Growth Regulation, vol. 87, no. 3, pp. 403--412, April 2019. doi: 10.1007/s10725-019-00480-8. Abstract
     Cadmium (Cd) is~considered as a non-essential heavy metal with substantial toxicity on environment. Withania somnifera, a reputed therapeutic herb exhibits~vast pharmacological activities due to the presence of steroidal lactones-withanolides. The present study deals with reactive oxygen species (ROS) management through primary and secondary metabolism as adaptive response, on exogenous Cd exposure of W. somnifera. Increased invertase enzyme activity resulted in higher reducing sugars in plant under Cd stress to provide additional carbon source and NADH or NADPH. Higher activities of proline metabolic pathway enzymes such as ornithine aminotransferase (OAT) and pyrroline-5-corboxylate synthase (P5CS) resulted in elevated proline accumulation. The considerable participation of phenylpropanoid metabolism also found dominantly upregulated. Increased enzyme activities of phenylalanine ammonia lyase (PAL), shikimate dehydrogenase (SKDH), glucose-6-phosphate dehydrogenase (G6PDH), and cinnamyl alcohol dehydrogenase (CADH) resulted in phenolics and flavonoids accumulation under higher Cd stress. Upregulation of glutathione-S-transferase (GST) activity conferred its role in Cd chelation. In addition, glutamate oxaloacetate transaminase (GOT), amyl esterase (EST) and diaphorase (DIA) established their participation in Cd tolerance mechanism. Thus, present study elucidated major role of proline and phenylpropanoid metabolism in providing the osmoticum and antioxidants to the plants under Cd stress. The information may be helpful in developing stress resistant plants by targeting these pathways using conventional and molecular approaches.
141. Nefzi, Ahlem and Abdallah, Rania Aydi Ben and {Jabnoun-Khiareddine}, Hayfa and Ammar, Nawaim and {Daami-Remadi}, Mejda, Ability of Endophytic Fungi Associated with Withania Somnifera L. to Control Fusarium Crown and Root Rot and to Promote Growth in Tomato, Brazilian Journal of Microbiology, vol. 50, no. 2, pp. 481--494, April 2019. doi: 10.1007/s42770-019-00062-w. Abstract
     Fusarium crown and root rot (FCRR), caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL), is a soilborne tomato disease of increased importance worldwide. In this study, Withania somnifera was used as a potential source of biological control and growth-promoting agents. Seven fungal isolates naturally associated with W. somnifera were able to colonize tomato seedlings. They were applied as conidial suspensions or a cell-free culture filtrate. All isolates enhanced treated tomato growth parameters by 21.5–90.3\% over FORL-free control and by 27.6–93.5\% over pathogen-inoculated control. All tested isolates significantly decreased by 28.5–86.4\% disease severity over FORL-inoculated control. The highest disease suppression, by 86.4–92.8\% over control and by 81.3–88.8\% over hymexazol-treated control, was achieved by the I6 isolate. FORL radial growth was suppressed by 58.5–82.3\% versus control when dual cultured with tested isolates and by 61.8–83.2\% using their cell-free culture filtrates. The most active agent was identified as Fusarium sp. I6 (MG835371), which displayed chitinolytic, proteolytic, and amylase activities. This has been the first report on the potential use of fungi naturally associated with W. somnifera for FCRR suppression and for tomato growth promotion. Further investigations are required in regard to mechanisms of action involved in disease suppression and plant growth promotion.
142. Singh, Ruchi and Gupta, Pankhuri and Khan, Furqan and Singh, Susheel Kumar and {Sanchita} and Mishra, Tripti and Kumar, Anil and Dhawan, Sunita Singh and Shirke, Pramod Arvind, Modulations in Primary and Secondary Metabolic Pathways and Adjustment in Physiological Behaviour of Withania Somnifera under Drought Stress, Plant Science, vol. 272, pp. 42--54, July 2018. doi: 10.1016/j.plantsci.2018.03.029. Abstract
     In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH2+) for CO2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway.
143. Mishra, Aradhana and Singh, Satyendra Pratap and Mahfooz, Sahil and Singh, Surendra Pratap and Bhattacharya, Arpita and Mishra, Nishtha and Nautiyal, C. S., Endophyte-Mediated Modulation of Defense-Related Genes and Systemic Resistance in Withania Somnifera (L.) Dunal under Alternaria Alternata Stress, Applied and Environmental Microbiology, vol. 84, no. 8, pp. e02845-17, April 2018. doi: 10.1128/AEM.02845-17. Abstract
     Endophytes have been explored and found to perform an important role in plant health. However, their effects on the host physiological function and disease management remain elusive. The present study aimed to assess the potential effects of endophytes, singly as well as in combination, in Withania somnifera (L.) Dunal, on various physiological parameters and systemic defense mechanisms against Alternaria alternata. Seeds primed with the endophytic bacteria Bacillus amyloliquefaciens and Pseudomonas fluorescens individually and in combination demonstrated an enhanced vigor index and germination rate. Interestingly, plants treated with the two-microbe combination showed the lowest plant mortality rate (28\%) under A. alternata stress. Physiological profiling of treated plants showed improved photosynthesis, respiration, transpiration, and stomatal conductance under pathogenic stress. Additionally, these endophytes not only augmented defense enzymes and antioxidant activity in treated plants but also enhanced the expression of salicylic acid- and jasmonic acid-responsive genes in the stressed plants. Reductions in reactive oxygen species (ROS) and reactive nitrogen species (RNS) along with enhanced callose deposition in host plant leaves corroborated well with the above findings. Altogether, the study provides novel insights into the underlying mechanisms behind the tripartite interaction of endophyte-A. alternata-W. somnifera and underscores their ability to boost plant health under pathogen stress. IMPORTANCE W. somnifera is well known for producing several medicinally important secondary metabolites. These secondary metabolites are required by various pharmaceutical sectors to produce life-saving drugs. However, the cultivation of W. somnifera faces severe challenge from leaf spot disease caused by A. alternata. To keep pace with the rising demand for this plant and considering its capacity for cultivation under field conditions, the present study was undertaken to develop approaches to enhance production of W. somnifera through intervention using endophytes. Application of bacterial endophytes not only suppresses the pathogenicity of A. alternata but also mitigates excessive ROS/RNS generation via enhanced physiological processes and antioxidant machinery. Expression profiling of plant defense-related genes further validates the efficacy of bacterial endophytes against leaf spot disease.
144. Mishra, Aradhana and Singh, Satyendra Pratap and Mahfooz, Sahil and Bhattacharya, Arpita and Mishra, Nishtha and Shirke, Pramod Arvind and Nautiyal, C. S., Bacterial Endophytes Modulates the Withanolide Biosynthetic Pathway and Physiological Performance in Withania Somnifera under Biotic Stress, Microbiological Research, vol. 212--213, pp. 17--28, July 2018. doi: 10.1016/j.micres.2018.04.006. Abstract
     Despite the vast exploration of endophytic microbes for growth enhancement in various crops, knowledge about their impact on the production of therapeutically important secondary metabolites is scarce. In the current investigation, chitinolytic bacterial endophytes were isolated from selected medicinal plants and assessed for their mycolytic as well as plant growth promoting potentials. Among them the two most efficient bacterial endophytes namely Bacillus amyloliquefaciens (MPE20) and Pseudomonas fluorescens (MPE115) individually as well as in combination were able to modulate withanolide biosynthetic pathway and tolerance against Alternaria alternata in Withania somnifera. Interestingly, the expression level of withanolide biosynthetic pathway genes (3-hydroxy-3-methylglutaryl co-enzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductase, farnesyl di-phosphate synthase, squalene synthase, cytochrome p450, sterol desaturase, sterol Δ-7 reductase and sterol glycosyl transferases) were upregulated in plants treated with the microbial consortium under A. alternata stress. In addition, application of microbes not only augmented withaferin A, withanolide A and withanolide B content (1.52–1.96, 3.32–5.96 and 12.49–21.47 fold, respectively) during A. alternata pathogenicity but also strengthened host resistance via improvement in the photochemical efficiency, normalizing the oxidized and non-oxidized fraction, accelerating photochemical and non-photochemical quantum yield, and electron transport rate. Moreover, reduction in the passively dissipated energy of PSI and PSII in microbial combination treated plants corroborate well with the above findings. Altogether, the above finding highlights novel insights into the underlying mechanisms in application of endophytes and emphasizes their capability to accelerate biosynthesis of withanolides in W. somnifera under biotic stress caused by A. alternata.
145. Ankad, Gireesh M. and Hiremath, Jagadishchandra and Patil, R. T. and Pramod, H. J. and Hegde, Harsha V., Evaluation of Kunapa Jala and Pancha Gavya on Plant Quality, Physiology, Biochemistry, Yield and Growth Attributes – A Case Study of Withania Somnifera Dun., Journal of Ayurveda and Integrative Medicine, vol. 8, no. 4, pp. 247--251, October 2017. doi: 10.1016/j.jaim.2017.01.004. Abstract
     Background ‘Vrikshayurveda’, an ancient science of plant life described by Surapala, has clearly outlined a systematized agricultural practice that insisted on the use of Kunapa jala (KJ) and Pancha gavya (PG) to enhance the yield and quality of plants. Objective An experiment was conducted to evaluate the effect of KJ and PG on growth, physiological, biochemical, quality attributes and yield of Withania somnifera Dun. Materials and methods The effect of KJ and PG was evaluated in comparison with control, organic (farmyard manure and humic acid) and inorganic (NPK) fertilizer at 60, 90, 120 and 150 days after sowing (DAS). The study was conducted in randomized complete block design method. Results KJ group were higher in, total leaf area (1707.89~cm2) at 120 DAS, leaf area index (3.795) at 120 DAS, crop growth rate (0.256~g~m−2~day−1) at 60–90 DAS, leaf area duration (101.909) at 120–150 DAS, relative growth rate (0.0170~g~g−1~day−1) at 60–90 DAS, net assimilation rate (0.0537~g~m−2~day−1) at 60–90 DAS. Leaf area ratio was higher in PG group (37.937~m2~g−1) at 60 DAS. The higher levels of chlorophyll a, b and carotenoids were in KJ group~(1.877, 0.745 and 1318.14~mg~g−1 respectively) at 90 DAS. Yield and quality attributes at harvest (150 DAS) indicated, higher dry root yield (5.93 quintal hectare−1), root length (15.66~cm) were higher in KJ group and root diameter was higher in PG group (1.36~cm). Conclusion Vrikshayurveda practices viz. Kunapa jala and Pancha gavya were effective on studied parameters of W. somnifera. Economical and eco-friendly Vrikhayurveda practices can be initiated for sustainable agriculture.
146. Singh, Akanksha and Gupta, Rupali and Srivastava, Madhumita and Gupta, M. M. and Pandey, Rakesh, Microbial Secondary Metabolites Ameliorate Growth, in Planta Contents and Lignification in Withania Somnifera (L.) Dunal, Physiology and Molecular Biology of Plants, vol. 22, no. 2, pp. 253--260, April 2016. doi: 10.1007/s12298-016-0359-x. Abstract
     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.
147. Gupta, Rupali and Singh, Akanksha and Srivastava, Madhumita and Gupta, M. M. and Pandey, Rakesh, Augmentation of Systemic Resistance and Secondary Metabolites by Chitinolytic Microbes in Withania Somnifera against Meloidogyne Incognita, Biocontrol Science and Technology, vol. 26, no. 12, pp. 1626--1642, December 2016. doi: 10.1080/09583157.2016.1230729. Abstract
     Ashwagandha (Withania somnifera L. Dunal), a therapeutically imperative herb is known for its useful steroidal lactones (withanolide and withaferin) and acyl steryl glycosides. The plant is a highly susceptible host for the plant parasitic nematode, Meloidogyne incognita. Selected chitinolytic microbes, namely Cellulosimicrobium cellulans MTN13, Flavobacterium johnsoniae MTN 20, Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14, alone and in combination for M. incognita management and enhancement of secondary metabolites in W. somnifera cv. Poshita were evaluated. A significant enhancement in biomass yield (1.9-fold) and disease diminution (2.7-fold) was found in the dual microbial treatment Streptomyces sp. and Chitiniphilus sp. with respect to the untreated inoculated control plants. A significant augmentation (2.7- and 2.0-fold, respectively) in withanolide A and withaferin A was also found in the same treatment. The studies revealed increment of defence variables 1.1- to 1.3-fold and 1.6- to 2.1-fold in single- and dual-microbe treatments, respectively, than the untreated inoculated plants. The stimulation of the phenylpropanoid pathway and phenolics accumulation was the maximum at 5 days post inoculation (dpi), whereas antioxidant enzymes activities were the highest at 7 dpi. The results thus highlight a possible new function of chitinolytic microbes alone and in combinations that can effectively manage M. incognita-induced stress along with enhanced active molecules of W. somnifera.
148. Anuroopa, N. and Bagyaraj, D.J., Influence of Different AM Fungi on the Growth, Nutrition and Withanolide Concentration of Withania Somnifera, Medicinal Plants - International Journal of Phytomedicines and Related Industries, vol. 7, no. 4, pp. 272, 2015. doi: 10.5958/0975-6892.2015.00040.4.
149. Singh, Anupama and Lawrence, K. and Pandit, Swati and Lawrence, R. S., Response of Leaves, Stems and Roots of Withania Somnifera to Copper Stress., International Journal of Plant, Animal and Environmental Sciences, vol. 4, no. 3, pp. 60--67, 2014. url: https://www.cabdirect.org/cabdirect/abstract/20143288363. Abstract
     The effects of Cu+2 stress (0, 10, 20, 50, 100 and 200 mM as CuSO4.5H2O) on growth and biochemical parameters of Withania somnifera L. Dunal, grown in a field pot experiment were studied. Increasing Cu+2 concentrations led to decreased stem length, root elongation and leaf area. Copper (Cu+2) stress decreased chlorophyll and carotenoids content in leaves and stems as compared to controls; this...
150. Hosamani, P. A. and Lakshman, H. C. and Sandeepkumar, K. and Kadam, M. A. and Kerur, A. S., Role of Arbuscular Mycorrhizae in Conservation of Withania Somnifera., Bioscience Discovery Journal, vol. 2, no. 2, pp. 201--206, 2011. url: https://www.cabdirect.org/cabdirect/abstract/20113222949. Abstract
     An experiment was undertaken to study the selected medicinal plant and their therapeutic value with the association of Arbuscular mycorrhizal fungi. Rhizospheric soil samples were collected from the local places of Dharwad, which is geographically located between 14°15′ and 15°50′ North longitudes and 74°48′ and 76°20′ east latitude of northwestern part of Karnataka state, India. The present...
151. Elango, S. Rajasekar* {and} R., Effect of Microbial Consortium on Plant Growth and Improvement of Alkaloid Content in Withania Somnifera (Ashwagandha), Current Botany, 2011. url: https://updatepublishing.com/journal/index.php/cb/article/view/1371. Abstract
     The effect of microbial consortium consisting Plant Growth Promoting Rhizobacteria (PGPR) like Azospirillum, Azotobacter, Pseudomonas and Bacillus were tested separately and in combination on Withania somnifera for two consecutive years (2009 and 2010). The combinations of above mentioned PGPR strains significantly increased plant height, root length, and alkaloid content in Withania somnifera when compared to the uninoculated control. Plant growth promoting rhizobacteria (PGPR) exhibit direct and indirect mechanisms as plant growth promoters and biological control agents. Direct mechanism by PGPR, include the provision of bio-available phosphorus for plant uptake, nitrogen fixation for plant. The results of this study suggest that the PGPR applied in combination have the potential to increase the plant growth, alkaloid content of Withania somnifera.
152. Kaur, Mohinder and Sharma, Sapna and Mishra, Atul, Influence of Phosphate Solubilizing Pseudomonas and Bacillus Strains on the Growth of Ashvagandha (Withania Somnifera), Indian Journal of Agricultural Research, vol. 45, no. 2, pp. 128--133, 2011. url: https://arccjournals.com/journal/indian-journal-of-agricultural-research/. Abstract
     Twenty seven bacterial strains of both wenty seven bacterial strains of both wenty seven bacterial strains of both wenty seven bacterial strains of both wenty seven bacterial strains of both P PP PPseudomonas seudomonas seudomonas seudomonas seudomonas and and and and and Bacillus Bacillus Bacillus Bacillus Bacillus sp. from the rhizospher sp. from the rhizospher sp. from the rhizospher sp. from the rhizospher sp. from the rhizosphere ee ee of Ashvagandha were isolated and screened out for phosphate solubilization and production of of Ashvagandha were isolated and screened out for phosphate solubilization and production of of Ashvagandha were isolated and screened out for phosphate solubilization and production of of Ashvagandha were isolated and screened out for phosphate solubilization and production of of Ashvagandha were isolated and screened out for phosphate solubilization and production of other types of plant growth promoting activities affecting dir other types of plant growth promoting activities affecting dir other types of plant growth promoting activities affecting dir other types of plant growth promoting activities affecting dir other types of plant growth promoting activities affecting directly or indir ectly or indir ectly or indir ectly or indir ectly or indirectly ectly ectly ectly ectly. Out of twenty . Out of twenty . Out of twenty . Out of twenty . Out of twenty seven strains six strains , three of seven strains six strains , three of seven strains six strains , three of seven strains six strains , three of seven strains six strains , three of Bacillus Bacillus Bacillus Bacillus Bacillus sp. (WsNB-2, WsNB-6, and WsNB-13) and three of sp. (WsNB-2, WsNB-6, and WsNB-13) and three of sp. (WsNB-2, WsNB-6, and WsNB-13) and three of sp. (WsNB-2, WsNB-6, and WsNB-13) and three of sp. (WsNB-2, WsNB-6, and WsNB-13) and three of P PP PPseudomonas seudomonas seudomonas seudomonas seudomonas sp. (WsCP sp. (WsCP sp. (WsCP sp. (WsCP sp. (WsCP-1, WsCP -1, WsCP -1, WsCP -1, WsCP -1, WsCP-5 and WsCP -5 and WsCP -5 and WsCP -5 and WsCP -5 and WsCP-6) wer -6) wer -6) wer -6) wer -6) were selected to study the effect of phosphate e selected to study the effect of phosphate e selected to study the effect of phosphate e selected to study the effect of phosphate e selected to study the effect of phosphate solubilizing strains on the growth of rooted cutting of Ashvagandha and on other soil parameters solubilizing strains on the growth of rooted cutting of Ashvagandha and on other soil parameters solubilizing strains on the growth of rooted cutting of Ashvagandha and on other soil parameters solubilizing strains on the growth of rooted cutting of Ashvagandha and on other soil parameters solubilizing strains on the growth of rooted cutting of Ashvagandha and on other soil parameters like root colonization and phosphorous content of soil before and after the experiment. The like root colonization and phosphorous content of soil before and after the experiment. The like root colonization and phosphorous content of soil before and after the experiment. The like root colonization and phosphorous content of soil before and after the experiment. The like root colonization and phosphorous content of soil before and after the experiment. The selected strains significantly enhanced plant height after two months of plantation. Rhizosphere selected strains significantly enhanced plant height after two months of plantation. Rhizosphere selected strains significantly enhanced plant height after two months of plantation. Rhizosphere selected strains significantly enhanced plant height after two months of plantation. Rhizosphere selected strains significantly enhanced plant height after two months of plantation. Rhizosphere bacterial population incr bacterial population incr bacterial population incr bacterial population incr bacterial population increased significantly eased significantly eased significantly eased significantly eased significantly, which has r , which has r , which has r , which has r , which has resulted in nutrient mobilization and esulted in nutrient mobilization and esulted in nutrient mobilization and esulted in nutrient mobilization and esulted in nutrient mobilization and ultimately resulted in enhanced plant growth.
153. Mir, Bilal Ahmad and Mir, Shabir Ahmad and Khazir, Jabeena and Tonfack, Libert Brice and Cowan, Don A. and Vyas, Dhiraj and Koul, Sushma, Cold Stress Affects Antioxidative Response and Accumulation of Medicinally Important Withanolides in Withania Somnifera (L.) Dunal, Industrial Crops and Products, vol. 74, pp. 1008--1016, November 2015. doi: 10.1016/j.indcrop.2015.06.012. Abstract
     Withania somnifera (L.) Dunal (Indian ginseng) is a high value medicinal plant. It synthesizes a large array of biologically active withanolides. In this study, two month old seedlings of AGB002 (wild genotype) and AGB025 (cultivated genotype) of W. somnifera were subjected to cold stress (4°C) under controlled environment. Plants were analyzed for three medicinally important secondary metabolites (withanolide A, withanone and withaferin A), lipid peroxidation (MDA), cell injury, superoxide radical (O2−) accumulation and anti-oxidative enzymes activities such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). Increases in the titers of superoxide anion and MDA were observed from day 1 to day 7 in both genotypes, although the increase on the first day of exposure was significantly higher. Enzymatic activities of SOD, CAT, APX and GR also showed an increasing trend in both genotypes and reached a maximum on day 7 of the cold temperature exposure; however, this increase was higher in AGB002 than AGB025. Withanolide A (WS-1) in the roots of both genotypes significantly decreased on the first day of cold exposure and then showed a recovery until day 7. WS-1 was not detected in the leaves of either genotype. Withanone (WS-2) content in the leaves also decreased towards the end of the cold period in both genotypes. Cold stress also elicited the accumulation of WS-2 in AGB025, but was not detectable in control seedlings. At maturity, WS-2 was also detected in control plants. Furthermore, a significant increase in the leaf withaferin A (WS-3) content was recorded from day 1 to day 7 of the cold exposure in both the genotypes, suggesting the possible involvement of withanolides in cold-protection. AGB002 showed comparatively higher accumulation of antioxidant enzymes and selected marker withanolides than AGB025, indicating that AGB002 is better adapted to cold than AGB025. It could be inferred from these observations that cold stress induces bioactive withanolide accumulation in W. somnifera as a mechanism for scavenging reactive oxygen species (ROS). These studies also provide an impetus for enhancing the withanolide accumulation in W. somnifera using controlled environment technology.
154. Singh, Gaurav and Tiwari, Manish and Singh, Surendra P. and Singh, Ruchi and Singh, Surendra and Shirke, Pramod A. and Trivedi, Prabodh K. and Misra, Pratibha, Sterol Glycosyltransferases Required for Adaptation of Withania Somnifera at High Temperature, Physiologia Plantarum, vol. 160, no. 3, pp. 297--311, 2017. doi: 10.1111/ppl.12563. Abstract
     Heat is a major environmental stress factor that confines growth, productivity, and metabolism of plants. Plants respond to such unfavorable conditions through changes in their physiological, biochemical and developmental processes. Withania somnifera, an important medicinal plant, grows in hot and dry conditions, however, molecular mechanisms related to such adaptive properties are not known. Here, we elucidated that members of the sterol glycosyltransferases (SGT) gene family play important roles in the survival of W. somnifera under adverse conditions through maintaining the integrity of the membrane. SGTs are enzymes involved in sterol modifications and participate in metabolic flexibility during stress. Silencing of WsSGT members, for instance WsSGTL1, WsSGTL2 and WsSGTL4, was inimical for important physiological parameters, such as electron transport rate, photochemical quantum yield, acceptor side limitation, non-photochemical quenching (NPQ), Fv/Fm and net photosynthetic rate, whereas stomatal conductance, transpiration rate and dark respiration rates (Rds) were increased. Decreased NPQ and increased Rds helped to generate significant amount of ROS in the Wsamisgt lines. After heat stress, H2O2, lipid peroxidation and nitric oxide production increased in the Wsamisgt lines due to high ROS generation. The expression of HSPs in Wsamisgt lines might be involved in regulation of physiological processes during stress. We have also observed increased proline accumulation which might be involved in restricting water loss in the Wsamisgt lines. Taken together, our observations revealed that SGTL enzyme activity is required to maintain the internal damages of the cell against high temperature by maintaining the sterol vs sterol glycosides ratio in the membranes of W. somnifera.
155. Takshak, Swabha and Agrawal, S. B., Secondary Metabolites and Phenylpropanoid Pathway Enzymes as Influenced under Supplemental Ultraviolet-B Radiation in Withania Somnifera Dunal, an Indigenous Medicinal Plant, Journal of Photochemistry and Photobiology B: Biology, vol. 140, pp. 332--343, November 2014. doi: 10.1016/j.jphotobiol.2014.08.011. Abstract
     The present study aims to investigate the effects of supplemental ultraviolet B (3.6kJm−2day−1 above ambient) radiation on secondary metabolites and phenylpropanoid pathway enzymes of Withania somnifera under field conditions at 40, 70, and 100days after transplantation. Secondary metabolites’ (alkaloids, anthocyanins, carotenoids, flavonoids, lignin, phytosterols, saponins, and tannins) concentrations were analysed at the end of the treatments. Activities of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumarate-CoA ligase (4CL), chalcone–flavanone isomerase (CHI), and dihydroflavonol reductase (DFR) were also determined. In treated plants, secondary metabolite-concentrations generally increased (higher concentrations being recorded in roots compared to leaves). Anomalies were recorded for lycopene in roots and phytosterols in leaves (all sampling ages); β-carotene declined in leaves at third sampling age. s-UV-B-treated plants depicted decrease in withanolide A content with concomitant increase in withaferin A (two major alkaloids analysed by HPLC) compared to their respective controls. Phenylpropanoid pathway enzyme-activities increased in leaves and roots under s-UV-B treatment, the latter showing greater increase. The study concludes that s-UV-B is a potent factor in increasing the concentrations of secondary metabolites and their biosynthetic pathway enzymes in W. somnifera.
156. Takshak, S. and Agrawal, S. B., Effect of Ultraviolet-B Radiation on Biomass Production, Lipid Peroxidation, Reactive Oxygen Species, and Antioxidants in Withania Somnifera, Biologia Plantarum, vol. 58, no. 2, pp. 328--334, June 2014. doi: 10.1007/s10535-014-0390-0. Abstract
     The present study was aimed at understanding the effects of long term supplemental UV-B (3.6 kJ m−2 d−1) on biomass production, accumulation of reactive oxygen species, lipid peroxidation, and enzymatic antioxidants in leaves and roots of Withania somnifera (an indigenous medicinal plant). Under the UV-B treatment, a reduction in biomass and an increased malondialdehyde content (a characteristic of lipid peroxidation) were observed in both the shoots and roots. Amongst ROS, H2O2 content increased under UV-B in the leaves, whereas it decreased in the roots, and superoxide radical production rate decreased in both the plant parts. The activities of all enzymatic antioxidants tested (ascorbate peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, and superoxide dismutase) increased under the UV-B treatment, the increase being greater in the roots.
157. Chandan, Jyoti and Gupta, Suruchi and Babu, Vikash and Singh, Deepika and Singh, Ravail, Comprehensive Analysis of~Codon Usage Pattern in Withania Somnifera and Its Associated Pathogens: Meloidogyne Incognita and Alternaria Alternata, Genetica, vol. 150, no. 2, pp. 129--144, April 2022. doi: 10.1007/s10709-022-00154-w. Abstract
     Meloidogyne incognita (Root-knot nematode)~and Alternaria alternata (fungus) were among the dominant parasites of the medicinal plant Withania somnifera. Despite the fatal nature of their infection, a comprehensive study to explore their evolution and adaptation is lacking. The present study elucidates evolutionary and codon usage bias analysis of W. somnifera (host plant), M. incognita (root-knot nematode) and A. alternata (fungal parasite). The results of the present study revealed a weak codon usage bias prevalent in all the three organisms. Based on the nucleotide analysis, genome of W. somnifera and M. incognita was found to be A-T biased while A. alternata had GC biased genome. We found high similarity of CUB pattern between host and its nematode pathogen as compared to the fungal pathogen. Inclusively, both the evolutionary forces influenced the CUB in host and its associated pathogens. However, neutrality plot indicated the pervasiveness of natural selection on CUB of the host and its pathogens. Correspondence analysis revealed the dominant effect of mutation on CUB of W. somnifera and M. incognita while natural selection was the main force affecting CUB of A. alternata. Taken together the present study would provide some prolific insight into the role of codon usage bias in the adaptability of pathogens to the host’s environment for establishing parasitic relationship.
158. Kumar, Ashutosh and Kumar, S. P. Jeevan and Chintagunta, Anjani Devi and Agarwal, Dinesh K. and Pal, Govind and Singh, Arvind Nath and {Simal-Gandara}, Jesus, Biocontrol Potential of Pseudomonas Stutzeri Endophyte from Withania Somnifera (Ashwagandha) Seed Extract against Pathogenic Fusarium Oxysporum and Rhizoctonia Solani, Archives of Phytopathology and Plant Protection, vol. 55, no. 1, pp. 1--18, January 2022. doi: 10.1080/03235408.2021.1983384. Abstract
     Agricultural crops are susceptible to abiotic and biotic factors. Among biotic factors, plant pathogens attacks are prominent and significantly reduce the crop productivity and production. Long term application of synthetic pesticides in agriculture not only causes resistance development in pathogens but also deteriorates the soil quality and pollutes the ecosystem. In the current study, an investigation has been made with an objective to isolate an endophytic bacterium from Withania somnifera seed and evaluate its potential biocontrol activity against soil-borne pathogens like Fusarium oxysporum var. ciceri and Rhizoctonia solani. The molecular approach of bacterium identification using 16S rRNA sequencing methods includes kit based DNA extraction and PCR amplification and the isolated endophytic bacterium was identified as Pseudomonas stutzeri. In dual culture assay, the endophytic bacterium P. stutzeri PSIISS-1 effectively suppressed growth of pathogenic fungi F. oxysporum var. ciceri (61.0\%) and R. solani (53.3\%), respectively. In vivo studies were conducted with initial microbial inoculum size of 108cell/ml and the studies revealed that the bacterium P. stutzeri PSIISS-1 co-inoculated with R. solani had significantly inhibited the sheath blight disease with the efficacy of 76\% in susceptible paddy MTU-7029. Simultaneously, co-inoculation of P. stutzeri PSIISS-1 along with the pathogen F. oxysporum var. ciceri in chickpea JG-14 variety resulted in reduction of wilt symptom up to 80.45\%. Bacterial inhibitions against the pathogens were corroborated with chitinase, protease and lipase enzymatic activities. Based on the results obtained from the study, P. stutzeri showed biocontrol activity against plant pathogens which can be used as potential bio-pesticides.
159. Gaur, Sandeep Kumar and Kumar, Krishna, Toxicity and Insect Growth Regulatory Effects of Root Extract from the Medicinal Plant, Withania Somnifera (Linnaeus) against Red Flour Beetle, Tribolium Castaneum (Coleoptera: Tenebrionidae), Archives of Phytopathology and Plant Protection, vol. 53, no. 17-18, pp. 856--875, October 2020. doi: 10.1080/03235408.2020.1802566. Abstract
     The insect growth regulatory effects of the medicinal plant, Withania somnifera (Family: Solanaceae) on the moulting and metamorphosis of economically important stored grain pest, Tribolium castaneum (Coleoptera: Tenebrionidae) were assessed under laboratory conditions. Seventh instar larvae of the red flour beetle, T. castaneum were topically treated with different doses of root extract of W. somnifera. Treatment with root extract of W. somnifera disrupted growth, development, moulting and metamorphosis in T. castaneum. The results included mortality, prolongation of larval-pupal and pupal-adult ecdysis, ecdysial stasis, development of larval-pupal and pupal-adult intermediates and adultoids, eclosion inhibition, reduced pupation and adult emergence. The LC50 value in topical application of root extract of W. somnifera was 0.5850 µg·µL−1 against last instar larvae of T. castaneum. These results are similar to those observed by the application of juvenile hormone analogues and may be due to interference with the normal hormonal mechanism of moulting and metamorphosis.
160. Gaur, Sandeep Kumar and Kumar, Krishna, Toxicity and Insect Growth Regulatory Activities of Medicinal Plant, Withania Somnifera, in Flesh Fly, Sarcophaga Ruficornis (Diptera: Sarcophagidae), The Journal of Basic and Applied Zoology, vol. 81, no. 1, pp. 30, May 2020. doi: 10.1186/s41936-020-00154-2. Abstract
     The flesh fly Sarcophaga ruficornis is well known for its medical and veterinary importance in causing myiasis in humans and animals. The conventional use of chemical pesticides for controlling insect pests has resulted in environmental pollution besides posing serious hazards to non-target organisms and the development of pest resistance against these compounds. Considering the various harmful and adverse effects of chemical pesticides, an alternative and safe approach for the pest management has to be explored. The plant extracts derived from plants are eco-friendly in nature, easily biodegradable, and can be used as botanical pesticides.
161. Gaur, Sandeep Kumar and Kumar, Krishna, Sensitivity of Tobacco Caterpillar, Spodoptera Litura, to Extract from a Medicinal Plant, Withania Somnifera, International Journal of Vegetable Science, vol. 26, no. 1, pp. 62--78, January 2020. doi: 10.1080/19315260.2019.1605556. Abstract
     The larval stage of a polyphagous moth Spodoptera litura (Lepidoptera: Noctuidae), commonly known as tobacco caterpillar, causes loss in many vegetables. Use of chemical pesticides to control this pest has resulted in environmental pollution, is hazardous to non-target organisms, and contributes to development of pest resistance against these compounds. Alternatives for pest management need to be developed. Extracts derived from plants can function as insect growth regulators and are easily biodegradable. Topical administration of root extracts from a medicinal plant, Ashwagandha (Withania somnifera L. Dunal), to sixth instar larvae and freshly molted pupae (0–2 h old) of S. litura caused prolongation of larval-pupal and pupal-adult ecdysis duration, mortality, reduced pupation, and adult emergence. Root extracts of W. somnifera produced the morphological and developmental abnormalities like ecdysial failure, formation of larval-pupal and pupal-adult intermediates, abnormal pupae and adultoids. Treatment with root extracts of W. somnifera was more toxic to pupae compared to larvae of S. litura. The results may be due to interference with normal hormonal mechanism eventually leading to disruption of molting and metamorphosis.
162. Gaur, Sandeep Kumar and Kumar, Krishna, A Comparative Bioefficacy of Seed and Root Extracts of a Medicinal Plant, Withania Somnifera When Administered to Prepupae of Lepidopteran Insects, Spodoptera Litura (Lepidoptera: Noctuidae) and Pericallia Ricini (Lepidoptera: Arctiidae), The Journal of Basic and Applied Zoology, vol. 80, no. 1, pp. 37, May 2019. doi: 10.1186/s41936-019-0107-1. Abstract
     The lepidopterans, Spodoptera litura and Pericallia ricini, are polyphagous pests of agricultural importance. The unscrupulous and non-judicious use of chemical pesticides for controlling the insect pests has resulted into severe environmental hazards, threatening non-target organisms and human health.
163. Akhtar, R. and Javaid, A., Biological Management of Basal Rot of Onion by {\emph{Trichoderma Harzianum}} and {\emph{Withania Somnifera}}, Planta Daninha, vol. 36, March 2018. doi: 10.1590/S0100-83582018360100009. Abstract
     ABSTRACT: Onion is attacked by destructive soil-borne fungal plant pathogen Fusarium oxysporum f. sp. cepae, resulting in basal rot disease. In the present study, three Trichoderma species (T. pseudokoningii, T. harzianum and T. reesei) and leaves of solanaceous weed Withania somnifera were used for management of this disease. The in vitro interaction study revealed T. harzianum as the most effective biocontrol agent against the pathogen. In a pot trial, dried leaf material of W. somnifera (1\%, 2\% and 3\% w/w) and inoculum of T. harzianum were mixed in the pot soil previously inoculated with the pathogen. The highest incidence of the disease (87\%) was found in positive control (pathogen inoculation without any amendment). Different rates of dry leaf material reduced the incidence of the disease to 41-66\%. T. harzianum in combination with leaf material reduced the incidence of the disease to 20-53\%. In a laboratory bioassay, the dry leaf extract of W. somnifera was prepared in methanol and partitioned with n-hexane, chloroform, ethyl acetate and n-butanol. The highest concentration (200 mg mL-1) of all except for the n-butanol fraction significantly decreased fungal biomass over control. This study concludes that basal rot of onion can be controlled by combined application of W. somnifera dry leaf material and biological control agent T. harzianum.
164. Gaur, S. K. and Kumar, K., Withania Somnifera Acts as a Potential Insect Growth Regulator in the Polyphagous Pest, Pericallia Ricini, Journal of Plant Protection Research, vol. 57, no. 4, 2017. doi: 10.1515/jppr-2017-0052.
165. Singh, V. and Singh, B. and Sharma, A. and Kaur, K. and Gupta, A.p. and Salar, R.k. and Hallan, V. and Pati, P.k., Leaf Spot Disease Adversely Affects Human Health-Promoting Constituents and Withanolide Biosynthesis in Withania Somnifera (L.) Dunal, Journal of Applied Microbiology, vol. 122, no. 1, pp. 153--165, 2017. doi: 10.1111/jam.13314. Abstract
     Aims The present work investigates the implication of leaf spot disease on the antioxidant potential and commercial value of pharmaceutically important constituents of Withania somnifera, a high-valued medicinal plant. Methods and Results Leaf spot disease was induced in W. somnifera by inoculating Alternaria alternata (Fr.) Keiss. pathogen. Total polyphenolic content and antioxidant potential showed a significant decrease during leaf spot disease. Evaluation of pharmaceutically active constituents withaferin A, withanone and withanolide A utilizing high-performance liquid chromatography showed a significant decrease in diseased samples as compared to healthy ones. Quantitative expression of major genes involved in withanolide biosynthesis also showed down-regulation in diseased samples. Alterations in the ultra-structure of chloroplasts were also analysed under transmission electron microscopy to get a better insight into the changes of withanolide biosynthesis in leaf during disease infestation. Conclusions The present work suggests that when the pathogenic fungus invades the host plants, it evokes multiple responses, which could be studied at various levels. The knowledge gained from this work will provide appropriate rationale for controlling the bio-deterioration of the pharmaceutically active metabolites in W. somnifera and development of suitable strategies against leaf spot disease. Significance and Impact of the Study This is the first study to investigate the effect of leaf spot disease on the human health-promoting constituents and withanolide biosynthesis in this high-valued medicinal plant.
166. Singh, Varinder and Singh, Baldev and Joshi, Robin and Jaju, Puneet and Pati, Pratap Kumar, Changes in the Leaf Proteome Profile of Withania Somnifera (L.) Dunal in Response to Alternaria Alternata Infection, PLOS ONE, vol. 12, no. 6, pp. e0178924, June 2017. doi: 10.1371/journal.pone.0178924. Abstract
     Withania somnifera is a high value medicinal plant which is used against large number of ailments. The medicinal properties of the plant attributes to a wide array of important secondary metabolites. The plant is predominantly infected with leaf spot pathogen Alternaria alternata, which leads to substantial biodeterioration of pharmaceutically important metabolites. To develop an effective strategy to combat this disease, proteomics based approach could be useful. Hence, in the present study, three different protein extraction methods tris-buffer based, phenol based and trichloroacetic acid-acetone (TCA-acetone) based method were comparatively evaluated for two-dimensional electrophoresis (2-DE) analysis of W. somnifera. TCA-acetone method was found to be most effective and was further used to identify differentially expressed proteins in response to fungal infection. Thirty-eight differentially expressed proteins were identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry (MALDI TOF/TOF MS/MS). The known proteins were categorized into eight different groups based on their function and maximum proteins belonged to energy and metabolism, cell structure, stress and defense and RNA/DNA categories. Differential expression of some key proteins were also crosschecked at transcriptomic level by using qRT-PCR and were found to be consistent with the 2-DE data. These outcomes enable us to evaluate modifications that take place at the proteomic level during a compatible host pathogen interaction. The comparative proteome analysis conducted in this paper revealed the involvement of many key proteins in the process of pathogenesis and further investigation of these identified proteins could assist in the discovery of new strategies for the development of pathogen resistance in the plant.
167. Singh, Anup Kumar and Kumar, Sarma Rajeev and Dwivedi, Varun and Rai, Avanish and Pal, Shaifali and Shasany, Ajit K. and Nagegowda, Dinesh A., A WRKY Transcription Factor from Withania Somnifera Regulates Triterpenoid Withanolide Accumulation and Biotic Stress Tolerance through Modulation of Phytosterol and Defense Pathways, New Phytologist, vol. 215, no. 3, pp. 1115--1131, 2017. doi: 10.1111/nph.14663. Abstract
     Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W. somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense.
168. Singh, Gaurav and Tiwari, Manish and Singh, Surendra Pratap and Singh, Surendra and Trivedi, Prabodh Kumar and Misra, Pratibha, Silencing of Sterol Glycosyltransferases Modulates the Withanolide Biosynthesis and Leads to Compromised Basal Immunity of Withania Somnifera, Scientific Reports, vol. 6, no. 1, pp. 25562, May 2016. doi: 10.1038/srep25562. Abstract
     Sterol glycosyltransferases (SGTs) catalyse transfer of glycon moiety to sterols and their related compounds to produce diverse glyco-conjugates or steryl glycosides with different biological and pharmacological activities. Functional studies of SGTs from Withania somnifera indicated their role in abiotic stresses but details about role under biotic stress are still unknown. Here, we have elucidated the function of SGTs by silencing SGTL1, SGTL2 and SGTL4 in Withania somnifera. Down-regulation of SGTs by artificial miRNAs led to the enhanced accumulation of withanolide A, withaferin A, sitosterol, stigmasterol and decreased content of withanoside V in Virus Induced Gene Silencing (VIGS) lines. This was further correlated with increased expression of WsHMGR, WsDXR, WsFPPS, WsCYP710A1, WsSTE1 and WsDWF5 genes, involved in withanolide biosynthesis. These variations of withanolide concentrations in silenced lines resulted in pathogen susceptibility as compared to control plants. The infection of Alternaria alternata causes increased salicylic acid, callose deposition, superoxide dismutase and H2O2 in aMIR-VIGS lines. The expression of biotic stress related genes, namely, WsPR1, WsDFS, WsSPI and WsPR10 were also enhanced in aMIR-VIGS lines in time dependent manner. Taken together, our observations revealed that a positive feedback regulation of withanolide biosynthesis occurred by silencing of SGTLs which resulted in reduced biotic tolerance.
169. Saroj, A. and Kumar, A. and Srivastava, A. K. and Khaliq, A. and Absar, N. and Alam, M. and Samad, A., New Report of Black Leaf Spot Mold (Pseudocercospora Fuligena) on Withania Somnifera from India, Plant Disease, vol. 98, no. 9, pp. 1275--1275, September 2014. doi: 10.1094/PDIS-03-14-0231-PDN. Abstract
     Withania somnifera (family solanaceae) commonly known as ashwagandha and Indian ginseng, originated in India is one of the most powerful medicinal plants for more than 3,000 years (1). It is commercially cultivated for its roots, a natural rich source of glycowithanolides, tannins, potassium nitrate, etc., which are an anti-inflammatory, anti-tumor, anti-oxidant, anti-ulcer, and regulator of the nervous system and sleep (2). During the monsoon of July 2011, black spots on the leaves of infected plants were observed in the ashwagandha growing Lucknow, Raibareilly, and adjoining areas of Uttar Pradesh province with 10 to 20\% disease incidence. Early stage of disease were characterized by the presence of light chlorotic spots on both sides of old leaves that later turned into dark black spots resulting in early defoliation. About 27 samples were collected from different locations of the fields for isolation of the causal organism and microscopic studies. Infected leaves were cut into small pieces, surface sterilized with 1\% sodium hypochlorite for 1 min, rinsed thrice with sterilized distilled water, and placed onto potato dextrose agar (PDA) plates. After 21 days of dark incubation at 25°C, 8- to 10-mm grayish-brown colonies were observed. Microscopic studies at early and mature stages of infection showed production of conidia in conidiophores. Conidiophores were mostly 5 to 9, few dense pale brown, simple unbranched, septate, geniculate and 14 to 55 × 3 to 5.5 μm. Conidia were subhyline, obclavate to cylindrical, some were straight to slightly curved, multiseptate, base long obconic to long obconically truncate, and 12 to 85 × 3.5 to 5 μm. On the basis of cultural and morphological studies, the pathogen was identified as Pseudocercospora fuligena (3). The pathogen identity was further confirmed at molecular level using universal primers ITS1/ITS4 through PCR (4). An amplification of the expected size (\textasciitilde 550 bp) was generated, eluted from agarose gel by QIAquick gel extraction kit (Qiagen), cloned into pGEM-T Easy vector (Promega), sequenced, and deposited in GenBank (Accession No. KF881898). NCBI BLASTn showed 99\% identity with P. fuligena (GU214675) strain CPC 12296, isolated from Lycopersicon sp. Pathogenicity test was carried out on 10 plants of W. somnifera cv. Poshita through two approaches, one using mycelia from culture and another using spore suspension from naturally infected leaves. In the first approach, fungal mycelia were applied onto the healthy ashwagandha leaves, whereas in the second approach, infected leaves were washed with distilled water and spore suspension of 106 spores/ml was sprayed on healthy plants. Plants sprayed with sterilized distilled water served as controls. Inoculated plants were placed in a growth chamber at 28°C under 90\% humidity for 3 days. After, pots were placed in the glasshouse at 27 ± 2°C with 70 to 80\% humidity for 21 days. Initial symptoms appeared on the 7th day while typical symptoms appeared on all the inoculated plants after 12 to 17 days. Control plants remained free of infection. Re-isolation of the pathogen on PDA fulfilled Koch's postulates. Black leaf mold caused by P. fuligena has been reported on tomato (5). This is the first report of black leaf mould caused by P. fuligena on W. somnifera from India. P. fuligena has the potential to reduce yield of W. somnifera. References: (1) Anonymous. Alt. Med Rev. 9:211, 2004. (2) B. D. Basu and K. R. Kirtikar. Indian Medicinal Plants: Plates, vol. 1-4. Bishen Singh Mahendra Pal Singh, Dehradun, India, 1991. (3) T. C. Wang et al. Plant Dis. 79:661, 1995. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (5) S. Yamada. Ann. Phytopathol. Soc. Jpn. 15:13, 1951.
170. Kumar, Susheel and Kaushik, Nutan and Proksch, Peter, Identification of Antifungal Principle in the Solvent Extract of an Endophytic Fungus Chaetomium Globosum from Withania Somnifera, SpringerPlus, vol. 2, no. 1, pp. 37, February 2013. doi: 10.1186/2193-1801-2-37. Abstract
     Extracts of Chaetomium globosum EF18, isolated as endophytic fungus from Withania somnifera, were found effective against Sclerotinia sclerotiorum. Ethyl acetate and methanol extracts were more effective than hexane extract showing {$>$}80\% growth inhibition. Bioactive compound (antibiotic Sch 210971, m/z 445 and λmax 290) having antifungal activity against S. sclerotiorum has been isolated in pure form from the ethyl acetate extract following bioassay guided fractionation. Apart from this compound other fractions of polar to medium polarity were also found effective. Fraction no. VIII from VLC (Vacuum liquid chromatography) column of ethyl acetate extract was most active having IC50 value 35.4 μg/ml.
171. Baghel, G. and Naqvi, Q.A. and Snehi, S.K. and Khan, M.S. and Raj, S.K., Molecular Identification of Three Isolates of Jatropha Mosaic India Virus Associated with Mosaic Disease of Withania Somnifera in India, Archives of Phytopathology and Plant Protection, vol. 45, no. 17, pp. 2114--2119, October 2012. doi: 10.1080/03235408.2012.721683. Abstract
     The association of begomovirus with mosaic disease of Withania somnifera was detected at three locations in India by PCR using begomovirus-specific degenerate primers. The resulting amplicons of ∼1.2 kb from three locations (Aligarh, Lucknow and Hindaun City) were sequenced. The begomovirus isolates of three locations shared 96–97\% identities among them and 91\% identities with Jatropha mosaic India virus (JMIV). Based on highest sequence identities and close phylogenetic relationships with JMIV, these begomovirus isolates associated with mosaic disease of W. somnifera were identified as isolates of JMIV.
172. Saroj, A. and Kumar, A. and Qamar, N. and Alam, M. and Singh, H. N. and Khaliq, A., First Report of Wet Rot of Withania Somnifera Caused by Choanephora Cucurbitarum in India, Plant Disease, vol. 96, no. 2, pp. 293--293, February 2012. doi: 10.1094/PDIS-09-11-0801. Abstract
     Withania somnifera (Ashwagandha) is native to India and commercially cultivated for the production of root withanolides that have anticarcinogenic properties. A disease appeared on plantings of W. somnifera during the 2010 monsoon at the CIMAP and in adjoining areas of northern India. Symptoms first appeared as water-soaked lesions on leaves and stems that progressed to a wet rot. Mature lesions harbored black fructifications of the suspect pathogen. Pathogen isolations were done by placing pieces of infected tissues on potato dextrose agar. A fungus tentatively identified as a Choanephora sp. that produced white aerial mycelia that later turned pale yellow was consistently isolated from infected plant parts. Mycelia were hyaline and nonseptate. Sporangiophores bearing sporangiola were erect, hyaline, unbranched, apically dilated to form a clavate vesicle from which arose dichotomously branched distally clavate secondary vesicles. Sporangiola were indehiscent, ellipsoid, brown to dark brown with distinct longitudinal striations, and measured 12 to 20 × 6 to 12 μm. Sporangia were multispored, spherical, initially white to yellow and pale brown to dark brown at maturity, and measured 40 to 160 μm. Sporangiospores from sporangia were ellipsoid to broadly ellipsoid, brown to dark brown, indistinctly striate with fine hyaline polar appendages, and measured 16 to 20 × 8 to 12 μm. On the basis of the cultural as well as morphological characteristics and description in the monograph by Kirk (2), the fungus was identified as a Choanephora sp. The identification was also confirmed by IMTECH, Chandigarh, India with Accession No. MTCC-10731. The species was later characterized as Choanephora cucurbitarum (Berk. \& Ravenel) Thaxt (GenBank Accession No. AB470642) by using universal primers ITS-1 and ITS-4. Its sequence comprising of 18S rRNA partial, complete ITS 1, 5.8S rRNA, ITS 2, and 28S rRNA partial was submitted to NCBI GenBank with Accession No. JN639861. Pathogenicity of the fungus was established on five healthy plants by artificial inoculation with spray of an aqueous spore suspension containing 106 spores/ml. Plants sprayed with sterile distilled water were used as controls. Both inoculated and control plants were kept in a humidity chamber (96\%) for 3 days and thereafter placed in the glasshouse at 28 ± 2°C. Initial symptoms developed in 2 to 3 days while typical disease symptoms appeared on all the inoculated plants after 7 to 10 days. Control plants were free from infection. The reisolation from artificially infected plants again yielded a Choanephora sp., thus fulfilling Koch's postulates. W. somnifera cultivation has been affected by root rot and wilt caused by Fusarium solani and leaf spot caused by Alternaria dianthicola (3). The occurrence of a Choanephora sp. was reported on periwinkle, petunia (1), and Boerhavia diffusa (4). However, to our knowledge, incidence of this pathogen on W. somnifera has not been reported so far. Thus, wet rot of W. somnifera caused by C. cucurbitarum is a new report from India and worldwide. References: (1) G. E. Holcomb. Plant Dis. 87:751, 2003. (2) P. M. Kirk. Mycol. Pap. 152:1, 1984. (3) C. K. Maiti et al. Plant Dis. 91:467, 2007. (4) N. Singh et al. New Dis. Rep. 23:29, 2011.
173. Sharma, Ashutosh and Kumar Pati, Pratap, First Record of the Carmine Spider Mite, {\emph{Tetranychus Urticae}} , Infesting {\emph{Withania Somnifera}} in India, Journal of Insect Science, vol. 12, no. 50, pp. 1--4, April 2012. doi: 10.1673/031.012.5001.
174. Sharma, A. and Sharma, I. and Pati, P.K., Post-Infectional Changes Associated with the Progression of Leaf Spot Disease in Withania Somnifera, Journal of Plant Pathology, vol. 93, no. 2, pp. 397--405, 2011. url: https://www.jstor.org/stable/41999011. Abstract
     Leaf spot caused by Alternaria alternata is a prevalent disease of Withania somnifera, a high value medicinal plant. The severity of infection is closely related with the progression of the disease. In the present work, various biochemical parameters such as free proline, total chlorophyll, total protein, lipid peroxidation, ion leakage and the activity of some antioxidant enzymes were analyzed to assess the post infectional changes associated with infection. As disease progressed, a decrease of chlorophyll and total protein content was observed. The lowest level of total chlorophyll (16 fold decrease) was observed when the severity of infection was at its peak. A significant increase in free proline content, lipid peroxidation and ion leakage was also observed. Antioxidant enzymes behaved in a differential way. Catalase, ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase specific activities showed a significant increase concomitantly with disease progression. By contrast, glutathione reductase and monodehydroascorbate reductase decreased significantly (2.5 and 6.5 folds, respectively) as disease progressed. Besides Superoxide dismutase, the other antioxidant enzymes analysed did not have a significant correlation with the number of spots/cm² leaf area. Although the present study suggests a complex pattern of post infectional changes, the information obtained is useful for a better understanding of host pathogen interaction.
175. Sharma, Ashutosh and Pati, Pratap Kumar, First Report of Withania Somnifera (L.) Dunal, as a New Host of Cowbug (Oxyrachis Tarandus, Fab.) In Plains of Punjab, Northern India, pp. 3, 2011. Abstract
     During February-June 2009-10, cowbugs were found on aerial apical parts of Ashwagandha (Withania somnifera) plants in the Amritsar District of Punjab. The cowbugs fed on apical portions of the stem, making them rough and woody in appearance, brown in colour that gradually dried and apical leaves were shed off. These cowbugs were identified as Oxyrachis tarandus [1] (Hemiptera: Membracidae). To the best of our knowledge, this is the first report of Withania somnifera (L.) Dunal as a new host for O. tarandus in Punjab province of Northern India.
176. Gaur, Ratna and Kumar, Krishna, Insect Growth-Regulating Effects of Withania Somnifera in a Polyphagous Pest, Spodoptera Litura, Phytoparasitica, vol. 38, no. 3, pp. 237--241, July 2010. doi: 10.1007/s12600-010-0092-x. Abstract
     Treatment of sixth instar larvae and pupae of the polyphagous pest Spodoptera litura Fabr. (Lepidoptera: Noctuidae) with an acetone extract of leaves of Ashwagandha, Withania somnifera L. (Solanaceae), caused toxicity, molt disturbances, formation of larval–pupal, pupal–adult intermediates and adultoids. Our results suggest that W. somnifera acts as an insect growth regulator causing disruption of the endocrine mechanism regulating molting and metamorphosis.
177. Javaid, Arshad and Shafique, Shazia and Shafique, Sobiya, Herbicidal Activity of Withania Somnifera against Phalaris Minor, Natural Product Research, vol. 24, no. 15, pp. 1457--1468, September 2010. doi: 10.1080/14786410903169292. Abstract
     Herbicidal activity of Withania somnifera (L.) Dunal. was studied against Phalaris minor Retz., one of the most problematic weeds of wheat in Pakistan. In laboratory bioassays the aqueous, methanol and n-hexane extracts of 5, 10 and 15\% w/v (fresh weight basis) of the roots and shoots of W. somnifera were applied. Extracts in the different solvents exhibited markedly variable herbicidal activities against germination and seedling growth of the target weed species. The methanol extracts showed the highest toxicity. Different concentrations of methanol shoot and root extracts declined the germination of P. minor by 21–71\%, its shoot length by 40–72\%, its root length by 50–99\% and the plant biomass by 32–83\%. The aqueous extracts proved to be comparatively less toxic than the methanol extracts, where generally the highest concentration of 15\% exhibited pronounced toxicity against the target weed species. There was up to 48, 51, 99 and 55\% suppression of the weed's germination, shoot length, root length and plant biomass, respectively, due to the 15\% aqueous root and shoot extracts. Generally, the n-hexane extracts of both roots and shoots exhibited insignificant or stimulatory effects against weed shoot length and plant biomass. In a foliar spray bioassay, aqueous and methanol shoot extracts of 10\% w/v (dry weight basis) concentration were sprayed on one- and two-week old pot grown P. minor seedlings. Two subsequent sprays were carried out at five day intervals each. The aqueous extract significantly reduced the shoot and root dry biomass of one-week old P. minor plants. In a residue incorporation bioassay, crushed shoots of W. somnifera were incorporated in the soil at 1, 2, … 5\% w/w. Phalaris minor seeds were sown one week after residue incorporation and plants were harvested 45 days after sowing. The lower concentrations of 2 and 3\% significantly reduced, while higher concentrations of 4 and 5\% of residue incorporation completely arrested, the germination of P. minor. The present study concludes that both roots and shoots of W. somnifera contain herbicidal constituents against P. minor.
178. Atri, Neelam and Rai, Nilesh and Singh, Anurag Kumar and Verma, Madhu and Barik, Suvakanta and Gautam, Vibhav and Singh, Santosh Kumar, Screening for Endophytic Fungi with Antibacterial Efficiency from Moringa Oleifera and Withania Somnifera, Journal of scientific research, vol. 64, no. 01, pp. 127--133, 2020. doi: 10.37398/JSR.2020.640118. Abstract
     Plants have a great potential to grow while carrying inside them fungi and bacteria known as endophytes. These endophytes have a potential to produce bioactive molecules which are of immense pharmaceutical importance. In the current study, plants having medicinal properties such as Moringa oleifera and Withania somnifera were explored for studying endophytic fungi associated with them. Moringa oleifera and Withania somnifera are widely known for their nutritional and medicinal values. The endophytic fungal associations of these plants have revealed their efficacy in therapeutic field. In the current study, endophytic fungal strains were extracted by incubating the dried plant samples on potato dextrose agar media, supplemented with streptomycin at 28 °C for 5-10 days. A total of 21 and 24 endophytes were isolated from herbal plant Moringa oleifera and Withania somnifera, respectively. Microscopic study revealed four active strains of M. oleifera i.e. Colletotrichum sp. (MO-S2), Cladosporium sp. (MO-S4, MO-L3) and Fusarium sp. (MO-R1), and two active strains of W. somnifera i.e. Alternaria sp. (WS-S8) and Fusarium sp. (WS-R5). Active strains of endophytic fungi were further screened for their antibacterial activity against pathogenic gram negative bacterial strain of Escherichia coli (ATCC 25922) and gram positive strain of Staphylococcus aureus (ATCC25323). Our results showed that fungal isolates of M. oleifera (MO-S2) and W. somnifera (WS-S8) had antibacterial activity against both the bacterial strains. However, three fungal strains of M. oleifera (MO-L3, MO-S4, and MO-R1) and one of W. somnifera (WS-R5) showed antibacterial activity against gram negative bacterial strain of E. coli.
179. George, Tijith K. and Houbraken, Jos and Mathew, Linu and Jisha, M. S., Penicillium Setosum, a New Species from Withania Somnifera (L.) Dunal, Mycology, vol. 10, no. 1, pp. 49--60, January 2019. doi: 10.1080/21501203.2018.1555868. Abstract
     Medicinal plants are considered as sources of novel and unexplored groups of endophytic microorganisms. A study on endophytic fungal species from the medicinal plant Withania somnifera (L.) Dunal resulted in the isolation of a Penicillium isolate (WSR 62) with antibiotic activity. Phylogenetic analysis showed that the isolate belongs to section Lanata-divaricata, and it is most closely related to P. javanicum. Subsequent detailed phylogenetic analyses using partial β-tubulin (BenA), calmodulin (CaM) and DNA-dependent RNA polymerase II (RPB2) gene sequences of a larger number of related strains revealed the distinctiveness of the isolate in the P. javanicum-clade. The isolate grows fast on Czapek yeast autolysate agar (CYA) and malt extract agar (MEA) incubated at 25°C, 30°C and 37°C. The obverse colony colour is dominated by the conspicuous production of cleistothecia and is greyish yellow on CYA and yellowish brown on MEA. Production of cleistothecia containing prominent spinose ascospores was present on all tested agar media. Based on the phylogenetic analysis and the phenotypic characterisation, strain WSR 62 from Withania is described here as a novel species named Penicillium setosum.
180. Saema, Syed and ur Rahman, Laiq and Singh, Ruchi and Niranjan, Abhishek and Ahmad, Iffat Zareen and Misra, Pratibha, Ectopic Overexpression of WsSGTL1, a Sterol Glucosyltransferase Gene in Withania Somnifera, Promotes Growth, Enhances Glycowithanolide and Provides Tolerance to Abiotic and Biotic Stresses, Plant Cell Reports, vol. 35, no. 1, pp. 195--211, January 2016. doi: 10.1007/s00299-015-1879-5. Abstract
     Overexpression of sterol glycosyltransferase (SGTL1) gene ofWithania somniferashowing its involvement in glycosylation of withanolide that leads to enhanced growth and tolerance to biotic and abiotic stresses.
181. {Sanchita} and Singh, Ruchi and Mishra, Anand and Dhawan, Sunita S. and Shirke, Pramod A. and Gupta, Madan M. and Sharma, Ashok, Physiological Performance, Secondary Metabolite and Expression Profiling of Genes Associated with Drought Tolerance in Withania Somnifera, Protoplasma, vol. 252, no. 6, pp. 1439--1450, November 2015. doi: 10.1007/s00709-015-0771-z. Abstract
     Physiological, biochemical, and gene expression responses under drought stress were studied in Withania somnifera. Photosynthesis rate, stomatal conductance, transpiration rate, relative water content, chlorophyll content, and quantum yield of photosystems I and II (PSI and PSII) decreased in response to drought stress. Comparative expression of genes involved in osmoregulation, detoxification, signal transduction, metabolism, and transcription factor was analyzed through quantitative RT–PCR. The genes encoding 1-pyrroline-5-carboxylate synthetase (P5CS), glutathione S-transferase (GST), superoxide dismutase (SOD), serine threonine-protein kinase (STK), serine threonine protein phosphatase (PSP), aldehyde dehydrogenase (AD), leucoanthocyanidin dioxygenase/anthocyanin synthase (LD/AS), HSP, MYB, and WRKY have shown upregulation in response to drought stress condition in leaf tissues. Enhanced detoxification and osmoregulation along with increased withanolides production were also observed under drought stress. The results of this study will be helpful in developing stress-tolerant and high secondary metabolite yielding genotypes.
182. {Bilal Ahmad Mir}, Reproductive Behaviour and Breeding System of Wild and Cultivated Types of Withania Somnifera (L.) Dunal, Journal of Medicinal Plants Research, vol. 6, no. 5, February 2012. doi: 10.5897/JMPR11.1303. Abstract
     The Indian germplasm of Withania somnifera (L.) Dunal shows remarkable genetic variability both in the cultivated and the wild populations. The utilization of this variability through conventional breeding requires a clear understanding of its reproductive biology and breeding system. It is an amphimictic species practicing open pollination. However, a proximal placement of the stigma and the anther and a synchrony between the receptivity and dehiscence of anthers strongly predispose the species to self pollination and selfing. However open pollination results in equally high percentage of fruit and seed set as on controlled selfing indicating that the species shows facultative autogamy. This raises the probability of genetic improvement through hybridization. The somatic complements revealed a diploid number of 2n=48 in all the accessions tested. The absence of karyomorphological differences indicated that numerical and structural changes do not have a role in controlling the genetic variability of the species. Experimental crosses between the cultivated and the wild accessions produced viable seeds. A significantly higher fruit set and seed germ inability in crosses involving the cultivated types as the seed parent point to the existence of maternal effect.
183. {R. K. Sharma}, Evaluation of Antioxidant Activities of Withania Somnifera Leaves Growing in Natural Habitats of North-West Himalaya, India, Journal of Medicinal Plants Research, vol. 6, no. 5, February 2012. doi: 10.5897/JMPR11.257. Abstract
     Evaluation of antioxidant properties of medicinal plants from Indian Himalayan region has been very rarely carried out. Withania somnifera L. is one of the commercially available and most preferred medicinal plants in the Himalayan region due to its aphrodisiac property and potential to cure various diseases. The review of literature has indicated that the antioxidant activities of W. somnifera have not been carried out for so long. Therefore, in the present study an attempt was made to evaluate the antioxidant properties of W. somnifera collected from two different habitats that is, forest and roadside at Kullu, north-west Himalaya. The total phenolic and flavonoid contents and DPPH (1, 1-Diphenyl-2pycrylhydrazyl) scavenging potential of leaves extract of W. somnifera varied significantly between the habitat (p{$<$}0.05). The results further showed that the DPPH scavenging potential of leaves extract at forest site was found significantly higher that is 51\% over the roadside. The study indicates that antioxidant activities of leaves of W. somnifera L. varies habitat to habitat and antioxidant properties decrease if plants are exposed to vehicular pollution. Therefore, plantation and cultivation of this species in vehicular pollution free areas are suggested.
184. Pandey, Shiv S. and Singh, Sucheta and Pandey, Harshita and Srivastava, Madhumita and Ray, Tania and Soni, Sumit and Pandey, Alok and Shanker, Karuna and Babu, C. S. Vivek and Banerjee, Suchitra and Gupta, M. M. and Kalra, Alok, Endophytes of Withania Somnifera Modulate in Planta Content and the Site of Withanolide Biosynthesis, Scientific Reports, vol. 8, no. 1, pp. 5450, April 2018. doi: 10.1038/s41598-018-23716-5. Abstract
     Tissue specific biosynthesis of secondary metabolites is a distinguished feature of medicinal plants. Withania somnifera, source of pharmaceutically important withanolides biosynthesizes withaferin-A in leaves and withanolide-A in roots. To increase the in planta withanolides production, a sustainable approach needs to be explored. Here, we isolated endophytes from different parts of W. somnifera plants and their promising role in in planta withanolide biosynthesis was established in both in-vivo grown as well in in-vitro raised composite W. somnifera plants. Overall, the fungal endophytes improved photosynthesis, plant growth and biomass, and the root-associated bacterial endophytes enhanced the withanolide content in both in-vivo and in-vitro grown plants by modulating the expression of withanolide biosynthesis genes in leaves and roots. Surprisingly, a few indole-3-acetic acid (IAA)-producing and nitrogen-fixing root-associated endophytes could induce the biosynthesis of withaferin-A in roots by inducing in planta IAA-production and upregulating the expression of withanolide biosynthesis genes especially MEP-pathway genes (DXS and DXR) in roots as well. Results indicate the role of endophytes in modulating the synthesis and site of withanolides production and the selected endophytes can be used for enhancing the in planta withanolide production and enriching roots with pharmaceutically important withaferin-A which is generally absent in roots.
185. Mir, B. A. and Koul, S. and Kumar, A. and Kaul, M. K. and Soodan, A. S. and Raina, S. N., Assessment and Characterization of Genetic Diversity in Withania Somnifera (L.) Dunal Using RAPD and AFLP Markers, African Journal of Biotechnology, vol. 10, no. 66, pp. 14746--14756, 2011. doi: 10.4314/ajb.v10i66. Abstract
     Genetic diversity of 23 accessions of Withania somnifera collected from different geographical regions of India was estimated by employing Random Amplification of Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) markers. Eighteen RAPD primers and six AFLP primer combinations revealed 37.82 and 43.94\% polymorphism, respectively, among 163 and 286 genetic loci amplified. The AFLP assay revealed higher levels of polymorphism among the tested W. somnifera accessions compared to the RAPD. Mean genetic diversity based on Shannon index ranged from 1.33 (RAPD) to 5.13 (AFLP). Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. The matrix reveals, two main clusters, wild accessions formed one cluster and the cultivated accessions formed the other. The cultivated accessions are well separated from the wild ones at a low similarity value of 0.3, indicating that cultivated and wild accessions are highly distinct. Morphologically cultivated accessions were also quite distinct from the wild ones and the cluster analysis of RAPD and AFLP fingerprints clearly discriminated the five cultivated accessions of W. somnifera. A strong correlation was observed between morphology and molecular marker systems. Identification of specific markers to wild as well as cultivated accessions is yet another important finding in the present study. Such genetic diversity is useful in facilitating the development of large number of new varieties through hybridization, transfer of useful genes, thus maximizing the use of such available germplasms as genetic resource materials for breeders. The present input, first of its kind in Ashwagandha, will thus assist the marker assisted crop improvement programme.Key words: Withania somnifera, genetic diversity, RAPD, AFLP, polymorphism, Shannon index.
186. Anandakumar, T. M. and Kumar, Devendra and Shivanna, B. and Kumar, Rakesh, Physical Properties of Ashwagandha Seeds (Withania Somnifera L.) – A Medicinal Crop, Industrial Crops and Products, vol. 186, pp. 115233, October 2022. doi: 10.1016/j.indcrop.2022.115233. Abstract
     Ashwagandha (Withania somnifera L.) is a potential medicinal plant belongs to the Solanaceae family, offers various health benefits, such as enhancement of memory, improved blood sugar, and inhibition of inflammation, stress and anxiety, and boosted in muscle strength and fertility. Hence, the demand for the roots of ashwagandha is very high in the Indian market, thus the ashwagandha crop needs to be cultivated in a larger area to mitigate the demand of roots in Indian as well as foreign markets. To cultivate this crop in larger areas with less time and manpower consumption, mechanization of sowing operation is very much essential. In this regard, this study has been conducted to determine the physical properties of ashwagandha seeds (Five cultivars, namely NMITLI-101, NMITLI-118, CIM-Pushti, Pratap and Poshitha) for designing and developing of seed sowing equipment. The data collected were analyzed on R software based web application developed by department of agricultural statistics, college of agriculture, Vellayani, Keral Agricultural University, Vellanikkara, Kerala. Results revealed that cv. CIM-Pushti seed has significantly higher values as compared with other cultivars in major, minor, intermediate diameter, arithmetic mean diameter, and geometric mean diameters. Similarly, in the case of physical properties like sphericity, surface area, sample volume, and elongation ratio, cv. CIM-Pushti showed significantly higher values as compared with other cultivars whereas in the case of aspect ratio and flakiness ratio, cv. Poshita showed significantly higher values. In the case of gravimetric properties, cv. Pratap and NMITLI-118 resulted in higher values of test weight (1000 seeds weight) as compared with other cultivars. Further, cv. Pratap and CIM-Pushti have significantly higher values of bulk density and true density over other cultivars, respectively. These results are likely to be very much helpful for agricultural engineers to design various seed handling tools/machines like conveying machines, storage containers, processing machines and sowing equipment for Ashwagandha seeds.
187. Kumar, Arun and Mir, Bilal A. and Sehgal, Deepmala and Dar, Tanvir H. and Koul, Sushma and Kaul, Maharaj K. and Raina, Soom N. and Qazi, Ghulam N., Utility of a Multidisciplinary Approach for Genome Diagnostics of Cultivated and Wild Germplasm Resources of Medicinal Withania Somnifera, and the Status of New Species, W. Ashwagandha, in the Cultivated Taxon, Plant Systematics and Evolution, vol. 291, no. 3, pp. 141--151, February 2011. doi: 10.1007/s00606-010-0372-4. Abstract
     Realizing the inconsistencies that exist in the extent and nature of differentiation in the Withania somnifera genetic resources in India, the 21 cultivated and wild accessions, and the two hybrids (cultivated~×~wild accessions and vice versa) were investigated for morphological, cytogenetical, chemical profiling, and crossability features. Their nuclear and chloroplast genomes were also assayed at the nucleotide sequence level, and by use of DNA markers. Chloroplast DNA diversity and somatic chromosome number (2n~=~48) were not helpful in identifying the differences. Other approaches, on the other hand, especially restriction endonuclease digests, types and sequence length composition of ITS 1 and ITS 2 of nuclear ribosomal DNA, AFLP fingerprinting, and crossability barriers unambiguously provided startling discrete differences between the cultivated and wild accessions, indicating a clear division of W. somnifera into two distinct lineages. These data, therefore, are indicative of the fact that because of the unique characteristics of its nuclear genome, and strong crossability barriers vis-à-vis wild accessions of W. somnifera, the cultivated accessions should be relegated to the rank of the separate species, W. ashwagandha.
188. Tripathi, Niraj and Saini, Navinder and Mehto, Vandana and Kumar, Sunil and Tiwari, Sharad, Assessment of Genetic Diversity among Withania Somnifera Collected from Central India Using RAPD and ISSR Analysis, Medicinal and Aromatic Plant Science and Biotechnology, vol. 6, no. 1, pp. 33--39, 2012. url: https://www.researchgate.net/publication/235285945_Assessment_of_Genetic_Diversity_among_Withania_somnifera_Collected_from_Central_India_using_RAPD_and_ISSR_Analysis. Abstract
     Withania somnifera is a multipurpose plant of immense therapeutic value and wide geographic distribution exhibiting extensive phenotypic and chemical variability. Characterization of plants using molecular markers is an ideal approach for improvement and conservation of plant genetic resources. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular fingerprinting markers were employed as genetic markers to assay the genetic relationship of 16 accessions of W. somnifera collected from different locations of India. Randomly selected 25 decamer primers amplified 204 RAPD marker loci out of which 152 bands (74.5\%) were polymorphic. Twenty eight ISSR primers were used to generate fingerprints and a total of 186 alleles were amplified, out of which 151 were polymorphic (81.1\%). ISSR markers were more informative than the RAPD markers. Several ISSR markers amplified genotype-specific alleles that can be used for authentication and detection of adulteration in plant material. Similarity matrices were generated from RAPD and ISSR marker data separately using Jaccard’s coefficient and dendrograms were constructed based on UPGMA clustering. Genotypes were clustered into two groups and the grouping was similar for both marker systems.
189. Kumar, Ashish and Lal, R. K. and Gupta, A. K. and Jnanesha, A. C. and Jhang, Tripta and Srivastava, Jyotsana and Kumari, Manisha, Exegesis of Cultivars-Multi-Year/Environment Interactions for Agro Morphological Traits in Ashwagandha (Withania Somnifera (L.) Dunal), South African Journal of Botany, vol. 151, pp. 523--531, December 2022. doi: 10.1016/j.sajb.2022.10.039. Abstract
     A randomized block design was used to examine the important medicinal plant ashwagandha in three replications throughout the late Kharif seasons of 2018, 2019, and 2020. The cultivars of Ashwagandha (Withania somnifera Dunal), CIMAP Chetak, CIMAP Pratap, NMITLI-101, NMITLI-118, and Poshita were used to evaluate the quality of dry root yield for a better quality of five variations over the years in south India and variety recommendations. The years I and III were the two years with the most discrimination, whereas year II had the least discrimination. A lower AEA angle in a test year is more typical of subsequent test years. The first and second years are the most representative, whereas the third and fourth are the least representative. The year I was discriminating and representative during the test year, making them suitable candidates for selecting adapted varieties. The separation of stable and unstable types was made easier in environments that were discriminating or non-representative of test years. There was much diversity for stability in the various traits, as reflected by the character means. A line that passes through the biplot origin and measures performance over a year determines the rank. Four varieties, CIMAP-Pratap, Poshtita, CIMAP-Chetak, and NMITLI-118, were highly stable and good rankers for the dry root yield, root starch (\%), starch and fiber ratio, and total alkaloid, among other economic traits (\%). These varieties were highly stable for dry root yield and root starch (\%), starch and fiber ratio, and total alkaloid, based on the mean performance of major economic features and stability measures (\%). As a result, these types are recommended for further cultivation in southern India.
190. Joshi, Nilesh R. and Patel, M. A. and Patel, K. N. Prajapati {and} A. D., Genetic Variability, Correlation and Path Analysis in Ashwagandha [Withania Somnifera (L.) Dunal], Electronic Journal of Plant Breeding, vol. 5, no. 4, pp. 875--880, December 2014. url: https://www.ejplantbreeding.org/index.php/EJPB/article/view/309. Abstract
     Forty diverse genotypes of Ashwagandha [Withania somnifera (L.)Dunal] were evaluated for 12 quantitative characters. Higher magnitude of genotypic and phenotypic coefficient of variation was recorded for leaf width, number of primary branches per plant, number of secondary branches per plant, root length, dry weight of root, withanoloide content and starch content. High heritability in conjuction with high genetic advance were observed for dry weight of root, root length, diameter of root at collar region, withanoloide content and starch content which indicated that selection could be effective for these traits. The estimates of correlation coefficient revealed that the genotypic correlations were higher than their corresponding phenotypic correlation for all characters. It was observed that root length, diameter of root at collar region, plant height, leaf length, leaf width, days to flower initiation and days to maturity showed significant positive association with dry weight of root at genotypic as well as phenotypic levels. Path analysis revealed that plant height, days to maturity, diameter of root at collar region and leaf width were major characters having positive direct effect and significant association with dry root yield per plant. Hence selection for these trait would be quite effective to improve dry root yield in Ashwagandha.
191. Samaddar, Tapojita and Nath, Sayantani and Halder, Mihir and Sil, Bipradut and Roychowdhury, Dipasree and Sen, Sumitra and Jha, Sumita, Karyotype Analysis of Three Important Traditional Indian Medicinal Plants, Bacopa Monnieri, Tylophora Indica and Withania Somnifera, The Nucleus, vol. 55, no. 1, pp. 17--20, April 2012. doi: 10.1007/s13237-012-0048-2. Abstract
     Chromosome characters of three indigenous medicinal plants of high repute were studied. Their karyotypes were commonly mono-modal, decreasing in length from the longest to the shortest chromosomes. The chromosome complement in centromeric positions was 6sm+34M+24m for Bacopa monnieri (L.) Wettst. (2n\,=\,64), 2m.st+12M+6sm+2m for Tylophora indica (Burm.f.) Merrill (2n\,=\,22) and 2m.st+4m.sm+4M+18m+20sm for Withania somnifera (L.) Dunal (2n\,=\,48).
192. Srivastava, Chhamta, Taxonomic and Ethnobiological Status of {\emph{Withania Somnifera}} (L.) Dunal: The Indian Ginseng, Medicinal Plants - International Journal of Phytomedicines and Related Industries, vol. 1, no. 2, pp. 129, 2009. doi: 10.5958/j.0975-4261.1.2.019.
193. Singhal, Vijay and Kumar, Puneet, Cytomixis during Microsporogenesis in the Diploid and Tetraploid Cytotypes of Withania Somnifera (L.) Dunal, 1852 (Solanaceae), Comparative cytogenetics, vol. 2, pp. 85-92., January 2008. Abstract
     Withania somnifera (L.) Dunal, 1852, popularly known as “Ashwagandha” shows considerable morphogenetic diversity in Indian populations. Both the diploid (n=12) and tetraploid (n=24) cytotypes presently reported in the populations from Punjab, Rajasthan and Himachal Pradesh show the phenomenon of cytomixis which is a first record for the species. The inter PMC (Pollen mother cell) transfer of chromatin material in the diploid cytotype is present in 40.76\% PMCs involving 2-6 PMCs. However, among tetraploids frequency of cytomixis is much less (15.80-24.32\%) and involves only 2-3 PMCs. Chromatin transfer is noticed during the first and second meiotic division in the diploid as compared with the tetraploid where it is observed only during the first meiotic division. The percentage of meiotic abnormalities such as chromosome stickiness, lagging of chromatin material during anaphases and telophases is also higher in the diploid compared with the tetraploid. The microsporogenesis is also abnormal in the diploid resulting into the formation of polyads and tetrads with micronuclei whereas it is normal in the tetraploid. Cytomixis also seems to have had a greater effect on the apparent pollen fertility in the diploid compared with the tetraploid. Furthermore, cytomixis have also resulted into pollen grains of three different sizes in the diploid and only of two sizes in the tetraploid.
194. Srivastava, Abhilasha and Gupta, Anil K. and Shanker, Karuna and Gupta, Madan M. and Mishra, Ritu and Lal, Raj K., Genetic Variability, Associations, and Path Analysis of Chemical and Morphological Traits in Indian Ginseng [Withania Somnifera (L.) Dunal] for Selection of Higher Yielding Genotypes, Journal of Ginseng Research, vol. 42, no. 2, pp. 158--164, April 2018. doi: 10.1016/j.jgr.2017.01.014. Abstract
     Background The study was carried out to assess the genetic variability present in ashwagandha and to examine the nature of associations of various traits to the root yield of the plant. Methods Fifty-three diverse genetic stocks of ashwagandha (Withania somnifera) were evaluated for 14 quantitative characteristics. Analysis of variance, correlation, and path coefficient analysis were performed using the mean data of 2 years. Results Analysis of variance revealed that the genotypes differed significantly for all characteristics studied. High heritability in conjunction with high genetic advance was observed for fresh root weight, 12 deoxywithastramonolide in roots, and plant height, which indicated that selection could be effective for these traits. Dry root weight has a tight linkage with plant height and fresh root weight. Further, in path coefficient analysis, fresh root weight, total alkaloid (\%) in leaves, and 12 deoxywithastramonolide (\%) in roots had the highest positive direct effect on dry root weight. Conclusion Therefore, these characteristics can be exploited to improve dry root weight in ashwagandha genotypes and there is also scope for the selection of promising and specific chemotypes (based on the alkaloid content) from the present germplasm.
195. Bolleddula, Jayaprakasam and Fitch, William and Vareed, Shaiju K. and Nair, Muraleedharan G., Identification of Metabolites in Withania Sominfera Fruits by Liquid Chromatography and High-Resolution Mass Spectrometry, Rapid Communications in Mass Spectrometry, vol. 26, no. 11, pp. 1277--1290, 2012. doi: 10.1002/rcm.6221. Abstract
     RATIONALE Withania somnifera is a rich source of biologically active secondary metabolites. Our earlier investigations on the fruits of this plant have yielded novel compounds, withanamides, with potent antioxidant activity and protective effect on β-amyloid-induced cytotoxicity in vitro. However, several minor compounds present in the fruits have not been characterized previously which may contribute to the observed biological activities. METHODS Liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) with data-dependent and targeted MS/MS experiments were conducted to elucidate the structure of observed metabolites. RESULTS A total of 62 metabolites identified included 32 withanamides, 22 withanolides, 3 steroidal saponins, 2 lignanamides, feruloyl tyramine, methoxy feruloyl tyramine and a diglucoside of hydroxyl palmitic acid. The structures of these compounds were proposed based on accurate masses of the molecular and fragment ions. Several of these new compounds identified from the profile were derived from withanamides with variations in aliphatic and/or glycosyl moieties. In addition, six new withanolides, a new hydroxy fatty acid diglucoside and several known compounds in the extract were identified. CONCLUSIONS The current study revealed the presence of several new and known compounds in Withania somnifera fruits. High-resolution MS and MS/MS experiments provide an extremely effective approach to derive the structures of plant secondary metabolites including isomeric compounds. Copyright © 2012 John Wiley \& Sons, Ltd.
196. Ghimire, B. and Heo, K., Embryology of Withania Somnifera (L.) Dunal (Solanaceae), Acta Biologica Cracoviensia. Series Botanica, vol. 54, no. 2, 2012. doi: 10.2478/v10182-012-0027-6.
197. Ghimire, Balkrishna and Ghimire, Bimal Kumar and Heo, Kweon, Seed Characteristics of Withania Somnifera (Solanaceae), Korean Journal of Plant Taxonomy, vol. 41, no. 2, pp. 103--107, 2011. doi: 10.11110/kjpt.2011.41.2.103. Abstract
     가지과 Withania somnifera (L.) Dunal의 종자 해부학적 특징을 알기 위하여 광학현미경과 주사전자 현미경을 사용하여 관찰하였다. 종자의 색깔은 노랑색이고, 종자의 형태는 신장형이며, 크기가 2−3 mm 정도이다. 종자는 종의를 가지고 있지 않았으며, 배유가 잘 발달한 유배유종자였다. 종피형태는 외종피외층형으로 나타났다. 종피는 외종피만으로 구성되는 단주피성 이었으며, 한 층의 외종피외층, 여러 층의 외종피중층, 그리고 한 층의 외종피내층으로 구성되었다. 그러나 종자가 완전히 성숙했을 때는 외종피중층이 모두 압착되었으며, 외종피외층은 특징적으로 파상형의 후벽세포로 발달하였다. 종피의 표면은 망상구조를 가지며, 수층 벽구조는 파상형으로 발달하였고, 표면에 여러 개의 구멍이 발달하는 특징이 관찰되었다. The seed characteristics of Withania somnifera were studied using light and scanning electron microscopy in order to determine the specific features of this species. The seed color is yellow, and the seed shape is reniform measuring between two to three millimeters. The seed of W. somnifera is exarillate and albuminous. The seed coat type is exotestal. The seed coat develops from a single integument. The young seed coat consists of single-layered exotesta, multi-layered mesotesta and single-layered endotesta. However, parenchymatous mesotesta layers are completely compressed at maturity. Therefore, the seed coat was represented by sclerenchymatous exotesta. The primary sculpture on the seed surface is reticulate, and cells are irregular in shape with undulating anticlinal walls. In addition, the seed surface has several characteristic holes between the anticlinal walls.
198. Ha, Ji Won and Yu, Jae Sik and Lee, Bum Soo and Kang, Dong-Min and Ahn, Mi-Jeong and Kim, Jung Kyu and Kim, Ki Hyun, Structural Characterization of Withanolide Glycosides from the Roots of Withania Somnifera and Their Potential Biological Activities, Plants, vol. 11, no. 6, pp. 767, January 2022. doi: 10.3390/plants11060767. Abstract
     Withania somnifera (Solanaceae), commonly known as “ashwagandha”, is an ayurvedic medicinal plant that has been used for promoting good health and longevity. As part of our ongoing natural product research for the discovery of bioactive phytochemicals with novel structures, we conducted a phytochemical analysis of W. somnifera root, commonly used as an herbal medicine part. The phytochemical investigation aided by liquid chromatography-mass spectrometry (LC/MS)-based analysis led to the isolation of four withanolide glycosides (1–4), including one new compound, withanoside XII (1), from the methanol (MeOH) extract of W. somnifera root. The structure of the new compound was determined by nuclear magnetic resonance (NMR) spectroscopic data, high-resolution (HR) electrospray ionization (ESI) mass spectroscopy (MS), and electronic circular dichroism (ECD) data as well as enzymatic hydrolysis followed by LC/MS analysis. In addition, enzymatic hydrolysis of 1 afforded an aglycone (1a) of 1, which was identified as a new compound, withanoside XIIa (1a), by the interpretation of NMR spectroscopic data, HR-ESIMS, and ECD data. To the best of our knowledge, the structure of compound 2 (withagenin A diglucoside) was previously proposed by HRMS and MS/MS spectral data, without NMR experiment, and the physical and spectroscopic data of withagenin A diglucoside (2) are reported in this study for the first time. All the isolated compounds were evaluated for their anti-Helicobacter pylori, anti-oxidant, and anti-inflammatory activities. In the anti-Helicobacter pylori activity assay, compound 2 showed weak anti-H. pylori activity with 7.8\% inhibition. All the isolated compounds showed significant ABTS radical scavenging activity. However, all isolates failed to show inhibitory activity against nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. This study demonstrated the experimental support that the W. somnifera root is rich in withanolides, and it can be a valuable natural resource for bioactive withanolides.
199. Ghoshal, Sautik and Ghule, Chetana and Mirgal, Amit and Girme, Aboli and Hingorani, Lal, Recent Findings by High-Performance Thin-Layer Chromatographic Separation for a Comprehensive Analysis of Withania Somnifera by Densitometry and Mass Spectrometry: An Assessment to Quality and Adulteration, JPC – Journal of Planar Chromatography – Modern TLC, vol. 35, no. 5, pp. 439--451, November 2022. doi: 10.1007/s00764-022-00187-z. Abstract
     Withania somnifera (L.) Dunal (WS), also known as Ashwagandha, is an Indian herb classified as Rasayana in Ayurveda owing to its rejuvenating and health-promoting activities. A sensitive and robust high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of withanoside IV (WS4), withanoside V (WS5), withaferin A (WFA), and kaempferol-based glucoside (KRG) in the roots and aerial parts of WS. The present rapid HPTLC method reports the separation and simultaneous quantification of three diverse classes of WS as withanolide (WFA), withanosides (WS4 and WS5) with an analytical marker flavonoid glycoside (KRG) for effective detection, of the aerial parts separating them from roots. In normal-phase TLC plates, these markers showed good resolution in ethyl acetate‒chloroform‒methanol‒water (8:3:4.4:1.8, V/V)~solvent system. Densitometric analysis was performed for KRG (at 254~nm), WFA, WS4, and WS5 (at 540~nm after derivatization) with characterization by high-performance thin-layer chromatography‒tandem mass spectrometry (HPTLC‒MS/MS). The method was found linear (R2\,{$>$}\,0.99) for KRG and WFA (200‒1000~ng/band) and WS4‒WS5 (400‒2000~ng/band) with excellent recoveries (80‒100\%) for all compounds. In this HPTLC based assessment, three compounds of WS could be rapidly detected for quality control distinguishing from other Withania and plant species reported as adulterants. Additionally, this method separated pigment-based compounds at 366~nm in chemical fingerprinting in WS aerial parts samples. Therefore, the presence of KRG as an analytical marker with these pigment zones can identify aerial parts, leaves, stems, fruits with calyx, and fruits in the HPTLC analysis, separating them from root samples. Hence, this HPLTC method was found robust and economical for the estimation and surety of the quality of WS samples in quantitative and qualitative analysis.
200. Bargale, Sushant Sukumar and Tripathy, T. B. and Shashirekha, H. K., Phyto Physicochemical Profile of Withania Somnifera Dunal (Solanaceae), Journal of Drug Delivery and Therapeutics, vol. 9, no. 3-s, pp. 263--268, June 2019. doi: 10.22270/jddt.v9i3-s.3008. Abstract
     Introduction- The present article deals with study of phytochemical analysis of Withania somnifera Dunal roots. Withania somnifera also known as Ashwagandha or winter cherry. Various preparations of Ashwagandha (WS) are available in the market used in the treatment of many clinical conditions in India. Objective- Evolution of Physico-chemical values and phytochemical analysis of Ashwagandha Churna. ~Materials and Methods- The current investigation deals with extraction and detection or screening of active phytochemical compounds from different extracts of Withania somnifera root. Pharmacognostic studies, Physicochemical studies, Preliminary phytochemical studies was carried out. Result and conclusion - The result drown were 2\% foreign mater was determined. Loss on drying 1.6\%, total ash obtained was 9 \%, acid insoluble ash was 1\% and water soluble extractive was 12 \% and Alcohol soluble extractive was 13 \%. The phytochemical investigation revealed the presence of various phytochemical constituents such as alkaloids, flavonoids, carbohydrate, Steroids and Saponin Glycoside. Keywords: Ashwagandha, Withania Somnifera, Phytochemical.
201. Molchanova, Ann and Kekina, Helene and Logvinenko, Lidia and Caruso, Gianluca and Golubkina, Nadezhda and Shevchuk, Oksana and Khlipenko, Ludmila, Antioxidant Properties and Elemental Composition of Withania Somnifera L., Agriculture and Food, 2019. url: https://www.scientific-publications.net/get/1000034/1564599629890995.pdf. Abstract
     Withania somnifera (L.) Dunal is popular in the traditional medicine of many areas of the world due to its high biological activity. Research was carried out in order to characterize the plants of this species in terms of antioxidant content and activity, as well as macro- and microelements concentration in leaves and roots plants grown along Crimea sea coast, which receive an intensive element transfer from the sea surface. The leaves of Withania somnifera showed higher levels of polyphenols, flavonoids and total antioxidant activity compared to roots (7.52 mg eq. GA g-1 d.w., 2.30 mg eq. quercetin g-1 d.w. and 38.2 mg eq. GA g-1 d.w. in leaves; 3.83 mg eq. GA g-1 d.w; 0.48 mg eq. quercetin g-1 d.w. and 10.8 mg eq. GA g-1 d.w. in the roots). Withanolides content in roots reached 0.62 \%. The determination of 25 macro- and microelements revealed that Withania somnifera intensively accumulated Ca, K, Mg, P, B, I, Li, Mn, Mo, Si and Zn in leaves, whereas the concentration of V, Cr, Fe and Al in roots exceeded those recorded in leaves by 5.5, 5.86, 4.35 and 5.26 times respectively. The highest difference between leaves and roots concentration was detected for Li whose content in leaves was 10 times higher than in roots. Among other cultivated herbs (14 species belonging to Artemisia and Myrthus), Withania somnifera showed the highest levels of Li which makes it suppose this species is a good Li source for humans. Moreover, the leaves of W. somnifera contain high levels of iodine.
202. Barnes, D. A. and Barlow, R. and Nigam, Poonam S. and {Owusu-Apenten}, Richard K., Antioxidant, Anticancer and Antibacterial Activity of Withania Somnifera Aqueous Root Extract, November 2015. doi: 10.9734/JABB/2016/22523. Abstract
     Aims: To evaluate total antioxidant capacity, anticancer activity and antibacterial effects Withania somnifera aqueous-root extracts. Study Design: In vitro study. Place of Study: School of Biomedical Sciences, Ulster University, UK. Methodology: Total antioxidant capacity (TAC) of whole powder and freeze dried W. somnifera aqueous-root extracts was determined using FRAP, DPPH, Folin and ABTS assays. Anticancer activity was accessed using MDA-MB-231 breast cells and Sulforhodamine B staining for cell viability. Antibacterial activity was by disk diffusion assay with penicillin, amoxicillin and streptomycin as positive controls. Results: The TAC for W. somnifera extract was 86, 47, 195,or 443 gallic acid equivalents per 100g dry basis (mgGAE/ 100 g) using FRAP, DPPH, Folin or ABTS assays, respectively. Corresponding TAC values for freeze dried W. somnifera aqueous-root extract were, 418, 553, 1898 or, 1770 (mgGAE/100 g). W. somnifera aqueous-root extract inhibited MDA-MB-231 cell proliferation in a dose-dependent manner with IC50 = 0.19 mg/ml (21 µM GAE). Nil antibacterial effects were detected for freeze dried W. somnifera extract (0-1 mg/ml) across six species of bacteria tested. Conclusion: Withania somnifera root water extract showed significant antioxidant and anticancer activity for MDA-MB-231 breast cancer cells but no antibacterial activity under the conditions of this study.
203. Ramachandran, A. and Kumar, M. S., FT-IR, UV and Antimicrobial Activity with Ania Somnifera and Withania Obtusifolia., International Journal of Pharma and Bio Sciences, vol. 5, no. 4, 2014. url: https://www.cabdirect.org/globalhealth/abstract/20153064150. Abstract
     The present study was to compare the Phytochemical and Antimicrobial analysis of W. somnifera and W. obtusifolia. In the Ethanolic leaf and root extract of Withania species were screened for phytochemical compounds by qualitative and FT-IR, UV method. Withania species have been used for remedies of human pathogen because they contain therapeutic compounds. This research work was mainly focused to...
204. Devkar, Santosh T. and Jagtap, Suresh D. and Katyare, Surendra S. and Hegde, Mahabaleshwar V., Estimation of Antioxidant Potential of Individual Components Present in Complex Mixture of Withania Somnifera (Ashwagandha) Root Fraction by Thin-Layer Chromatography—2,2-Diphenyl-1-Picrylhdrazyl Method, JPC – Journal of Planar Chromatography – Modern TLC, vol. 27, no. 3, pp. 157--161, June 2014. doi: 10.1556/JPC.27.2014.3.2. Abstract
     A thin-layer chromatography–2,2-diphenyl-1-picrylhdrazyl (TLC—DPPH) method has been developed for the quantification of withanolides and simultaneous estimation of antioxidant potential of each constituent present in a complex mixture. High-performance thin-layer chromatography (HPTLC) of withanolide-rich fraction of Withania somnifera root was performed on Si 60F254 HPTLC plates with dichloromethane-toluene-methanol-acetone-diethyl ether (6.5:7:4:1.5:1, v/v) as a mobile phase for the separation of withanolides and on the basis of RF values 0.07, 0.55, 0.61, 0.63, 0.66, and 0.77 for withanoside V, withaferine A, 1,2-deoxywithastramonolide, withanone, withanolide A, and withanolide B, respectively. The concentrations of withanoside V, withaferine A, 1,2-deoxywithastramonolide, withanone, withanolide A, and withanolide B were 0.86, 12.9, 1.92, 1.52, 5.24, and 4.52 mg g−1 respectively. A TLC-DPPH rapid test was used to assess the free-radical scavenging activity of constituents in withanolide-rich fraction for the first time. In withanolides, withanoside V and withanolide B show the highest antioxidant potential. In unknown compounds, peak No. 17 at RF 0.92 has the highest antioxidant potential and peak No. 16 at RF 0.87 has recorded the lowest antioxidant potential. It is also found that some of the unidentified components had significant antioxidant potential. This technique has several advantages; therefore, this method will be useful for rapidly validating the Ayurvedic products containing W. somnifera.
205. Joshi, Pallavi and Misra, Laxminarain and Siddique, Amreen A. and Srivastava, Monica and Kumar, Shiv and Darokar, Mahendra P., Epoxide Group Relationship with Cytotoxicity in Withanolide Derivatives from Withania Somnifera, Steroids, vol. 79, pp. 19--27, January 2014. doi: 10.1016/j.steroids.2013.10.008. Abstract
     Withania somnifera is one of the highly reputed medicinal plants of India. Its steroidal constituents exist in the form of two major substitution patterns, viz. withaferin A (1) and withanone (5). Withaferin A with oxidation at carbons 4, 5, and 6 is considered as an active type, especially as anticancer, whereas the withanones with oxidation at carbons 5, 6, and 7 rarely show any activity. We prepared a series of derivatives with modifications at carbons 5, 6, and 7 in ring B of these withanolides to study the role of the epoxide group towards the cytotoxic property of these bioactive steroids. We have converted withanolides into the respective thiiranes, amino alcohols and alcohols by selective reactions at the epoxide ring and were evaluated for in vitro anticancer activity against four cancer cell lines to study the structure activity relationships. The transformations of the epoxide group in withanolides of the withaferin A type showed moderate reduction in their cytotoxicity whereas the almost inactive withanones have shown some improvements in their alcohol derivatives.
206. Vinotha, S. and Ira, T. and Sri Ranjani, S., Phytochemical Screening of Various Extracts of Root of Withania Somnifera (L.) Dunal, 2013. url: http://repo.lib.jfn.ac.lk/ujrr/handle/123456789/6258. Abstract
     Withania somnifera (L) Dunal (Solanaceae), commonly known as Aswagandha, is one of the most valued medicinal plants with a number of pharmaceutical applications. Root extracts of W. somnifera are commonly used as a remedy for variety of ailments and a general tonic for overall health and longevity in the Traditional medicine system. It is also reported to have anti-inflammatory, anti-arthritic, antitumor, immunomoduatory and antioxidant effects. Although the phytochemical screening of W. somnifera has been already published, the aim of this study is to compile its phytochemical constituents in various extracts, which were carried out using standard laboratory procedures. Quantification of some of the active constituents like alkaloids, flavonoids and saponins were also carried out. The preliminary phytochemical screening of hot and cold ethanol, methanol and water extracts showed the presence of alkaloids, saponins, flavonoids, steroids, tannins, proteins, reducing sugar and coumarins and absence of quinones or anthraquinones. Cold and hot water extracts indicated the presence of fat and fixed oil. The total alkaloid and flavonoid content were found to be 0.81± 0.01\% and 14.43±0.40\% respectively, and total saponin content was (Foaming Index) FI {$<$} 100. The findings are consistent with the presence of biologically active constituents in the polar extracts of W. somnifera.
207. Devkar, Santosh and Badhe, Yogesh and Jagtap, Suresh and Hegde, Mahabaleshwar, Quantification of Major Bioactive Withanolides in Withania Somnifera (Ashwagandha) Roots by HPTLC for Rapid Validation of Ayurvedic Products, JPC - Journal of Planar Chromatography - Modern TLC, vol. 25, no. 4, pp. 290--294, July 2012. doi: 10.1556/jpc.25.2012.4.2. Abstract
     The high-performance thin-layer chromatography (HPTLC) method has been developed for the simultaneous quantification of withaferine A, 1,2 deoxy-withastramonolide, withanolide A, and withanolide B for the validation of Withania somnifera (Ashwagandha roots) as raw material and Ashwagandha-containing finished Ayurvedic products. HPTLC of W. somnifera methanolic extracts was performed on Si 60F254 (10 × 10 cm) HPTLC plates with dichloromethane-toluene-methanol-acetone-diethyl ether (7.5:7.5: 3:1:1 v/v) as a mobile phase. Upon separation, quantitative evaluation of these withanolides was performed in the absorption reflection mode at 235 nm. The method was validated for precision, reproducibility, and accuracy. On the basis of RF values of 0.58, 0.61, 0.68, and 0.79 for withaferine A, 1,2 deoxy-withastramonolide, withanolide A, and withanolide B, respectively, were identified. On the basis of linear calibration curves for all withanolides in the range of 0.2-1.2 μg, an average recovery of withaferine A, 1,2 deoxy-withastramonolide, withanolide A, and withanolide B was 98\%, 99.5\%, 98\%, and 99\%, respectively. The method is very simple, precise, specific, sensitive, accurate, and economical for rapidly validating the Ayurvedic products containing W. somnifera (Ashwagandha).
208. Oza, Vishal P. and Trivedi, Shraddha D. and Parmar, Pritesh P. and Subramanian, R. B., Withania Somnifera (Ashwagandha): A Novel Source of L-Asparaginase, Journal of Integrative Plant Biology, vol. 51, no. 2, pp. 201--206, 2009. doi: 10.1111/j.1744-7909.2008.00779.x. Abstract
     Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L-asparaginase enzyme, Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 °C. The Km value for the enzyme is 6.1 × 10−2 mmol/L. This is the first report for L-asparaginase from W. somnifera, a traditionally used Indian medicinal plant.
209. Naik, Pralhad D and L, Mishra R, Quantitative Analysis of Secondary Metabolites of Withania Somnifera and Datura Stramonium - Count Search, International Journal of Science and Research, vol. 4, no. 3, pp. 300--303, March 2015. url: https://www.ijsr.net/. Abstract
     Medicinal plants are importance resource of drugs of traditional system of medicine. Secondary metabolites are the chemical constituents present in the plants and are important in determining medicinal properties of the plant. The present paper deals with the methanol extract, n - Hexane extract of Withania somnifera, Datura stramonium were screened for the quantitative analysis by standard procedure and subjected to analysis by UV Spectrophotometer. Results shows that Steroids and Alkaloids are in higher amount than the phenols \& flavonoids. This indicate that seasonal variation played a key role in increasing secondary metabolites rather than the phenological stage of the plant. - Count Search
210. Agarwal, Aditya Vikram and Gupta, Parul and Singh, Deeksha and Dhar, Yogeshwar Vikram and Chandra, Deepak and Trivedi, Prabodh Kumar, Comprehensive Assessment of the Genes Involved in Withanolide Biosynthesis from Withania Somnifera: Chemotype-Specific and Elicitor-Responsive Expression, Functional \& Integrative Genomics, vol. 17, no. 4, pp. 477--490, July 2017. doi: 10.1007/s10142-017-0548-x. Abstract
     Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C28-steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C30-terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ∆-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.
211. Hidayat, Topik and Priyandoko, Didik and Wardiny, Putri Yunitha and Islami, Dina Karina, Molecular Phylogenetic Screening of Withania Somnifera Relative From Indonesia Based on Internal Transcribed Spacer Region, HAYATI Journal of Biosciences, vol. 23, no. 2, pp. 92--95, April 2016. doi: 10.1016/j.hjb.2016.02.002. Abstract
     Withania somnifera (family Solanaceae), known commonly as Ashwaganda, is one of the important medicinal plants, and recent studies reported that Withanone, one of the chemical components in this plant, has ability to kill cancer cell. Because of endemic state of this plant to South Asia, exploring plant species under the same family which grow well in Indonesia has been of interest. The purpose of this study was to screen the Indonesian plant which has strong phylogenetic relationship with Ashwaganda. Thus, phylogenetic analysis using DNA sequences of internal transcribed spacer (ITS) region was conducted. Thus, 19 species of Solanaceae and two species of Convolvulaceae as outgroup were examined. Five ITS regions of Ashwaganda retrieved from GenBank were included in the phylogenetic analysis. Parsimony analysis showed that Indonesia Solanaceae comprises seven groups which is consistent with the global Solanaceae relationship as previously reported. Furthermore, our study revealed that two species, Physalis angulata and Physalis peruviana, are relative to W. somnifera. Morphologically, they share characters of flower and fruit. This result indicated that these two species are potential to have similar chemical properties as Ashwaganda, thus we can have new variants of Withanone originated from Indonesia with similar effect.
212. Gupta, Parul and Goel, Ridhi and Agarwal, Aditya Vikram and Asif, Mehar Hasan and Sangwan, Neelam Singh and Sangwan, Rajender Singh and Trivedi, Prabodh Kumar, Comparative Transcriptome Analysis of Different Chemotypes Elucidates Withanolide Biosynthesis Pathway from Medicinal Plant Withania Somnifera, Scientific Reports, vol. 5, no. 1, pp. 18611, December 2015. doi: 10.1038/srep18611. Abstract
     Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania.
213. Bhatia, Anil and Bharti, Santosh K. and Tewari, Shri K. and Sidhu, Om P. and Roy, Raja, Metabolic Profiling for Studying Chemotype Variations in Withania Somnifera (L.) Dunal Fruits Using GC–MS and NMR Spectroscopy, Phytochemistry, vol. 93, pp. 105--115, September 2013. doi: 10.1016/j.phytochem.2013.03.013. Abstract
     Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plant with several pharmaceutical and nutraceutical applications. Metabolic profiling was performed by GC–MS and NMR spectroscopy on the fruits obtained from four chemotypes of W. somnifera. A combination of 1H NMR spectroscopy and GC–MS identified 82 chemically diverse metabolites consisting of organic acids, fatty acids, aliphatic and aromatic amino acids, polyols, sugars, sterols, tocopherols, phenolic acids and withanamides in the fruits of W. somnifera. The range of metabolites identified by GC–MS and NMR of W. somnifera fruits showed various known and unknown metabolites. The primary and secondary metabolites observed in this study represent MVA, DOXP, shikimic acid and phenylpropanoid biosynthetic metabolic pathways. Squalene and tocopherol have been rated as the most potent naturally occurring compounds with antioxidant properties. These compounds have been identified by us for the first time in the fruits of W. somnifera. Multivariate principal component analysis (PCA) on GC–MS and NMR data revealed clear distinctions in the primary and secondary metabolites among the chemotypes. The variation in the metabolite concentration among different chemotypes of the fruits of W. somnifera suggest that specific chemovars can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.
214. Udayakumar, R. and Kasthurirengan, S. and Mariashibu, T. S. and Sudhakar, B. and Ganapathi, A. and Kim, E. J. and Jang, K. M. and Choi, C. W. and Kim, S. C., Analysis of Genetic Variation among Populations of Withania Somnifera (L.) in South India Based on RAPD Markers., European Journal of Medicinal Plants, vol. 3, no. 2, pp. 266--280, 2013. doi: 10.9734/EJMP/2013/2801. Abstract
     Aim: The present study was carried out to analyze the genetic variations among 20 different populations of Withania somnifera (L.) Dunal collected from different habitats (locations) by RAPD analysis. Methodology: DNA was isolated from the fresh leaf samples collected from the field by Bernatsky and Tankley method. Isolated genomic DNA was purified by phenol: chloroform: isoamyl alcohol (25:24:1)...
215. Rana, Satiander and Dhar, Niha and Bhat, Wajid W. and Razdan, Sumeer and Khan, Shabnam and Dhar, Rekha S. and Dutt, Prabhu and Lattoo, Surrinder K., A 12-Deoxywithastramonolide-Rich Somaclonal Variant in Withania Somnifera (L.) Dunal—Molecular Cytogenetic Analysis and Significance as a Chemotypic Resource, In Vitro Cellular \& Developmental Biology - Plant, vol. 48, no. 5, pp. 546--554, October 2012. doi: 10.1007/s11627-012-9458-8. Abstract
     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.
216. Namdeo, Ajay G. and Sharma, Ajay and Yadav, Kavita N. and Gawande, Rupali and Mahadik, Kakasaheb R. and {Lopez-Gresa}, Maria Pilar and Kim, Hye Kyong and Choi, Young Hae and Verpoorte, Robert, Metabolic Characterization of Withania Somnifera from Different Regions of India Using NMR Spectroscopy, Planta Medica, vol. 77, no. 17, pp. 1958--1964, November 2011. doi: 10.1055/s-0031-1279997. Abstract
     Thieme E-Books \& E-Journals
217. Bharti, S. K. and Bhatia, Anil and Tewari, S. K. and Sidhu, O. P. and Roy, Raja, Application of HR-MAS NMR Spectroscopy for Studying Chemotype Variations of Withania Somnifera (L.) Dunal, Magnetic Resonance in Chemistry, vol. 49, no. 10, pp. 659--667, 2011. doi: 10.1002/mrc.2817. Abstract
     Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plants with a number of pharmaceutical and nutraceutical applications. Metabolic profiling has been performed by HR-MAS NMR spectroscopy on fresh leaf and root tissue specimens from four chemotypes of W. somnifera. The HR-MAS NMR spectroscopy of lyophilized defatted leaf tissue specimens clearly distinguishes resonances of medicinally important secondary metabolites (withaferin A and withanone) and its distinctive quantitative variability among the chemotypes. A total of 41 metabolites were identified from both the leaf and root tissues of the chemotypes. The presence of methanol in leaf and root tissues of W. somnifera was detected by HR-MAS NMR spectroscopy. Multivariate principal component analysis (PCA) on HR-MAS 1H NMR spectra of leaves revealed clear variations in primary metabolites among the chemotypes. The results of the present study demonstrated an efficient method, which can be utilized for metabolite profiling of primary and secondary metabolites in medicinally important plants. Copyright © 2011 John Wiley \& Sons, Ltd.
218. Abraham, A. and Kirson, I. and Glotter, E. and Lavie, D., A Chemotaxonomic Study of Withania Somnifera (L.) Dun., Phytochemistry, vol. 7, no. 6, pp. 957--962, June 1968. doi: 10.1016/S0031-9422(00)82182-2. Abstract
     Three chemical types of Wittania somnifera (L.) Dun., Solanaceae, each containing different steroidal lactones of the withanolide type, have been found to occur in Israel; they have been called types I, II and III. Morphological differences could not be detected between the three types, although each of them has a definite and separate area of distribution. No qualitative ontogenetic changes in the withanolide content could be observed.
219. Shilpashree, H. B. and Sudharshan, S. J. and Shasany, Ajit K. and Nagegowda, Dinesh A., Molecular Characterization of Three CYP450 Genes Reveals Their Role in Withanolides Formation and Defense in Withania Somnifera, the Indian Ginseng, Scientific Reports, vol. 12, no. 1, pp. 1602, January 2022. doi: 10.1038/s41598-022-05634-9. Abstract
     The medicinal properties of Ashwagandha (Withania somnifera) are attributed to triterpenoid steroidal lactones, withanolides, which are proposed to be derived from phytosterol pathway, through the action of cytochrome P450 (CYP450) enzymes. Here, we report the characterization of three transcriptome-mined CYP450 genes (WsCYP749B1, WsCYP76 and WsCYP71B10), which exhibited induced expression in response to methyl jasmonate treatment indicating their role in secondary metabolism. All three WsCYP450s had the highest expression in leaf compared to other tissues. In planta characterization of WsCYP450s through virus induced gene silencing (VIGS) and transient overexpression approaches and subsequent metabolite analysis indicated differential modulation in the accumulation of certain withanolides in W. somnifera leaves. While WsCYP749B1-vigs significantly enhanced withaferin A (\textasciitilde\,450\%) and reduced withanolide A (\textasciitilde\,50\%), its overexpression drastically led to enhanced withanolide A ({$>$}\,250\%) and withanolide B ({$>$}\,200\%) levels and reduced 12-deoxywithastramonolide (\textasciitilde\,60\%). Whereas WsCYP76-vigs led to reduced withanolide A (\textasciitilde\,60\%) and its overexpression increased withanolide A (\textasciitilde\,150\%) and reduced 12-deoxywithastramonolide (\textasciitilde\,60\%). Silencing and overexpression of WsCYP71B10 resulted in significant reduction of withanolide B (\textasciitilde\,50\%) and withanolide A (\textasciitilde\,60\%), respectively. Further, while VIGS of WsCYP450s negatively affected the expression of pathogenesis-related (PR) genes and compromised tolerance to bacteria P. syringae DC3000, their overexpression in W. somnifera and transgenic tobacco led to improved tolerance to the bacteria. Overall, these results showed that the identified WsCYP450s have a role in one or several steps of withanolides biosynthetic pathway and are involved in conferring tolerance to biotic stress.
220. Tripathi, Sandhya and Srivastava, Yashdeep and Sangwan, Rajender Singh and Sangwan, Neelam Singh, In Silico Mining and Functional Analysis of AP2/ERF Gene in Withania Somnifera, Scientific Reports, vol. 10, no. 1, pp. 4877, March 2020. doi: 10.1038/s41598-020-60090-7. Abstract
     Withania somnifera owing to its strong and remarkable stress tolerance property is a reliable candidate for the determination of genes involved in mechanism of adaption/tolerance of various stress conditions. 187 AP2/ERF gene related transcripts (GRTs) were identified during comprehensive search in W. somnifera transcriptome repertoire. Major hits in homology search were observed from the model plant Arabidopsis and members of Solanaceae family. Cloning, expression analysis~of the gene and genetic transient transformation with the gene (WsAP2) were performed to predict its functional role in planta. Enhanced expression of some of the pathway genes for terpenoid biosynthesis was observed in transformed tissues in comparison to the control tissues. It is speculated that WsAP2 gene crucially regulates the expression of GGPPS gene in addition to the regulation of~other important genes of terpenoid pathway via induction of expression of other genes such as HMGR, CAS, DXS and DXR. To the best of our knowledge, this is the first report representing detailed study of AP2/ERF gene family in W. somnifera. It is also suggested from the study that gene might have role in eliciting responses to combat stress and attribute the strong stress tolerant property associated with the plant.
221. Sharma, Arti and Rana, Satiander and Rather, Gulzar A. and Misra, Prashant and Dhar, Manoj K. and Lattoo, Surrinder K., Characterization and Overexpression of Sterol Δ22-Desaturase, a Key Enzyme Modulates the Biosyntheses of Stigmasterol and Withanolides in Withania Somnifera (L.) Dunal, Plant Science, vol. 301, pp. 110642, December 2020. doi: 10.1016/j.plantsci.2020.110642. Abstract
     Withanolides constitute an extensive and vital class of metabolites displaying wide array of structural and therapeutic properties with unique side-chain modifications. These show diversified scaffolds and are promising pharmaceutical molecules with well documented anti-inflammatory and anti-cancer properties. Sterols are dynamic class of compounds and essential molecules having structural and functional significance. These contribute to the synthesis of withanolides by providing structural precursors. In this context, we have characterized sterol Δ22-desaturase from Withania somnifera and also functionally validating it by confirming its desaturase nature in conjunction with quantitative real-time expression profiling and metabolite evaluation. Further, transgenic hairy roots of W. somnifera displayed a higher accumulation of stigmasterol and withanolides. The increase in chemical constituents was concomitant with an increased gene copy number predicted via Southern blotting. Additionally, transgenic lines of tobacco over-expressing WsCYP710A11 displayed a substantial increase in its expression, corroborating well with enhanced stigmasterol content. Characterization of CYP710A11 from W. somnifera and its homologous transgenic expression has demonstrated its role in the regulation of withanolides biosynthesis. It also exhibited a differential transcriptional profile in response to exogenous elicitations. These empirical findings suggest the crucial role of CYP710A11 in stigmasterol biosynthesis. This in turn has implications for the overproduction of withanolides via pathway channelling.
222. Tripathi, Sandhya and Sangwan, Rajender Singh and Mishra, Bhawana and Jadaun, Jyoti Singh and Sangwan, Neelam Singh, Berry Transcriptome: Insights into a Novel Resource to Understand Development Dependent Secondary Metabolism in Withania Somnifera (Ashwagandha), Physiologia Plantarum, vol. 168, no. 1, pp. 148--173, 2020. doi: 10.1111/ppl.12943. Abstract
     Withania somnifera (Ashwagandha) is considered as Rasayana in Indian systems of medicine. This study reports a novel transcriptome of W. somnifera berries, with high depth, quality and coverage. Assembled and annotated transcripts for nearly all genes related with the withanolide biosynthetic pathway were obtained. Tissue-wide gene expression analysis reflected almost similar definitions for the terpenoid pathway in leaf, root and berry tissues with relatively higher abundance of transcripts linked to steroid, phenylpropanoid metabolism as well as flavonoid metabolism in berries. The metabolome map generated from the data embodied transcripts from 143 metabolic pathways connected together and mediated collectively by about 1792 unique enzyme functions specific to berry, leaf and root tissues, respectively. Transcripts specific to cytochrome p450 (CYP450), methyltransferases and glycosyltransferases were distinctively located in a tissue specific manner and exhibited a complex network. Significant distribution of transcription factor genes such as MYB, early light inducible protein (ELI), minichromosome maintenance1, agamous, deficiens and serum response factor (MADS) and WRKY etc. was observed, as the major transcriptional regulators of secondary metabolism. Validation of the assembly was ascertained by cloning WsELI, which has a light dependent regulatory role in development. Quantitative expression of WsELI was observed to be considerably modulated upon exposure to abiotic stress and elicitors. Co-relation of over-expression of WsELI, may provide new aspects for the functional role of ELI proteins in plants linked to secondary metabolism. The study offers the first comprehensive and comparative evaluation of W. somnifera transcriptome data between the three tissues and across other members of Solanaceae (Nicotiana, Solanum and Capsicum) with respect to major pathways and their metabolome regulation.
223. Sharma, Arti and Rather, Gulzar A. and Misra, Prashant and Dhar, Manoj K. and Lattoo, Surrinder K., Jasmonate Responsive Transcription Factor WsMYC2 Regulates the Biosynthesis of Triterpenoid Withanolides and Phytosterol via Key Pathway Genes in Withania Somnifera (L.) Dunal, Plant Molecular Biology, vol. 100, no. 4, pp. 543--560, July 2019. doi: 10.1007/s11103-019-00880-4. Abstract
     Functional characterization of WsMYC2 via artificial microRNA mediated silencing and transient over-expression displayed significant regulatory role vis-à-vis withanolides and stigmasterol biosyntheses in Withania somnifera. Further, metabolic intensification corroborated well with higher expression levels of putative pathway genes. Additionally, copious expression of WsMYC2 in response to exogenous elicitors resulted in enhanced withanolides production.
224. Sharma, Arti and Rather, Gulzar A. and Misra, Prashant and Dhar, Manoj K. and Lattoo, Surrinder K., Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania Somnifera (L.) Dunal, Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.00842. Abstract
     Withania somnifera (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copious production. Previous investigations have revealed that P450 monooxygenases are pivotal enzymes involved in the biosynthetic machinery of various metabolites and assist in decorating their core skeletal structures. The present study entails the isolation and functional characterization of castasterone synthase (CYP85A69) from W. somnifera. The full length WsCYP85A69, having an open reading frame of 1413 bp, encodes 470 amino acid residues. Further, in vitro conversion of 6-deoxocastasterone into castasterone validated its oxidative functionality. Product formation was confirmed using LC-PDA-MS with a m/z value of 506 [M+ACN]+. In planta transient over-expression of WsCYP85A69 significantly enhanced castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Artificial micro-RNA mediated silencing of WsCYP85A69 resulted in the reduced accumulation of castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Altogether, these non-complementary approaches plausibly suggest a key role of WsCYP85A69 in the biosynthesis of castasterone and the accumulation of withanolides and stigmasterol. Furthermore, a promoter analysis of WsCYP85A69 resulted in the identification of several potential cis-regulatory elements. Elicitations, given on the basis of identified cis-regulatory elements, demonstrated methyl jasmonate as an effective inducer of WsCYP85A69. Overall, these empirical findings suggest that functional characterization of WsCYP85A69 may conceivably be helpful to unravel the mechanism of brassinosteroids biosynthesis and could also pave the way for targeted metabolic engineering.
225. Agarwal, Aditya Vikram and Singh, Deeksha and Dhar, Yogeshwar Vikram and Michael, Rahul and Gupta, Parul and Chandra, Deepak and Trivedi, Prabodh Kumar, Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania Somnifera, Plant and Cell Physiology, vol. 59, no. 2, pp. 262--274, February 2018. doi: 10.1093/pcp/pcx179.
226. Tripathi, Sandhya and Sangwan, Rajender Singh and Narnoliya, Lokesh Kumar and Srivastava, Yashdeep and Mishra, Bhawana and Sangwan, Neelam Singh, Transcription Factor Repertoire in Ashwagandha (Withania Somnifera) through Analytics of Transcriptomic Resources: Insights into Regulation of Development and Withanolide Metabolism, Scientific Reports, vol. 7, no. 1, pp. 16649, November 2017. doi: 10.1038/s41598-017-14657-6. Abstract
     Transcription factors (TFs) are important regulators of cellular and metabolic functions including secondary metabolism. Deep and intensive RNA-seq analysis of Withania somnifera using transcriptomic databases provided 3532 annotated transcripts of transcription factors~in leaf and root tissues, belonging to 90 different families with major abundance for WD-repeat (174 and 165 transcripts) and WRKY (93 and 80 transcripts) in root and leaf tissues respectively, followed by that of MYB, BHLH and AP2-ERF. Their~detailed comparative analysis with Arabidopsis thaliana, Capsicum annum, Nicotiana tabacum and Solanum lycopersicum counterparts~together gave interesting patterns. However, no homologs for WsWDR representatives, LWD1 and WUSCHEL, were observed in other Solanaceae species. The data extracted from the sequence read archives (SRA) in public domain databases were subjected to re-annotation, re-mining, re-analysis and validation for dominant occurrence of WRKY and WD-repeat (WDR) gene families. Expression of~recombinant LWD1 and WUSCHEL proteins in homologous system led to enhancements in withanolide content indicating their regulatory role in planta in the~biosynthesis. Contrasting expression~profiles of WsLWD1 and WsWUSCHEL provided tissue-specific insights for their participation in the regulation of developmental processes. The in-depth analysis provided first full-spectrum and comparative characteristics of TF-transcripts across plant species, in the perspective of integrated tissue-specific regulation of metabolic processes including specialized metabolism.
227. Mishra, Smrati and Bansal, Shilpi and Mishra, Bhawana and Sangwan, Rajender Singh and Asha and Jadaun, Jyoti Singh and Sangwan, Neelam S., RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania Somnifera, PLOS ONE, vol. 11, no. 2, pp. e0149691, February 2016. doi: 10.1371/journal.pone.0149691. Abstract
     Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.
228. Rana, Satiander and Bhat, Wajid Waheed and Dhar, Niha and Pandith, Shahzad A. and Razdan, Sumeer and Vishwakarma, Ram and Lattoo, Surrinder K., Molecular Characterization of Two A-Type P450s, WsCYP98A and WsCYP76A from Withania Somnifera (L.) Dunal: Expression Analysis and Withanolide Accumulation in Response to Exogenous Elicitations, BMC Biotechnology, vol. 14, no. 1, pp. 89, November 2014. doi: 10.1186/s12896-014-0089-5. Abstract
     Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, one of the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides.
229. Dasgupta, Modhumita Ghosh and George, Blessan Santhosh and Bhatia, Anil and Sidhu, Om Prakash, Characterization of Withania Somnifera Leaf Transcriptome and Expression Analysis of Pathogenesis – Related Genes during Salicylic Acid Signaling, PLOS ONE, vol. 9, no. 4, pp. e94803, April 2014. doi: 10.1371/journal.pone.0094803. Abstract
     Withania somnifera (L.) Dunal is a valued medicinal plant with pharmaceutical applications. The present study was undertaken to analyze the salicylic acid induced leaf transcriptome of W. somnifera. A total of 45.6 million reads were generated and the de novo assembly yielded 73,523 transcript contig with average transcript contig length of 1620 bp. A total of 71,062 transcripts were annotated and 53,424 of them were assigned GO terms. Mapping of transcript contigs to biological pathways revealed presence of 182 pathways. Seventeen genes representing 12 pathogenesis-related (PR) families were mined from the transcriptome data and their pattern of expression post 17 and 36 hours of salicylic acid treatment was documented. The analysis revealed significant up-regulation of all families of PR genes by 36 hours post treatment except WsPR10. The relative fold expression of transcripts ranged from 1 fold to 6,532 fold. The two families of peroxidases including the lignin-forming anionic peroxidase (WsL-PRX) and suberization-associated anionic peroxidase (WsS-PRX) recorded maximum expression of 377 fold and 6532 fold respectively, while the expression of WsPR10 was down-regulated by 14 fold. Additionally, the most stable reference gene for normalization of qRT-PCR data was also identified. The effect of SA on the accumulation of major secondary metabolites of W. somnifera including withanoside V, withaferin A and withanolide A was also analyzed and an increase in content of all the three metabolites were detected. This is the first report on expression patterns of PR genes during salicylic acid signaling in W. somnifera.
230. {Sanchita} and Singh, Swati and Sharma, Ashok, Bioinformatics Approaches for Structural and Functional Analysis of Proteins in Secondary Metabolism in Withania Somnifera, Molecular Biology Reports, vol. 41, no. 11, pp. 7323--7330, November 2014. doi: 10.1007/s11033-014-3618-3. Abstract
     Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway.~Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-d-xylulose-5-phosphate synthase, 1-Deoxy-d-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.
231. Sabir, Farzana and Mishra, Smrati and Sangwan, Rajender S. and Jadaun, Jyoti S. and Sangwan, Neelam S., Qualitative and Quantitative Variations in Withanolides and Expression of Some Pathway Genes during Different Stages of Morphogenesis in Withania Somnifera Dunal, Protoplasma, vol. 250, no. 2, pp. 539--549, April 2013. doi: 10.1007/s00709-012-0438-y. Abstract
     Withania somnifera Dunal is an important and extensively studied medicinal plant; however, there is no report available that relates withanolide content and its profile in relation to the expression of pathway genes during different morphogenic stages. In this study, withanolide A, withaferin A, and withanone, the major withanolides of W. somnifera, were measured in different in vitro stages during organogenesis, viz., shoot to root (direct rhizogenesis)/root to shoot (indirect via callus phase) transition vis-à-vis expression levels of key pathway genes involved in withanolide biosynthetic pathways. The morphogenic transitions were found to be tightly linked to the pattern of accumulation of withanolides. The high expression levels of most of the pathway genes in in vitro shoots in comparison to in vitro root and callus tissues exhibited a direct co-relation with the maximum withanolide content ({$>$}2.7~mg/gDW). The biogenesis of withaferin A, a major constituent of the leaves, was however found to be tightly linked to shoots/green tissue. In addition, we were also able to establish an efficient regeneration system from roots for their further utilization in biotechnological applications.
232. Singh, Somesh and Vishwakarma, {\relax Rishi}. K. and Kumar, R. J. Santosh and Sonawane, Prashant D. and {Ruby} and Khan, Bashir M., Functional Characterization of a Flavonoid Glycosyltransferase Gene from Withania Somnifera (Ashwagandha), Applied Biochemistry and Biotechnology, vol. 170, no. 3, pp. 729--741, June 2013. doi: 10.1007/s12010-013-0230-2. Abstract
     Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W. somnifera (UGT73A16) showing 85–92~\% homology with UGTs from other plants. UGT73A16 was expressed as a His6-tagged fusion protein in Escherichia coli. Several compounds, including flavonoids, were screened as potential substrates for UGT73A16. HPLC analysis and hypsochromic shift indicated that UGT73A16 transfers a glucose molecule to several different flavonoids. Based on kinetic parameters, UGT73A16 shows more catalytic efficiency towards naringenin. Here, we explored UGT73A16 of W. somnifera as whole cell catalyst in E. coli. We used flavonoids (genistein, apigenin, kaempferol, naringenin, biochanin A, and daidzein) as substrates for this study. More than 95~\% of the glucoside products were released into the medium, facilitating their isolation. Glycosylation of substrates occurred on the 7- and 3-hydroxyl group of the aglycone. UGT73A16 also displayed regiospecific glucosyl transfer activity towards 3-hydroxy flavone compound, which is the backbone of all flavonols and also for a chemically synthesized compound, not found naturally. The present study generates essential knowledge and molecular as well as biochemical tools that allow the verification of UGT73A16 in glycosylation.
233. Gupta, Parul and Agarwal, Aditya Vikram and Akhtar, Nehal and Sangwan, Rajender Singh and Singh, Surya Pratap and Trivedi, Prabodh Kumar, Cloning and Characterization of 2-C-Methyl-d-Erythritol-4-Phosphate Pathway Genes for Isoprenoid Biosynthesis from Indian Ginseng, Withania Somnifera, Protoplasma, vol. 250, no. 1, pp. 285--295, February 2013. doi: 10.1007/s00709-012-0410-x. Abstract
     Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-d-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428~bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon–intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.
234. Chaurasiya, Narayan D. and Sangwan, Neelam S. and Sabir, Farzana and Misra, Laxminarain and Sangwan, Rajender S., Withanolide Biosynthesis Recruits Both Mevalonate and DOXP Pathways of Isoprenogenesis in Ashwagandha Withania Somnifera L. (Dunal), Plant Cell Reports, vol. 31, no. 10, pp. 1889--1897, October 2012. doi: 10.1007/s00299-012-1302-4. Abstract
     Withanolides are pharmaceutically important C28-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of 13C label from 13C1-d-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of 13C abundance at each carbon of withaferin A from 13C1-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific 13C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids.
235. Jadhav, Santosh Kumar Ramachandra and Patel, Krunal Arvind and Dholakia, Bhushan Bhalchandra and Khan, Bashir Mohammad, Structural Characterization of a Flavonoid Glycosyltransferase from Withania Somnifera, Bioinformation, vol. 8, no. 19, pp. 943--949, October 2012. doi: 10.6026/97320630008943. Abstract
     Medicinal plants are extensively utilized in traditional and herbal medicines, both in India and around the world due to the presence of diverse low molecular weight natural products such as flavonoids, alkaloids, terpenoids and sterols. Flavonoids which have health benefits for humans are the large class of phenylpropanoid-derived secondary metabolites and are mostly glycosylated by UDP-glycosyltransferases (UGTs). Although large numbers of different UGTs are known from higher plants, very few protein structures have been reported till now. In the present study, the three-dimensional model of flavonoid specific glycosyltransferases (WsFGT) from Withania somnifera was constructed based on the crystal structure of plant UGTs. The resulted model was assessed by various tools and the final refined model revealed GT-B type fold. Further, to understand the sugar donors and acceptors interactions with the active site of WsFGT, docking studies were performed. The amino acids from conserved PSPG box were interacted with sugar donor while His18, Asp110, Trp352 and Asn353 were important for catalytic function. This structural and docking information will be useful to understand the glycosylation mechanism of flavonoid glucosides.
236. Rout, Jyoti Ranjan and Sahoo, Santi Lata, Morphological and Protein Profile Alterations in Withania Somnifera l. with Response to Iron Stress, Indian Journal of Life Sciences, vol. 2, no. 1, pp. 19--24, December 2012. url: https://go.gale.com/ps/i.do?p=AONE&sw=w&issn=22771743&v=2.1&it=r&id=GALE%7CA357968979&sid=googleScholar&linkaccess=abs. Abstract
     {$<$}em{$>$}Gale{$<$}/em{$>$} Academic OneFile includes Morphological and protein profile alterations in withan by Jyoti Ranjan Rout and Santi Lata Sahoo. Click to explore.
237. Oza, Vishal P. and Parmar, Pritesh P. and Patel, Darshan H. and Subramanian, R. B., Cloning, Expression and Characterization of l-Asparaginase from Withania Somnifera L. for Large Scale Production, 3 Biotech, vol. 1, no. 1, pp. 21--26, July 2011. doi: 10.1007/s13205-011-0003-y. Abstract
     l-Asparaginase (E.C. 3.5.1.1) is used as a therapeutic agent in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. In plants, l-asparaginase enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. An Indian medicinal plant, Withania somnifera was reported as a novel source of l-asparaginase. l-Asparaginase from W. somnifera was cloned and overexpressed in E. coli. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. The kinetic parameters of selected substrates were determined at various pH and the pH- and temperature-dependence profiles were analyzed. WA gene successfully cloned into E. coli BL21 (DE3) showed high asparaginase activity with a specific activity of 17.3~IU/mg protein.
238. Azhar, Muhammad Farooq and Naseer, Ubair and Aziz, Abida and Zafar, Shagufta and Qadir, Ihsan and Ahmad, Irfan and Anjum, Khayyam, Antioxidant and Phytochemical Composition of Leaves, Stem and Root Extracts of Withania Coagulans and Withania Somnifera, pp. 4, 2020. Abstract
     Different plant parts of Withania coagulans and Withania somnifera belonging to family Solanaceae were exploited for biochemical studies. These are wild plants and mostly considered as poisonous plants, although limitedly used in folk medicines in Pakistan. The extraction yield in menthol was also calculated for root, stem and leaves of both plants. The\,\% of extract yield in leaves of W. coagulans was recorded high (7.6\,\%) than stem (6.3\,\%) and root (6.3\,\%). The\,\% extract yield of all parts of W. coagulans was greater than the same parts of W. somnifera. The pattern of\,\% extract yield is similar for W. somnifera as in W. coagulans. In leaves its higher (5.5\,\%) and a decreases from stem (5.2\,\%) to roots (4.7\,\%). Flavonoids and total phenolics were determined by using Folin-Ciocalteu reagent and aluminum chloride reagent through spectrophotometric assay. Maximum value of total phenolics and flavonoids were observed in W. coagulans leaves (58.21 mg GEA/g and 47 mg RE/g). There is a similar decreasing pattern in total phenolics and flavonoids contents from leaves to stem and root extracts. Antioxidant activities in different parts extracts of both plants were calculated at different levels. All parts of W. coagulans exhibits more antioxidant activity than W. somnifera. These results reveal the presence of useful biomedical components in both species which could be used in formulation of modern medicines.
239. Misra, Laxminarain and Mishra, Priyanka and Pandey, Archana and Sangwan, Rajender S. and Sangwan, Neelam S., 1,4-Dioxane and Ergosterol Derivatives from Withania Somnifera Roots, Journal of Asian Natural Products Research, vol. 14, no. 1, pp. 39--45, January 2012. doi: 10.1080/10286020.2011.622719. Abstract
     The chemical investigation on the n-hexane extract of Withania somnifera roots has yielded octacosane, oleic and stearic fatty acids, stigmasterone, stigmasterol, sitostanone, oleanolic acid along with the ergosterol and 1,4-dioxane derivatives as new compounds. The isolation of alkenyl-1,4-dioxane compound is rare, whereas the ergosterol derivative may have biogenetic significance in the lactone formation in the E ring of withanolides. The presence of a 1,4-dioxane derivative in the nonpolar extract of roots assumes importance as this type of compound has not been reported earlier from W. somnifera. The structures of new compounds were elucidated by spectroscopic methods and chemical transformations.
240. Sidhu, O. P. and Annarao, Sanjay and Chatterjee, Sandipan and Tuli, Rakesh and Roy, Raja and Khetrapal, C. L., Metabolic Alterations of Withania Somnifera (L.) Dunal Fruits at Different Developmental Stages by NMR Spectroscopy, Phytochemical Analysis, vol. 22, no. 6, pp. 492--502, 2011. doi: 10.1002/pca.1307. Abstract
     Introduction Withania somnifera (Ashwagandha) is a high-value Ayurvedic medicinal plant and an important constituent of several dietary supplements. In order to substantiate the health claims, the herb has drawn considerable scientific attention. Objective The objective of the study was to investigate the alterations in primary and secondary metabolites of W. somnifera fruits during its maturity using NMR spectroscopy. Methodology Fruits at different stages of development from one week after fertilisation until maturity, classified in seven developmental stages, were analysed by a combined use of one- and two-dimensional NMR experiments. Results Seventeen metabolites were characterised and quantified from non-polar and polar extracts of different fruit development stages of W. somnifera. The principal component analysis of polar metabolites at different stages could be grossly classified into three metabolic phases, viz. initial phase, developmental phase and maturation phase. Conclusion Qualitative and quantitative analysis of metabolites in W. somnifera fruits indicated specific stages when fruits can be harvested for obtaining substantial bioactive ingredients for desirable pharmacological activity. This study potentially provides a complementary tool for quality control of herbal medicinal products when W. somnifera fruits are used. Copyright © 2011 John Wiley \& Sons, Ltd.
241. Ali, N. W. and Abouzid, S. and Nasib, A. and Khan, S. and Qureshi, J. and Choudhary, M. I., RP-HPLC Analysis of Withanolides in the Flowers, Leaves, and Roots of Withania Somnifera, Acta Chromatographica, vol. 22, no. 3, pp. 473--480, September 2010. doi: 10.1556/achrom.22.2010.3.10. Abstract
     Summary Ashwaghanda, Withania somnifera, is one of the most widely used herbs in Ayurvedic medicine. Leaves and roots are the traditionally used parts of the plant. An RP-HPLC method using a binary acetonitrile-water gradient containing 0.1\% acetic acid has been developed for analysis of withaferin A. The method was validated in accordance with ICH guidelines and used for analysis of the withanolide content of the flowers, leaves, and roots of W. somnifera. The withanolide content was highest in the flowers.
242. Praveen, N. and Naik, P. M. and Manohar, S. H. and Murthy, H. N., Distribution of Withanolide a Content in Various Organs of Withania Somnifera (L.) Dunal., International Journal of Pharma and Bio Sciences, vol. 1, no. 3, 2010. url: https://www.cabdirect.org/cabdirect/abstract/20113372347. Abstract
     Withanolide A is an important secondary metabolite in Withania somnifera, which is having a high medicinal value and possesses potent anti-tumor and antioxidant properties. Distribution of withanolide A in various organs of Withania somnifera was investigated by High Performance Liquid Chromatography (HPLC) method. The quantitative distribution of withanolide A was different in various organs...
243. Fernando, I. D. N. S. and Abeysinghe, D. C. and Dharmadasa, R. M., Determination of Phenolic Contents and Antioxidant Capacity of Different Parts of Withania Somnifera (L.) Dunal. from Three Different Growth Stages, Industrial Crops and Products, vol. 50, pp. 537--539, October 2013. doi: 10.1016/j.indcrop.2013.08.042. Abstract
     Withania somnifera (L.) Dunal. (Solanaceae) is a therapeutically important medicinal herb used in Ayurvedic and traditional systems of medicine for the treatment of an array of ailments. Diverse therapeutic properties reported from W. somnifera are mainly due to the high content of polyphenols and antioxidant activities present in different parts of the plant. Present study investigates the total phenolic content (TPC) and total antioxidant capacity (TAC) of different parts of three different growth stages of W. somnifera grown under three different spacing levels. The TAC and TPC were determined using Ferric Reducing Antioxidant Power assay and modified Folin–Ciocalteu colorimetric method respectively. Leaf extract exhibited significantly higher (p{$<$}0.05) TAC and TPC values for all three different growth stages. However, the highest TAC and TPC of leaf extract for all three spacing levels were observed just after flowering stage. The highest total phenolic content was exhibited in leaf extracts followed by flower, fruits, stem and roots respectively. With regard to the anti oxidant content, the highest amount was recorded from leaf followed by pods, flowers, stem and roots respectively. Presence of higher TPC and TAC just after flowering stage scientifically validates traditional claims of harvesting of W. somnifera after flowering stage. The higher content of TPC and TAC in leaf demonstrated the possibility of incorporation leaf for the development of newer, effective drugs instead of roots.
244. Meena, Lalit and Meena, A and Gupta, Janak and Patel, Mohammad and Khan, Sunil and {Kumar} and Gupta, Anil, Ashwagandha (Withania Somnifera L.), pp. 81--94, November 2020. Abstract
     Ashwagandha (Withania somnifera L.) is an annual medicinal plant belongs to family Solanaceae and known as winter cherry. Among the various medicinal plants, Withania somnifera (L.) Dunal (Winter cherry, Ashwagandha or Asgandh) is an important medicinal plant and its use in Ayurveda and Unani medicine extends back over 3000 to 4000 years.The Sanskrit name “ashva” meaning horse and “gandha” meaning smelling was given to this plant due to the smell of the roots resembling a sweating horse. Ashwagandha was first mentioned by sage Punarvasu Atreya over 4000 years ago (Rajeswara Rao et al., 2012)
245. Venugopal, S and Padma, M and Rajkumar, M and Seenivasan, N and Saidaiah, P and Sathish, G, Correlation and Path Analysis in Ashwagandha (Withania Somnifera L.) for Dry Root Yield, Journal of Plant Development Sciences, vol. 13, no. 7, pp. 445--452, 2021. Abstract
     The experiment was laid out in a completely Randomized Block Design with 29 ashwagandha access ions as treatments during Kharif, 2018 at M edicinal and Aromatic Plant Research Station, Sri Konda Laxman Telangana State Horticultural University, Rajendranagar, Hyderabad. Each treatment was randomly replicated thrice. The results on genotypic and phenotypic correlation reveal that mostly genotypic correlation coefficient is comparatively higher than the intensity of phenotypic correlation coefficient. This indicates less influence of environment in association studies. The positive and significant correlation was observed between dry root yield per plant with root diameter, main root length, leaf length, starch estimation, leaf width, fresh leaf weight, dry leaf weight, days to flower initiation, plant height and number of secondary roots per plant. Direct selection based on these traits would result in simultaneous improvement of aforesaid traits and dry root yield per se in ashwagandha. Although correlation coefficients indicate the nature of association among the characters, path analysis splits the correlation coefficients into measures of direct and indirect effects, thus providing an understanding on the direct and indirect contribution of each character towards yield. From the foregoing discussion, it can be concluded that main root length, root diameter, leaf weight and days to flower initiation had positive correlation and positive direct effect on dry root yield per plant. These are identified as superior yield components. Hence, the genotypes which exhibited better performance for these characters can be used in further improvement of ashwagandha.
246. Joshi, N. R. and Patel, M. A. and Prajapati, K. N. and Patel, J. R. Patel {and} A. D., Genetic Diversity In Ashwagandha (Withania Somnifera (L.) Dunal), Electronic Journal of Plant Breeding, vol. 6, no. 3, pp. 870--874, November 2015. url: https://ejplantbreeding.org/index.php/EJPB/article/view/204. Abstract
     Genetic divergence among 40 ashwagandha accessions of different geographic origin was assessed using Mahalanobis D2 statistics. Observations revealed significant genotypic differences and accordingly genotypes were classified into six clusters. Cluster I was the largest with thirty genotypes followed by II and III clusters which have four and three genotypes, respectively. Cluster IV, V and VI contained only one most divergent genotype. The maximum inter-cluster distance (D=1538.09) was found between cluster II and VI, followed by that between II and III (D=983.03). The minimum inter-luster distance was observed between cluster Iand II (D=285.09). Theintra-­cluster distance (D) ranged from 142.22 (cluster-III) to 192.10 (cluster-I). The three clusters (IV, V and VI) contained single genotype each and therefore, their intra-cluster distances were zero. The genotype of cluster VI was unique as it was having highest values for leaf width, diameter of root at collar region with high dry root yield. The cluster II was desirable in respect of days to flower initiation and days to maturity and also had highest value for number of primary branches per plant, withanoloide content and starch content. The cluster III exhibited highest value for plant height and leaf length. The cluster IV had highest mean values for number of secondary branches per plant and root length. Thus, hybridization among these genotypes can generate desirable transgressive segregants.
247. Dharmar, K and Britto, A John De, RAPD Analysis of Genetic Variability in Wild Populations of Withania Somnifera (L.) Dunal, pp. 5, 2011. Abstract
     Withania somnifera is extensively used as herbal medicine, because it contains clinically important compounds. In the present study, the genetic variability in W. somnifera among accessions of different geographical region in Tamil Nadu was assessed through Random Amplified Polymorphic DNA (RAPD) markers. Five accessions of W. somnifera were screened with ten primers of which six primers were found to be the most informative. These primers produced multiple band profiles with a number of amplified DNA fragments varying from 5 to 9. A total of 37 polymorphic bands were observed. The genetic distance between the population ranged from 0.2436 to 0.4754 and the genetic identity ranged from 0.6216 to 0.7838. The overall observed and effective number of alleles was about 1.6216 and 1.4271 respectively. Nei’s over all genetic diversity is 0.2465. The degree of percentage of polymorphism (83.78 \%) was found to be high in accession collected from Samykoundanpalayam in Tirupur District, Tamil Nadu. The information obtained here could be valuable for devising strategies for conservation this medicinal plant.
248. Bharathi, T. and Gnanamurthy, S. and Dhanavel, D. and Ariraman, M., Induced Physical Mutagenesis and Its Effect in Cytological Behavior of Ashwagandha ( Withania Somnifera (L.) Dunal), International Letters of Natural Sciences, vol. 17, pp. 152--161, June 2014. doi: 10.18052/www.scipress.com/ILNS.17.152. Abstract
     The mitotic effect of physical mutagen gamma rays was observed in the root tip cells of Ashwagandha. The Chromosome analysis has been showed as an important tool for establish variability of the plant seed by use of physical mutagen gamma rays. The gamma rays have of low wavelength and high penetrable power. The plant has tremendous medicinal values and it is known from ancient times. The dry and well matured seeds of ashwagandha were irradiated with different doses of gamma rays viz., 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 KR respectively. The chromosome number of control plant is 2n = 48. The gamma rays affect the normal cytological behavior of ashwagandha species. The chromosomal aberrations increase with increase in the doses of gamma rays to optimum level of 30KR, because it causes changes in the chromosome structure, cellular structure and metabolism of plants. The chromosome aberration like, Sticky metaphase, Precocious moment chromosome, Fragments, Anaphasic bridge, Anaphasic laggard, Telophasic laggard. The present investigation was carried out to study the cytogenetic analysis of the species Withania somnifera. The chromosomal aberration increases with increase in the doses to optimum level (50 KR) of physical mutagen gamma rays.
249. and P.Manivel, H. B. Deore*, Inheritance of Growth Habit and Berry Colour in Ashwagandha (Withania Somnifera (L.) Dunal) – A Medicinal Plant, Electronic Journal of Plant Breeding, vol. 5, no. 2, pp. 244--247, November 2014. url: https://www.ejplantbreeding.org/index.php/EJPB/article/view/126. Abstract
     Genetics studies were studied for the two qualitative character ivz., growth habit (Erect × Procumbent) and for berry (fruit) colour (Yellow × Red) in ashwagandha. The inheritance of the growth habit was controlled by single gene and the procumbent was dominant over the erect type. The gene symbol P for procumbent and p for erect is proposed. Inheritance studies of yellow versus red berry colour indicated that the trait was in control of classical duplicate recessive epitasis and followed the ratio of 9:7 (yellow: red). The berry colour in this cross is controlled by two genes (Y1 and Y2) with complementary recessive epitasis and gene symbols are proposed.
250. Gupta, B. M. and Ahmed, K. K. Mueen, Research on Withania Somnifera (Ashwaganda): A Scientometric Assessment of Global Publications Output during 1995-2018, International Journal of Pharmaceutical Investigation, vol. 9, no. 2, pp. 59--66, September 2019. doi: 10.5530/ijpi.2019.2.12. Abstract
     The present study examined 2008 global publications on Withania somnifera, as indexed and covered in international Scopus database during 1995-18, with a view to understand their publications growth rate, global publication share, citation impact, share of their international collaborative publications, distribution of publications by broad subjects, productivity and citation profile of top organizations and authors, preferred media of communication and bibliographic characteristics of high cited publications. The global publications registered 18.31\% annual average growth rate and its citation impact averaged to 18.55 citations per publication. The global share of top 10 countries ranged from 1.34\% to 70.42\%, with the largest share (70.42\%) from India, followed by USA (11.35\%) and other 8 countries from 1.34\% to 4.03\%. More than 100\% of the cumulative global publication and citation share comes from top 10 countries during 1995-18. Only four countries among top 10 registered relative citation index above the world average of 1.0: U.K. (2.48), USA (1.80), Germany (1.69) and South Korea (1.08) during 1995-18. Pharmacology, toxicology and pharmaceutics contributed the largest global publications share of 42.33\%, followed biochemistry, genetics and molecular biology (32.42\%), medicine (30.73\%), agricultural \& biological sciences (25.45\%), etc. during 1995-18. 377 global organizations and 422 global authors participated in global Withania somnifera research, of which the 15 most productive global organizations and authors together contributed 25.65\% and 14.59\% global publication share and 32.31\% and 23.82\% global citation share respectively during 1995-18. Amongst 1964 journal publications (in 313 journals) in global Withania somnifera research, the top 15 most productive journals together contributed 21.49\% global share of total journal publication output during 1995-18. Sixty Eight (68) publications were found to be high cited, as they registered citations from 100 to 601 during 1995-18 and they together received 11582 citations, averaging to 170.32 citations per publication.