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Summer Research Fellowship Programme of India's Science Academies

Assessment of salt stress on in-vitro grownWithania somnifera plantlets and green synthesis of silver nanoparticles for enhanced efficacy

Gauri Kundliya

Goa University, Taleigao Plateau, Goa 403206

Prof. Shashi Pandey Rai

Laboratory of Morphogenesis, Centre of Advanced Study in Botany, Department of Botany, Banaras Hindu University, Varanasi 221005

Abstract

Withania somnifera belongs to Solanaceae and is commonly known as Winter cherry, Indian ginseng or Ashwagandha. It is an important medicinal plant known to contain active ingredients like withanolides and flavonoids which have anti-inflammatory, anti-tumor, anti-stress, antioxidant, mind boosting, rejuvenating and antimicrobial properties. Being a highly important medicinal plant species and having low seed germination rate, there is a need to develop new propagation and conservation techniques for this plant. Plant tissue culture is an important technique by which large scale multiplication of plants can be achieved. In this study, in vitro regeneration of Withania somnifera was achieved using plain MS media. Further shoot multiplication was achieved using MS media supplemented with 1mg BAP. W. somnifera contains many bioactive materials such as triterpenoid steroidal lactones, alkaloids, flavonoids and tannin which may have a possible role in green synthesis of nanoparticles. In this study, green synthesis of silver nanoparticles was achieved using leaf extract and characterized by visible colour change. The AgNPs formed were further characterized using UV visible spectrophotometer which showed maximum peak at 450nm. Changing field environments such as drought, soil salinity, etc. can also hamper survival of this plant and somehow enhance the secondary metabolism. The effect of salt stress on agricultural crops is well studied but there is lack of information with respect to medicinal plants like W. somnifera. In this study, effect of salt stress induced by different NaCl concentrations( 50mM, 100mM, 150mM, 200mM, 250mM) supplemented Hoagland media was studied on growth, lipid peroxidation and total phenolics content. Salt stress inhibited growth and increased lipid peroxidation with increasing NaCl concentrations. Total phenolics content also increased due to salt stress with 150mM NaCl concentration inducing maximum secondary metabolism.

Keywords: Withania somnifera, micropropagation, silver nanoparticles, salt stress, abiotic stress, ROS

Abbreviations

Abbreviations
 AgNPsSilver Nanoparticles
 SPRSurface Plasmon Resonance
 ROSReactive Oxygen Species 
 MS  Murashige and Skoog 
 LPOLipid Peroxidation 
 MDAMalondialdehyde  
 BAP6-Benzylaminopurine 
 IBAIndole-3-butyric acid

INTRODUCTION

Background

Withania somnifera belongs to Solanaceae and is commonly known as Winter cherry, Indian ginseng or Ashwagandha. It is an important ancient plant used in home remedies for various ailments. It is a small evergreen shrub (35-75 cm) with a central stem from which branches extend radially in a star pattern. The flowers are small and green, while the fruit is orange-red [1].

It is widely distributed throughout the dry regions of India up to an attitude of 2000m in Himalayas[2]. Besides, it is also cultivated commercially in the states of Rajasthan, Punjab, Haryana, Uttar Pradesh, Gujarat, Maharashtra and Madhya Pradesh[1].

Withania somnifera is known to contain flavonoids and many active ingredients of the class withanolides. The medicinal properties of 12 alkaloids, 35 withanolides and many sitoindosides isolated from this plant species have been thoroughly studied. Two main withanolides, withferin A and withanolide found in leaves show pharmacological activity[2]. Its root is used as an anti-inflammatory drug for swellings, tumors, scrofula and rheumatism; and as a sedative and hypnotic in anxiety neurosis[1]. The genetic diversity of W. somnifera has become endangered because of overexploitation due to increased demand for production of medicines and a reduced span of viability and also low germination rate (21%) restricts its propagation through seeds. W. somnifera has been depleted from their natural habitat and is now included in the list of threatened species by The International Union for Conservation of Nature and Natural Resources because of large scale unrestricted overexploitation.

Therefore, there is a need for propagation, conservation and sustainable use of this plant species. With this regard many strategies have been developed amongst which tissue culture-based techniques are more important. Micropropagation is a technique used for propagation of plants which are difficult to be propagated by conventional methods. This technique is also used for mass multiplication of existing stocks of germplasm, for more biomass energy production and conservation of important, elite and rare plant species that are threatened or on the verge of extinction. It is an easy and economical way for obtaining large number of consistent, uniform and true-to-type plants. Although earlier attempts have been made for the propagation of W. somnifera through tissue culture but considerable effort is still required to make it more practicable [3].

W. somnifera, an important medicinal plant, contains phytochemicals such as triterpenoid steroidal lactones, alkaloids, flavonoids and tannin which have a possible role in the green synthesis of nanoparticles [4]. Nanotechnology involves designing of materials at nanoscale level having particular properties that are suitable for required applications. Nanoparticles have dimensions between 1-100nm and act as a bridge between bulk materials and atomic or molecular structures. Metal nanoparticles have known to be effective against various pathogenic microorganisms such as bacteria, fungi, algae, yeast and virus. This may be due to their small size, high surface to volume ratio with free reactive residues attributing high reactivity over bulk materials. Several types of metal nanoparticles have been developed like magnesium, iron, gold, copper, alginate, zinc and silver. However, silver nanoparticles (AgNPs) have sensible antimicrobial activity against microorganisms.

In ancient period, silver was used in several forms to preserve food products. Perhaps usage of silver utensils for eating and drinking and preservation of eatable and drinkable items was due to awareness of its antimicrobial action.

Recently, AgNPs have been widely explored by scientists due to their robust biocidal effects against various species of bacteria including E. coli, Staphylococcus and Streptococcus mutans. Studies have revealed that the antimicrobial activity of AgNPs is due to accumulation of AgNPs in the cell wall and formation of “pits” in the bacterial cell wall eventually leading to cell death. Thus, it can be an effective solution to the matter of multidrug resistance microorganisms. AgNPs have thus been used for bacterial diseases, incorporated into dental materials, treatment of skin burns and water purification, etc. Conventional physical and chemical methods for synthesis of AgNPs require use of heavy equipment, huge amount of energy input, highly toxic and dangerous compounds which can generate biological hazards and are not ecologically safe. Contrary to this plant-mediated synthesis is rapid, simple, non-toxic, economical and eco-friendly. Also plant mediated synthesis is beneficial over other biological synthesis methods which are associated with difficult procedures like maintaining microbial cultures and eliminates risk of handling microorganisms. Plant mediated synthesis can be easily scaled up for large scale synthesis. AgNPs synthesis have been achieved by use of various plant parts such as bark, root, stem, fruit, seed, callus, leaves and flowers [5].

Plant extract contains high levels of steroids, carbohydrates, sapogenins and flavonoids which causes bio-reduction of Ag and other phyto-constituents act as capping agents which provide stability to nanoparticles as they are formed [6]. W. somnifera contains several groups of chemical constituents such as triterpenoid steroidal lactones, alkaloids, flavonoids and tannin [4]. Hence this plant was used for synthesis of AgNPs. The biological activity of AgNPs depend on the size and shape of the particles. Smaller size and truncated -triangular particles are found to be effective and have superior properties. Green synthesis is advantageous over conventional chemical methods in ease in control of shape, size and distribution of AgNPs produced by optimization of the synthesis method and different parameters like temperature, pH, concentration of precursors, etc [7]. As the function of AgNPs depend on physico-chemical properties, the AgNPs produced need to be characterized.

Characterization can be done using various analytical techniques such as UV-vis spectroscopy, X-ray diffractometry (XRD) Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). UV-Vis Spectroscopy is a fast, simple, easy, reliable and sensitive technique for primary characterization of synthesized nanoparticles. AgNPs are known to have unique optical properties due to which they interact strongly with specific wavelengths of the incident light. The conduction band and valence band of AgNPs lie very close and thus electrons can move freely upon interaction with incident light. These electrons give rise to surface plasmon resonance (SPR) i.e., electrons show collective oscillation in resonance with the light wave. A spectrum is produced which can used to determine the particle size, shape, stability and monitor synthesis of nanoparticles. Accordingly, further characterization can be done after this [7].

Withania somnifera is thus an important medicinal herb with various therapeutic applications. Hence it is an important scientific research subject. The effect of salt stress on agricultural crops is well studied but there is lack of information with respect to medicinal plants like W. somnifera. Every year more and more land become unfavourable for plants due to increased salinity [8].

According to an estimate, about 40% of world’s land surface (i.e., about 800mha) is afflicted by salinity problems. In India, 6.74mha land surface face salinity problems. Salinized areas are increasing at the rate of 10% annually. By 2050, more than 50% arable land will be salinized. Salinity can be categorized into primary salinity (occurs naturally in soil) and secondary salinity (anthropogenic in nature). One of the major human activities leading to increased salinity is irrigated agriculture. Improper irrigation and poor drainage cause water logging. The irrigation water usually contains calcium (Ca2+), magnesium (Mg2+) and sodium (Na+). During evapotranspiration, water evaporates often causing Ca2+ and Mg2+ to precipitate, leaving Na+ dominant in the soil [9]. In Indo-Gangetic plains, soil salinity increases due to alternate wet and dry seasons. In wet months, water having products of weathering accumulate in low-lying areas. Following the wet season, in dry months, increased evaporation leads to concentration of soil solution which results in increase in Na+ ions along with increase in pH [10]. Increased salinity results in adverse effects on the growth and yield of plants.

The effect of salt stress can be characterized into short and long terms. Increased salinity in soil causes lowering of water potential making it more negative thereby lowering water potential gradient from soil to roots. Hence water uptake by plants from soil is reduced even when the soil has plenty of water. This falls under short term effect. Salt stress affect medicinal plants on different physiological stages. Seed germination is one of the most salt sensitive growth stages which show significant reduction under salt stress. At seedling stage, seedling height, number of leaves, leaf area, biomass may also be reduced. This could be due to slow or less mobilization of reserve foods, suspending cell division, enlarging and injuring hypocotyls. High salinity also causes hyperosmotic effects in plants leading to membrane disorganization and metabolic toxicity [8]. Salt stress also induces synthesis of abscisic acid, which when transported to guard cells, closes stomata. As a result of stomatal closure, photosynthesis declines, photoinhibition and oxidative stress occurs [11]. Oxidative stress includes excessive generation of reactive oxygen species (ROS) primarily produced from pathways such as photorespiration, from photosynthetic apparatus and from mitochondrial respiration. ROS causes oxidative damage such as pigment oxidation, lipid peroxidation, membrane destruction, protein denaturation, and DNA mutation. ROS causes lipid peroxidation where ROS remove electrons from the lipids in the cell membranes thereby damaging the cells [12]. These fall under long term effects.

Medicinal plants and crops show some common responses and mechanisms for salt tolerance such as protective mechanisms of antioxidants. Antioxidants can be divided into two classes: the low-molecular-mass nonenzymatic free radical scavengers such as proline, nitrogen-containing compounds such as amino acids, amides, proteins, polyamines and soluble sugars such as glucose, fructose, sucrose, and fructans. The other class includes enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)[13].Apart from these there is accumulation of different secondary metabolites such as isoprenoids, phenols, or alkaloids which have antioxidant properties. Phenylpropanoid derived phenols such as flavonoids, tannins, and hydroxycinnamate esters, act as important radical scavengers [14].

Secondary metabolites are organic compounds that are not involved in direct growth, development, or reproduction. Secondary metabolites are occasionally found in low concentrations and are not much significant for primary plant life. However, these compounds are important for plant’s survivability and defense strategies [15]. As mentioned above the distinctive characteristic of medicinal plants under stress conditions is accumulation of secondary metabolites which act as antioxidants to regulate the effects of stress. The antioxidant properties are mainly due to phenolic compounds. Plant phenolics are ubiquitous secondary metabolites and are most abundant class of secondary metabolites. Phenolics are characterized by at least one aromatic ring (C6) bearing one or more hydroxyl groups. Phenolic compounds may be increased or synthesized de novo as a response to various biotic and abiotic stress [13]. They play an important role in absorbing and neutralizing free radicals, quenching singlet oxygen, and decomposing peroxides. They act as potential radical scavengers by donating electrons to guaiacol peroxidases (GuPXs) for detoxification of high amounts of H2O2 produced under stress conditions[15]. Phenolic compounds also show various physiological properties such as anti-allergenic, anti-atherogenic, anti- inflammatory, anti- microbial, anti- thrombotic, cardioprotective and vasodilatory effects [16].

Objectives of the Research

  • In-vitro regeneration of Withania somnifera from seedlings.
  • Green synthesis of silver nanoparticles using W. somnifera leaf extract.
  • Assessment of salt stress on in vitro grown W. somnifera plantlets.

LITERATURE REVIEW

The concept of totipotency was established by the cell theory postulated by Schleiden in 1838 with respect to plants and by Schwann in 1839 with respect to plants and animals. The theory stated that cells are autonomic and capable of regeneration to give a complete plant. This theory led to the foundation of plant and cell tissue culture[17].

Haberlandt in 1902 was the first to attempt plant cell culture aseptically in a nutrient medium. He cultured pallisade cells, pith cells, stamen hairs and stomatal guard cells in a simple organically enriched medium containing glucose under aseptic conditions and was totally unsuccessful in all cases. The cells did not divide but continued to stay in living state for several weeks, increased in size and accumulated starch. He failed to recognize that meristematic cells of the plant body are heterotrophic and for dedifferentiation of these cells into a meristematic state requires the presence of plant growth regulators [18].

White in 1934 developed the first successful root and meristem cultures of Lycopersicon esculentum. He was able to establish continuous growing meristematic cell cultures of tomato on medium containing inorganic salts, yeast extract and sucrose and 3 vitamins B (pyridoxine, thiamine, nicotinic acid), thereby establishing the importance of additives [19]

Skoog in 1944 gave the first indication that in vitro organogenesis could be chemically reg­ulated to some extent. He found that addition of auxin in culture medium stimulated root formation whereas shoot formation was inhibited [20].

Skoog and Tsui in 1948 found that adenine sulphate promoted shoot initiation and reversed the inhibitory effect of auxin[21]. Skoog and Miller in 1957 put forward the concept of hormonal regulation of organ formation. They showed that the differentiation of roots and shoots in tobacco pith cultures was a function of auxin to cytokinin ratio. High auxin to cytokinin ratio promoted rooting whereas low auxin to cytokinin ratio promoted shoot formation. At equal concentrations of auxin and cytokinin, the tissue grew in an unorganised manner. This concept of organ formation is now applicable to most plant species [22].​

Murashige and Skoog in 1962 developed a revised White’s medium for rapid growth of tobacco tissue cultures later called the MS medium. They found that the promotion of growth was mainly though not entirely dependent on inorganic rather than organic nutrients in extract. Since then it has been widely used in various plant tissue culture techniques [23].

Murashige in 1974 proposed a three- stage method for plant propagation through tissue culture. These stages are:

I. Establishment of the aseptic culture;

II. Multiplication of propagula;

III. Preparation for re-establishment of plants in soil. [24]

Debergh and Maene in 1981 proposed a revised method for plant propagation through tissue culture. They stated that for better results healthy stock plants grown under controlled conditions must be used as starting material as most of the plants are systematically infected with bacterial and fungal pathogens. Hence, they introduced a stage 0 which involves preparation of healthy stock plants.

The stages are:

0. The preparation of stock plants under hygienic conditions;

I. The establishment of aseptic cultures;

II. The induction of meristematic centres, their development into buds and their rapid multiplication.

IIIa. The elongation of the buds to shoots and the preparation of uniform shoots for stage IIIb;

IIIb. The rooting and the initial growth of the in vitro produced shoots under in vivo conditions [25].

Sen and Sharma in 1991 successfully established in vitro shoot multiplication from shoot tips of aseptically germinated seedlings of Withania somnifera L. using MS basal media supplemented with low concentrations of 6-benzyladenine (BA). Rooting was achieved in excised shoots grown on growth regulator- free MS basal media. Further rooted shoots were successfully established in soil in a greenhouse [26]

Deka et al in 1999 achieved in vitro micropropagation from axillary buds and shoot tips of Withania somnifera L. on MS basal medium supplemented with BAP and kinetin. MS media supplemented with BAP and kinetin in the range 0.1-1mg /L exhibited 30% shoot regeneration within 15 days and upto 85% shoot regeneration within 60 days. Excised shoots developed rooting in growth regulator- free MS media [27]

Many other studies have reported in vitro propagation of Withania somnifera using different explants shoot tips ​[28]​ ​[29]​ ​[30]​ ​[31][32] [33] [34], axillary bud [35], hypocotyl [36], cotyledon [37], leaf [38] [39], seed [40], cotyledonary leaf segments [41], callus of leaf [42], shoot tip and root [43]and the nodal areas [44].

Withania somnifera is an important medicinal plant known to contain secondary metabolites that show pharmacological activity. In addition, these metabolites have shown to be effective in low cost green synthesis of nanoparticles. Hence, this plant can be used to synthesize and coat nanoparticles. These nanoparticles can be used for various biomedical applications as they pose none of the potential health hazards of more conventional chemical methods of nanoparticle synthesis.

In recent years, synthesis of nanomaterials of desired morphology and size using green chemistry which involves use of natural reducing, capping, and stabilizing agents have become an important area of research. Biological methods are being preferred over physical and chemical methods to avoid use of any harsh, toxic and expensive chemical substances. Biological methods include synthesis of nanomaterials using bacteria, fungi and plants [45]. Among these, plant mediated synthesis is beneficial over other biological methods as it eliminates the risk of handling and maintaining microbial and fungal cultures [5].

Shankar et al in 2003 demonstrated rapid synthesis of stable silver nanoparticles in high concentration using proteins/enzymes extracted from P. graveolens leaf. More than 90% of reduction was completed in 9h and the silver nanoparticles formed assembled into open, quasilinear superstructures. This was a significant advance in the field of green synthesis and provided further scope for decreasing the reduction time to make green synthesis an efficient alternative to conventional methods [46] .

Vilchis et al in 2008 used Camellia sinensis (green tea) extract as reducing and stabilizing agent for synthesis of silver nanoparticles. It was assumed that phenolic- acid type biomolecules present in C. sinensis extract, were responsible for the reduction and stabilization of the nanoparticles formed [47].

Begum et al demonstrated rapid formation of stable silver nanoparticles of various shapes: spheres, trapezoids, prisms and rods, using black tea leaf extracts. They proposed that the main biomolecules responsible for the nanoparticle synthesis were the polyphenols or flavonoids present in the leaf extracts [48].

Nagati et al in 2013 demonstrated the potential of leaf extract of the Withania somnifera in synthesis of silver nanoparticles with fairly well-defined dimensions in less time. The study demonstrated use of fresh leaves which is economical and easily available for synthesis. The nanoparticles formed were found to be highly toxic to multidrug resistant bacteria and could be used as an excellent source against multi drug resistant bacteria, enhancing wound healing process, and act as anticancer, anti-stress agent [49] .

Gregory Marslin et al in 2015 concluded that leaf extracts are suitable for the green synthesis of AgNPs with potent antimicrobial activity. They stated that this was highly relevant since the biomass of this plant is considered a waste product by the phytopharmaceutical industry and hence can be used for further economic processes. They also showed that the compounds catechin, p-coumaric acid, luteolin-7-glucoside, and a nonidentified withanolide derivative present in the WS aqueous leaf extract were responsible for green synthesis of AgNPs. The antimicrobial study concluded that AgNPs were 200 times more potent when compared to AgNO3 [50].

W. somnifera is thus an important medicinal plant with potential therapeautic applications. However, due to its increased demand for production of medicines and low germination rate, conservation of this plant is necessary. In addition to this, every year more and more land is affected by salinity problems. There is lack of information of salt stress on medicinal plants like W. somnifera.

According to Sreenivasulu et al, the effect of salt stress can be divided into three broad categories:

i) Reduction in osmotic potential of soil solution thereby reducing water availability to plants ultimately leading to osmotic stress in plants;

ii) Deterioration in the physical structure of the soil such that water permeability and soil aeration are diminished;

iii) Specific ion toxicity in plants which have inhibitory effect on plant metabolism and also causes mineral nutrient imbalances and deficiencies or

iv) a combination of these factors [51] .

Various studies have been conducted with respect to different medicinal plants. Very few studies have been conducted on effects and reponse of W. somnifera under salt stress. Studies based on effects of salt stress on various medicinal plants including W. somnifera have been reviewed below:

1) Morphological characteristics and development:

Many investigators have reported reduction in plant growth as an effect of salt stress in foeniculum vulgare subsp. vulgare [52]; majorana hortensis [53]; peppermint, pennyroyal, and apple mint [54] ; matricaria recutita [55]; thymus maroccanus [56]; geranium [57]; thymus vulgaris [58]; sweet fennel [59]; sage [60]; mentha pulegium [61]. Tabatabaie et al investigated salt stress on Mentha piperita var. officinalis and Lipia citriodora var. verbena and reported significant reduction in number of leaves, leaf area and leaf biomass under salt stress [62]. Najafi et al investigated on Satureja hortensis, and reported that leaf area, leaf and stem fresh weight, as well as dry weight of leaves, stems and roots were decreased in plants grown in different levels of NaCl [63].

Jaleel et al in 2008, reported reduction in vegetative growth, with respect to plant height and leaf area of W. somnifera under NaCl stress(40mM) [8]. Sabir et al in 2012, studied callus cultures and in vitro shoots of W. somnifera under salt stress and reported negative effect of different salt concentrations on growth of calli and shoots[13].

2) Total phenolics content:

Studies have reported increase in phenolic concentration with salt treatment in many medicinal and aromatic plants such as spearmint [64], Achillea fragratissima, Matricaria chamomilla, Nigella sativa [65], Mentha pulegium [61]and Withania somnifera [13].

3) Lipid peroxidation:

LPO has been associated with damages caused by various environmental stresses mediated by generation of ROS. The increase in LPO has been correlated with accumulation of ions and generation of ROS under salt stress in various studies [66] [67]. Lipid peroxidation is often expressed as as the level of malondialdehyde (MDA) content, which is a decomposition product of polyunsaturated fatty acid (PUFA), often considered a reflection of lipid peroxidation. Sabir et al 2012 reported a definite pattern of increase in the MDA content with increasing salt concentration in shoots and calli of W. somnifera under salt stress [13].

METHODOLOGY

In-vitro Regeneration of Withania somnifera from seedlings

Source and choice of explants:Withania somnifera explants were obtained from disease free seedlings grown under controlled conditions in Laboratory of Morphogenesis, Department of Botany, BHU, Varanasi.

Culture medium: The culture medium for plant tissue culture used was Murashige and Skoog Medium (MS medium) which was originally formulated by Murashige and Skoog in 1962. MS medium provides all necessary macronutrients, micronutrients, vitamins and amino acid.

Preparation of stock solutions: MS basal media was prepared by using four different stock solutions by weighing and dissolving various salts one by one in distilled water. The stock solutions prepared was stored in refrigerator and further used to prepare media for 2-3weeks.

Preparation of growth regulators: To prepare stock solution of growth regulator 6-Benzylaminopurine (BAP) of concentration 1mg/mL, BAP was measured on electronic balance and dissolved in minimum amount of 1N NaOH and final volume was maintained with distilled water.

Composition of stock solutions of MS Media:

Constituents (Chemical Name) Chemical formula Concentration (g/L) in stock medium
1.Inorganic nutrients
Stock solution I (Macro salts) 500mL with DW
Potassium Nitrate KNO3 19.00 g
Ammonium Nitrate NH4NO3 16.50 g
Calcium Chloride CaCl2.2H2O 4.40 g
Magnesium Sulphate MgSO4.7H2O 3.70 g
Potassium Dihydrogen Orthophosphate KH2PO4 1.70 g
Stock solution II (Micro salts) 250mL with DW
Potassium Iodide KI 0.0415 g
Boric Acid H3BO3 0.0310 g
Manganese Sulphate MnSO4.7H2O 1.115 g
Zinc Sulphate ZnSO4.7H2O 0.430 g
Copper Sulphate CUSO4.5H20 0.0125 g
Cobalt Chloride CoCl2.6H2O 0.00125 g
Sodium Molybdate Na2MoO4.2H20 0.00125 g
Stock solution III (Iron sources) 250mL with DW
Ferrous Sulphate FeSO4.7H2O 1.390 g
Na2EDTA C10H14N2Na2-O8.2H20 1.865 g
2. Organic nutrients
Stock solution IV 50 mL with DW
Nicotinic Acid C6H5NO2   0.005 g
Glycine C2H5NO2   0.20 g
Inositol C6H12O6 1.00 g

Media preparation:For preparation of full-strength MS medium, 50mL of stock solution I and 5mL each of stock solutions II, III, IV were mixed and final volume was maintained to 1L with distilled water. It was then mixed using stirrer for 10 min. The pH was adjusted to 5.8+0.2 with 0.5N NaOH and 0.1N HCl solutions. Agar 0.8% (w/v) and sucrose 3% (w/v) were then added. It was then again mixed using stirrer. To melt the agar medium was heated in microwave. Growth regulators are added prior to pouring media into culture tubes. The culture medium was poured in culture tubes (10-15mL). The tubes were marked and plugged with non-absorbent cotton plugs.

Sterilization of media:The plugged culture tubes containing media was then sterilized using autoclave at 121˚C for 15 min at 15psi. Then autoclaved media was allowed to cool and solidify in slant position at room temperature.

Culture room:In culture room, temperature, light intensity, relative humidity and photoperiod was maintained. The average temperature was maintained at 25 ˚C ± 2 ˚C.The light source was provided by 40 watt white fluorescent tube at a distance of 30-35cm in each rack which provided illumination between 2000-3000lux. Photoperiod of 8 hours was maintained.

 Experimental setup:Seedlings were cultured on full strength plain MS media and observed for growth and root initiation. One set of explants was cultured on MS media supplemented with 1mg/L BAP and observed for multiple shooting.

Green Synthesis of Silver Nanoparticles Using W. somnifera Leaf Extract

Preparation of plant leaf extract: Mature leaves of Withania somnifera were taken from Botanical Garden in Banaras Hindu University. It was washed under tap water and excess water was absorbed using blotting paper. The leaves were chopped into small pieces. 1g, 2g, 3g and 5g of chopped leaves were weighed and added in 100mL milliQ water separately and boiled for 6 min. The extract obtained was filtered using Whatman paper.

Preparation of AgNO3 solution: Weighing of AgNO3 for preparation of 1mM AgNO3 solution was done and dissolved in distilled water.

Green synthesis of nanoparticles and characterization: Mixing of plant extract and AgNO3 solution was done in the ratio 1:3 and kept in shaker until colour change was observed. It was further characterized using UV-Vis Spectrophotometer.

Assessment of salt stress on in vitro grown Withania somnifera plantlets

Source and choice of explant: The explants used for salt stress assessment were obtained from in vitro grown Withania somnifera plants cultured in liquid MS media supplemented with 1mg/L IBA. The plants were selected based on having similar morphology and rooting.

Culture medium: Hoagland media supplemented with different NaCl concentrations (50mM, 100mM, 150mM, 200mM, 250mM) were used.

Experimental setup: The explants were cultured in the media and kept in culture room in controlled conditions of temperature, light intensity, relative humidity and photoperiod. The average temperature was maintained at 25 ˚C ± 2 ˚C. The light source was provided by 40 watt white fluorescent tube at a distance of 30-35cm in each rack which provided illumination between 2000-3000lux. Photoperiod of 8 hours was maintained. After 7 days plants were harvested, and assessment of effects and response to salt stress was done.

Growth analysis: Fresh weight (FW) and dry weight (DW) of shoot and root was measured after the samples were dried at 70˚C for 72 hours.

Estimation of total phenolic content: Total phenolic content of W. somnifera leaf extract was determined using Folin- Ciocalteau spectrophotometric method. 100mg of fresh leaves were weighed from each sample and heated with 5mL of 1.2M HCl in 50% aqueous methanol (CH3OH) at 90 ˚C for 2 hours. The reaction tubes were then cooled and centrifuged at 10,000rpm for 10min. 20µL of supernatant was then mixed with 3.16mL water and 50µL FC reagent. The reaction mixture was allowed to stand for 2 min. 150µL of 1.9M Na2CO3 solution was then added and incubated at 40˚C for 30 min. After 30 min, absorbance was measured at 765nm.

Estimation of lipid peroxidation: LPO was estimated as the content of total 2-thiobarbituric acid reactive substances (TBARS) expressed as equivalents of malondialdehyde (MDA) by method given by Heath and Packer (1968) 0.15g of leaves was homogenized in 5mL solution containing 0.25% TBA and 10% TCA. The extract was then warmed at 100˚C for 45min and then allowed to cool. The reaction tubes were then centrifuged at 10000 ×g for 10min. Absorbance of supernatant was measured at 532nm and 600nm. m. After subtracting the non-specific absorbance at 600 nm, the TBARS content was calculated according to its extinction coefficient of 155mM-1 cm-1.

RESULTS AND DISCUSSION

In vitro regeneration of Withania Somnifera from Seedlings

height ms.png
    Effect on height of in vitro grown plantlet in plain MS media after 7 and 15 days.
    leaves ms.png
      Effect on number of leaves of in vitro grown plantlets in plain MS media.
      shoot bap.jpg
        Multiple shoot generation in in vitro grown plantlets in MS media supplemented with 1mg BAP.

        Seedlings grown in plain MS Media without any supplement of phytohormoes showed increase in height and number of leaves in 15 days of inoculation (Fig 1 and Fig 2). First emergence of the root was observed after 7 days of inoculation. Multiple shoot generation was observed in seedlings grown in MS media supplemented with 1mg BAP (Fig 3).

        Green synthesis of silver nanoparticles using W. somnifera leaf extract

        Nanoparticles.jpg
          Visible colour change of Withania somnifera leaf extract from greenish yellow to dark brown. 
          4_1.jpg
            UV Visible spectra of  reaction of different leaf extracts (1g, 2g, 3g and 5g) with 1mM AgNO3 solution in the ratio 1:3.

            Green synthesis of nanoparticles was achieved by reaction of Withania somnifera leaf extracts prepared by using different weights of leaves (1g, 2g, 3g and 5g) and 1mM AgNO3 in the ratio 1:3 . Synthesis of nanoparticles was primarily characterized by visible colour change from greenish yellow to dark brown. It was further characterized by using UV Visible spectrophotometer which showed 5g leaf extract having highest peak at 450nm.

            Assessment of Salt Stress on In vitro Grown Withania Somnifera Plantlets

            Growth analysis:

            fresh weight_2.png
              Effect of different doses of NaCl on fresh weight of in vitro grown plantlets after 1 week.
              dry weight_2.png
                Effect of different doses of NaCl on dry weight of in vitro grown plantlets after 1 week.

                Estimation of total phenolic content:

                total phenolics.png
                  Effect of differnt doses of NaCl on total phenolics content in in vitro grown plantlets after 1 week.

                  Estimation of lipid peroxidation:

                  LPO.png
                    Effect of different doses of NaCl on lipid peroxidation in invitro grown plantlets after 1 week. 

                    Salt stress on in vitro grown Withania somnifera plantlets showed detrimental effect on plant growth in terms of biomass. Biomass showed reduction with increasing NaCl concentrations in media(Fig 6 and Fig 7). Total phenolics content was also found to be increased with increasing NaCl concentrations indicating increased secondary metabolism under salt stress (Fig 8). Lipid peroxidation also increased with highest lipid damage in media supplemented with 150mM NaCl indicating the generation of ROS under salt stress (Fig 9).

                    CONCLUSION AND RECOMMENDATIONS

                    Withania somnifera is an important medicinal with high therapeutic value and therefore, at present it is being subjected to overexpolitation to meet the increasing demands. However, due to low germination rate and reduced span of viability, conservation and sustainable management of this plant is important. Micropropagation of W. somnifera can be done by using plant tissue culture techniques using MS media. Plain MS media showed satisfactory response for regeneration of plants from seedlings within 15 days and shoot multiplication was also achieved within 15 days by using MS media supplemented with 1mg BAP. Thus, plant tissue culture technology, apart from their use as a tool of research, can be of industrial importance for plant propagation and plant improvement using small pieces of tissue (explant) to produce large number of plants in a continuous process.

                    W. somnifera is known to contain secondary metabolites which have high medicinal value. These secondary metabolites can also be used for synthesis of nanoparticles. Synthesis of nanoparticles using extracts of fresh leaves is beneficial as it is readily available and economical. The biomass of this plant is considered waste in many pharmaceutical industries and thus it can be further used for large scale synthesis of nanoparticles. Green synthesis is also advantageoues in control of size and shape of nanoparticles formed. Thus, nanoparticles can be synthesised economically and with desired shape and size which can be furthur used for various biomedical applications. Silver nanoparticles are known to be beneficial against various pathogens including multidrug resistant pathogens. Moreover, green synthesis is eco-friendly and less tedious than other methods of synthesis including other biological methods using bacteria and fungi. In this study, leaf extract was mixed with 1mM AgNO3 solution in the ratio 1:3 and best response was seen in leaf extract prepared using 5g leaves.

                    Salinity is one of the major growing concerns in agriculture. About 40% of total land on earth is already afflicted with salinity problems. In addition, salt afflicted areas are increasing at the rate of 10% annually and is common in aird and dry areas. W. somnifera is commonly grown in dry and arid regions. Hence, it is important to study the effect and response of salt stress on this plant to develop resistance high value variety of this medicinally important plant. Salt stress is known to cause detrimental effect on growth and metabolism of plants. However, salt stress also induces increased secondary metabolism which can be exploited for production of high medicinal value plants. In this study, salt stress showed reduced growth and biomass which may be due to reduced nutrient supply and increased osmotic stress. Lipid peroxidation increased with increasing salt concentration in media indicating generation of ROS. Total phenolics content also showed increased response under increasing salt concentrations in media. This indicates increased secondary metabolism under salt stress. This may be in response to the increased ROS production as phenolic compounds have antioxidant properties.

                    ACKNOWLEDGEMENTS

                    I would take immense pleasure to record my deepest sense of gratitude towards my venerable supervisor Prof. Shashi Pandey-Rai for her incessent effort in getting my project accomplished. Her immense knowledge, conscientious guidance and amicable behavior encouraged me to accomplish this project and enlightened me the first glance of research.

                    I would like to extend my gratitude to Prof. R. S. Upadhyaya, Head of Department of Botany, BHU for providing all necessary facilities during the period of my summer training.

                    I place my sincere and heartfelt gratitude to the research scholars Ms Lakee Sharma, Ms Deepika Tripathi, Mr Bipin Maurya, Mr Niraj Kumar and Ms Nidhi Rai for their immense support, guidance and co-operation.

                    I owe a special thanks to Prof Savita Kerkar, Head of Department of Biotechnology, Goa University. for her valuable suggestions and support during allotment of summer training. I would like also like to thank IASc-INSA-NASI for giving me the opportunity and providing support to complete my summer training through Summer Research Fellowship Programme 2019.

                    Lastly, i would like to thank Mr. Hari Asare, lab attendant, Ms Lukeshwari Shyam, Mr Prantik Pahadi, Ms Ritambhara Singh, Ms Vandana Rai and Mr. Ashutosh Singh for their immense support and help.

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