Introduction

1. INTRODUCTION 1. Medicinal – Plants have been used as a source of medicine since time immemorial. The earliest documented evidence of the use of medicinal plants in classical Indian texts like the Rigveda, Atharvaveda, Charak Samhita and Sushruta Samhita dates back to about 5000 years along with Unani, Siddha and Amchi. Ayurveda, the traditional Indian medicine and the Chinese traditional medicine remain the most ancient yet living traditions with sound philosophical, experiential and experimental basis (Patwardhan et al., 2005). Thus, the use of herbal or traditional medicines has been derived from the rich traditions of the ancient civilizations and scientific heritage (Kamboj, 2000). This ancient wisdom has now become the basis of modern medicine and an important source for the future medicine and therapeutics. A number of medicinal plants have been and are being studied for their detailed chemical investigations for isolating pure bioactive molecules of interest leading to the discovery of new drugs. Plants produce a diverse array of organic compounds, which are mainly developed for their own defense against microbes, pathogens, insects and predators. These compounds are often referred to as “natural products” or “secondary products” or “secondary metabolites”. Medicinal plants produce secondary products which are the source of fine chemical compounds like pigments, alkaloids, isoprenoids, terpenes, waxes, drugs, dyes, flavours, fragrances, insecticides, etc. These secondary products or “secondary metabolites” are either derived from the whole plant or from different organs like the root, stem, bark, leaves, flower, seed etc. or are obtained from the excretory plant product such as gum, resins, latex, etc. The study of such medicinally important plants is carried out for the discovery of novel secondary metabolites which are presently of great interest for their potential use as pharmaceutical drugs, health products, food supplements, nutrients, food additives and cosmetics (Ferrari, 2010; Ahmed and Kim, 2010). Plant secondary metabolites provide an incredible resource for scientific and clinical researches and for the development of new drugs. 2. Secondary Metabolites: Definition, classification and biosynthesis – Plant cells produce a broad spectrum of chemical compounds which are necessary for the basic functions, viz. biochemical pathways for survival and propagation, and are

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Introduction known as “basic or primary metabolites” which results in respiration, differentiation, assimilation and transport. “Secondary metabolism” produces diverse and relatively less essential or non-essential byproducts which are not directly involved in the growth, development or reproduction of an organism are called as secondary products or “secondary metabolites” (Ahmed and Kim, 2010). A problem of a proper definition has been discussed by many researchers, but the chemical diversity and inadequate knowledge about the role of these secondary metabolites have hindered the attempts of accurately defining this group (Verpoorte, 2000). According to Verpoorte (2000), “Secondary metabolites are compounds with a restricted occurrence in taxonomic groups that are not necessary for a cell (organism) to live, but play a role in the interaction of the cell (organism) with its environment, ensuring the survival of the organism in its ecosystem.” In 1988 the NAPRALERT database contained a record of more than 88,000 secondary metabolites, and the every year there was introduction of approximately 4,000 newly reported secondary metabolites. Taking this estimate into consideration, there should now be more than 1,00,000 secondary metabolites identified (Wink, 2015). Also, many species are studied for a specific compound. Assuming that the existing trend of discovering novel secondary metabolites will continue for the existing plants, at least a million different compounds could be isolated. Each plant has its own unique and complex pattern of secondary metabolites which can exhibit differences at different developmental stages, and/or between individuals and/or between populations as well. Secondary metabolites have been categorized in several ways. Broadly, secondary metabolites from plants are classified into three main classes: terpenoids, alkaloids and phenolics (Kabera et al., 2014). According to Lattanzio (2013), secondary metabolites can be classified into several groups according to their biosynthetic routes and structural features. Secondary metabolites have also been categorized based on chemical characteristics, plant origin or biosynthetic origin. Based on chemical characteristics, the compounds can be divided in a number of groups based on a typical characteristic, example – alkaloids, which are characterized by a basic nitrogen function, or phenolics, which are characterized by aromatic ring systems having a phenolic-hydroxyl group, or presence of a particular type of basic skeleton e.g. anthracene, coumarine, quinone, indole, isoquinoline, etc. Based on plant origin secondary metabolites can be classified,

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Introduction example – Strychnos alkaloids, Atropa alkaloids, Digitalis cardenolides, etc. this category is mostly connected with pharmaceutical applications. The categorization based on biosynthetic origin has major examples, viz. the terpenoids, phenylpropanoids and polyketides which are derived from just a few building blocks; the isopentenyl diphosphate C5-unit, the phenylalanine/tyrosine-derived C9-unit (phenylpropanoids), the acetate C2-unit (polyketides) and some amino acids (Verpoorte, 1998). The pathways of biosynthesis are responsible for the occurrence of primary and secondary metabolites. Biosynthetic reactions need energy and this energy is obtained from the energy released through the citric acid cycle by glycolysis of carbohydrates. Oxidation of glucose, fatty acids and amino acids leads to the formation of adenosine triphosphate (ATP) which is a high energy molecule formed by catabolism of primary compounds. In fuel anabolic reactions involving intermediate molecules on the pathways ATP is recycled. Catabolism involves oxidation of starting molecules, biosynthesis or anabolism involves reduction reaction. So, a reducing agent or hydrogen donor, usually nicotinamide adenine dinucleotide phosphate (NADP) is needed. These catalysts are called as coenzymes and the most widely occurring is coenzyme A (CoA) made up of adenosine diphosphate (ADP) and phosphopantetheine (Kabera et al., 2014). Thus, generally the most common pathways accepted for biosynthesis are functioned through the pentose for glycosides, polysaccharides; shikimic acid pathway for phenols, aromatic alkaloids, tannins; acetate-malonate pathway for phenols and alkaloids; and mevalonic acid pathway for terpenes, steroids and alkaloids (Dewick, 2002). 3. Phenolic compounds – Phenolics are compounds have one or more hydroxyl groups attached directly to an aromatic ring i.e. a hydroxylated benzene ring. Phenolic compounds are the most widely distributed secondary metabolites in the plant kingdom. This is one of largest group of secondary metabolites occurring abundantly with more than 8,000 phenolic structures currently known, ranging from simple molecules such as phenolic acids to highly polymerized substances such as tannins (Jain et al., 2013). Phenolic compounds are uncommon in bacteria, fungi, and algae, but have been reported from bryophytes, pteridophtyes and gymnosperms and a wide range is present in the vascular plants. It is predicted that approximately 2% of all the carbon which is photosynthesized by the

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Introduction plants is converted into flavonoids or closely related compounds. Higher plants produce thousands of phenolic compounds and the number of newly characterized and identified ones is increasing rapidly. Common examples of phenolic acids are found in our daily diet which includes citrus fruits, apples, mangoes, onions, tea, coffee, wheat, rice, corn (Lattanzio, 2013; Jain et al., 2013) and many more derivatives are found in nature which are widely distributed and are a part of our nutrition. Apart from food, phenols have most diverse applications in agriculture, pharmaceuticals, cosmetics, health products, food supplements, etc. 3.1 Classification of phenolic compounds – The term “plant phenolics” comprises a highly diverse group of chemical compounds which have been classified in a number of ways. The phenolic compounds have been categorized into groups based on the number of carbons in the molecule (on the basis of their basic skeleton) (Harborne and Simmonds, 1964) (Table 1). Table 1: Classification of phenolic compounds (Harborne and Simmonds, 1964) Structure Class

C6 simple phenolics

C6 – C1 phenolic acids and related compounds

C6 – C2 acetophenones and phenylacetic acids cinnamic acids, cinnamyl aldehydes, cinnamyl C6 – C3 alcohols

C6 – C3 coumarins, isocoumarins, and chromones

C15 chalcones, aurones, dihydrochalcones

C15 Flavans

C15 Flavones

C15 Flavanones

C15 Flavanonols

C15 Anthocyanidins

C15 Anthocyanins

C30 Biflavonyls

C6–C1–C6, C6– benzophenones, xanthones, stilbenes C2–C6

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Introduction

C6, C10, C14 Quinones

C18 Betacyanins Lignans, dimers or oligomers neolignans Lignin Polymers Tannins oligomers or polymers Phlobaphenes Polymers

Swain and Bate-Smith (1962) used an alternative classification where they grouped the phenols into two categories: “common” and “less common”. Another classification of phenols was given by Ribéreau-Gayon (1972) where they grouped the phenols into three families viz. (i.) Widely distributed phenols - ubiquitous to all plants, or of importance in a specific plant (ii.) Phenols that are less widely distributed - limited number of compounds known (iii.) Phenolic constituents present as polymers. 3.2 Quinones – Quinones are a class of plant-derived secondary metabolites which are widely distributed in the plant kingdom. According to the number of benzene rings in the structural skeleton and fused, this class is mainly divided into four types namely, benzoquinone, naphthoquinone, phenanthrenequinone and anthraquinone (Lu et al., 2013). Quinones are oxygen containing compounds which are basically the oxidized homologous of aromatic derivatives and are classified by a 1,4-di keto cyclohexa-2, 5- diene pattern (paraquinones). In naturally occurring quinones, the dione conjugates to an aromatic nucleus (benzoquinones) or conjugates to condensed polycyclic aromatic systems like naphthalene (naphthoquinones), anthracene (anthraquinones), 1,2- benzaanthracene (anthrocyclinones), napthodianthrene (naphthodianthrones) which undergoes a reversible redox reaction in the presence of reductase enzymes and the reduced form of quinone (hydroquinone) (Jain et al., 2013). The natural quinone pigments exhibit great structural variation and shows a wide array of colours ranging from pale yellow to almost black. These pigments are mostly present in the bark, heartwood, root, leaves or other tissues where their colours are suppressed by other Page | 5

Introduction pigments and thus, very little do they contribute for the colours in higher plants. Quinones are extensively distributed in plants belonging to families like Polygonaceae, Rubiaceae, Leguminosae, Rhamnaceae, Labiatae, Boraginaceae (Lu et al., 2013), , Droseraceae, Myrsinaceae, Juglandaceae, Ebenaceae, Scrophulariaceae, Bignoniaceae, Verbenaceae (Seigler, 2012). 3.3 Naphthoquinones –

Naphthoquinones (C6–C4) belongs to the class of quinone pigments and have been reported from higher plant families like Avicenniaceae, Bignoniaceae, Boraginaceae, Droseraceae, Ebenaceae, Juglandaceae, Nepenthaceae, and Plumbaginaceae; from algae; fungi (Fusarium, Marasmius, Verticillium); lichens and bacteria (Streptomyces) (Babula et al., 2009). In higher plants naphthoquinones are biosynthesized by via a variety of pathways like direct incorporation of shikimate via succinyl-CoA combined pathway, or by shikimate/mevalonate pathway or by polyketide pathway (Durand and Zenk, 1974, Lattanzio, 2013). These are dark yellow pigments but apparently not as purpose for plant- colouring. These are frequently present in the heartwood or bark, where their presence is masked and when present in other living tissues, leaves, and roots they are generally in colourless form. The colour is produced only when the extracts are treated with an acid to bring about hydrolysis of sugar linkages and oxidation of quinol to quinone, example, as observed for plumbagin, an orange pigment identified in Plumbago capensis (Lattanzio, 2013). Naphthoquinones possess important pharmacological properties such as antioxidant activity, anti-inflammatory activity, anticancer activity, antimicrobial activity and antibacterial activity (Padhye et al., 2012). 3.4 Plumbagin –

Plumbagin (5-hydroxy-2-methyl-1, 4-napthoquinone; formula: C11H8O3) (Fig. 1) is an organic compound, a yellow dye derived from a simple hydroxy-naphthoquinone. It is named after the plant Plumbago, from which it was originally isolated from the roots and aerial parts of Plumbago pearsonii L. Bolus, Dyerophytum africanum (Lam.) Kuntze., and in roots of Plumbago auriculata Lam. (van der Vijver, 1972). Plumbagin has been reported from the families Plumbaginaeace (van der Vijver 1972), Drosophyllaceae (Crouch et al. 1990), Droseraceae (Jayaram and Prasad 2005), Nepenthaceae (Raj et al. 2011) and Ebenceae (Padhye et al., 2012).

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Introduction

Fig. 1: Structure of Plumbagin

Plumbagin has numerous therapeutic, traditional, ethnobotanical and pharmacological uses. Few activities are enlisted (Table 2) Table 2: Therapeutic activities of plumbagin anti-carcinogenic activity Eldhose et al., 2014, Srinivas et al., 2004 immuno-modulatory effect Checker et al., 2009 Anti-tumor properties Singh and Udupa, 1997; Yang et al., 2010 Antifungal activity Shin et al., 2007 anticancer and anti-proliferative Santhakumari and Rathinam, 1978 activity Anti-diabetic effect Sunil et al., 2012 Antifertility activity Premakumari et al., 1977 Anti-mutagenic activity Edenharder and Tang, 1997 anticancer, antibacterial, antifungal Krishnaswamy and Purushothaman, 1980 activity inhibit cell growth and induces Xu et al., 2013 apoptosis in human lung cancer antioxidant, cardio-protective, anti- Wang and Huang, 2005; Hsieh et al., malarial, antifungal, anti- 2006; (Mulabagal and Tsay, 2004) atherosclerotic, central nervous system stimulatory, anti-hyperglycemic, anti- inflammatory cytotoxic activity Srinivas et al., 2004, Yang et al., 2010; Ganeshan and Gani, 2013

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Introduction anticoagulant activity Ganeshan and Gani, 2013 Hyperlipidaemic activity Alpana, 1996

4. Family Plumbaginaceae - According to the database of “The Plant list” which has collaboration with the Royal Botanic Gardens, Kew and Missouri Botanical Garden, family Plumbaginaceae consists of 24 genera and 635 species. The genus Plumbago has 17 accepted species (http://www.theplantlist.org, http://www.catalogueoflife.org). In India, three species of Plumbago have been commonly recorded namely, P. indica L. (synonym: P. rosea L.), P. auriculata Lam. (synonym: P. capensis Thunb.) and P. zeylanica L. (synonym: P. scandens L.) (http://www.theplantlist.org). 4.1 Distribution of Plumbago species– Plumbago species are distributed throughout the tropical and subtropical regions of the world. In India commonly occurring Plumbago species are P. indica L. (scarlet/ red leadwort), P. auriculata Lam. (cape leadwort) and P. zeylanica L. (white leadwort/ ceylon leadwort) which are widely distributed in several parts of the country, growing as wild or in cultivation because of its medicinal properties. 4.1.1 Distribution of Plumbago zeylanica L. in India and Maharashtra State – India is one of the „mega-biodiversity‟ countries in the world harbouring more than 45,000 plant species. The diversity of India is unbeatable due to the presence of 16 different agro-climatic zones, 10 vegetative zones and 15 biotic provinces (Kamboj, 2000). P. zeylanica is a native of South Asia and is largely distributed in almost all parts of India, growing as wild or in cultivation because of its medicinal properties. It has several vernacular names because of its distribution (http://www.flowersofindia.net/catalog/slides/Chitrak.html) (Table 3). However the trade name remains to be „Chitraka‟. Thus, P. zeylanica is found growing in deciduous woodland, savannas‟ and scrublands from sea-level up to 2000 m altitude (Pant et al., 2012).

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Introduction

Table 3: P. zeylanica – list of vernacular names in India Language Vernacular name English white flowered leadwort, Ceylon Leadwort Sanskrit Chitra, Vahnisajnaka, Agni, Vahni Hindi Safed Chitrak, chitramol, Chita Marathi pandhra chitrak Bengali Safaid-sitarak Irula (Tamil Nadu) Ottuchedi Kannada Chitramulika Malayalam Vellakoduveli, Thumbakoduveli, Kottuveli Tamil Chithiramoolam, Karimai, Kodivaeli Telugu Chitramulamu Oriya Ogni Manipuri Telhidak Angouba Gujarati Chitaro Kashmiri Shatrajna, Chitra Assamese Boga agechita Manipuri Telhidak angouba Punjabi Chitrak

Maharashtra State is divided into 9 agro-climatic zones based on rainfall, soil type and vegetation (www.mahaagri.gov.in). P. zeylanica is found growing in all the agro- climatic zones. 4.1.2 Classification / systematic position / of Plumbago zeylanica L. – As Plumbaginaceae show affinities with both Centrospermae and Primulales, its taxonomic position is ambiguous. According to the Bentham and Hooker‟s classification, taxonomic profile of Plumbago zeylanica L. has been given as follows (Naik, 1984) – Kingdom – Plantae Division – Angiospermae Class – Dicotyledonae Sub-class – Gamopetalae

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Introduction

Order – Family – Plumbaginaceae Genus – Plumbago Species – zeylanica L. Fig. 2: P. zeylanica – a: habitat, b: inflorescence axis, c: flower

4.2 Active compounds from P. zeylanica – P. zeylanica is a widely accepted and important medicinal plant used in Ayurveda, Unani, Siddha and many traditional systems of medicines. The whole plant, especially the roots have been used in folk and traditional medicines for various treatments. A number of active compounds like the plumbagic acid glucosides, plumbagin, chitranone, flavonoids, sitosterol, stigmasterol, chloroplumbagin, biplumbagin, zeylinone, isozeylinone, plumbazeylanone, maritinone, elliptinone, lapachol, glucose, fructose, enzymes such as protease and invertase, coumarins like 5- mrthoxyseselin, seselin, suberosin, xanthyletin, xanthoxyletin have been reported from the roots and shoots of P. zeylanica (Bopaiah and Pradhan, 2001; Nile and Khobragade, 2010; Pant et al., 2012). Little or no plumbagin was present in leaves and stem, whereas naphthoquinones, sitosterol, lupeol, lupenylacetate, hentriacontane, and amino acids were reported from the aerial parts (Chen et al., 2011; Kumar et al., 2009). Zhang et al. (2007) isolated aspartic acid, tryptophan, tyrosine, threonine, alanine, histidine, glycine, methionine and hydroxyproline from the aerial parts of P. zeylanica. The earliest documented evidence of the use of P. zeylanica in Ayurveda, Unani, Siddha and other traditional medicine systems has now become the basis of modern

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Introduction medicine and an important source for the future medicine and therapeutics. It is evident from the literature survey that roots of P. zeylanica are a rich source of plumbagin which is high-value medicinal plant. The demand for the roots of P. zeylanica is increasing for plant-based medicines, health products, pharmaceuticals, cosmetics etc. in the national and international markets, unfortunately creating a heavy pressure on the natural plant populations in the wild due to over-harvesting the plant for roots. Also, uprooting of the plant has made the population vulnerable and in near future will soon be threatened. Besides this, the chemical synthesis of plumbagin is not economically feasible because of its compound structure, specific stereo-chemical requirement of the compound and high cost of chemicals (Ahmad et al., 2015). There is a need to pay attention on the conservation and cultivation of such medicinal plants used by the pharma-companies and industries considered to be endangered or threatened. Thus, there is a need to develop an alternative to the intact plant for production of plumbagin in order to make available plumbagin for the pharmaceutical industries without disturbing and depleting natural plant resources. 5. Strategies for increasing the secondary metabolites production – Plants produce secondary metabolites in very low quantities (less than 1% dry weight) and the secondary metabolite production is greatly influenced by the edapho- climatic parameters, physiological and developmental stage of the plant. Plant tissue culture is potentially valuable in order to study the biosynthesis of secondary metabolites and can ultimately provide an efficient way of producing commercially important and valuable plant metabolites. In the past two decades, plant biotechnology has grown as a promising area within the field of biotechnology which focuses mainly on the plant secondary metabolite production. The biotechnological production of secondary metabolites in plant cell and organ cultures is a reliable alternative to the extraction of the whole plant material (Ahmed and Kim, 2010). Many biotechnological strategies have been postulated and investigated for enhanced secondary metabolite production like screening of high yielding cell lines, media modification, precursor feeding, elicitation, large scale cultivation in bioreactor system, hairy-root culture, plant cell immobilization, biotransformation and many more (Namdeo, 2007).

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Introduction

An attractive alternative approach to the traditional methods of plantations is tissue culture which offers an organized supply of secondary metabolites with reliable product quality independent of the plant availability. These in vitro propagated medicinal plants also provide uniform, sterile and compatible plant material for biochemical characterization (Ahmad et al., 2015) and have many advantages in producing secondary metabolites in plant cell and organ culture as compared to the in vivo whole plant. The production of important metabolites can be generated continuously without any seasonal constraint. Plant tissue culturing comprises two types of culture systems: organized, viz. shoot culture and unorganized (cell suspension) culture systems. Both the culture systems have advantages and limitations to their credit. Organized culture system is genetically stable but is however slow growing, whereas unorganized culture system is fast growing but genetically unstable (Muffler et al 2011). Many medicinally important plants have been successfully propagated using both these culture systems (Vanisree et al., 2004; Karuppusamy, 2009; Ahsan et al., 2013; Sharma et al., 2010; Washimkar and Shende, 2016). In plants secondary metabolites are synthesized in response to pathogen attack, or when the plant cells detect a change in environmental condition such as stress or pathogen invasion. Plants react to it by producing certain biological responses through specific signal transduction. Elicitors are compounds from biotic or abiotic sources which when applied to the plant culture in small concentrations can trigger physiological and morphological responses and lead to metabolite accumulation. Based on „nature‟ elicitors are classified as biotic and abiotic elicitors, whereas based on „origin‟ elicitors are classified as exogenous and endogenous elicitors (Silja et al., 2014). Biotic elicitors are of biological origin such as polysaccharides from plant cell walls (e.g. yeast extract, cellulose, chitin and pectin) and micro-organisms. Abiotic elicitors are of non-biological origin which comprises physical (UV radiation, temperature, salinity stress), chemical (ethylene, different fungicides, antibiotics, heavy metals, high salt concentrations), and hormonal factors (jasmonic acid, salicylic acid and their derivatives, gibberellic acid) (Namdeo, 2007). Although plant tissue culture techniques are well-established, secondary

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Introduction metabolite production could yet be enhanced using elicitation strategies in plant cell and organ cultures. There lies a huge scope for large scale production of secondary metabolites using elicitors as an agent. 6. Genetic diversity study – India is one of the 12 mega-biodiversity countries in the world having diverse physical features and climatic conditions. P. zeylanica interestingly has adapted to these different types of environmental conditions and has been reported in many parts of the country. Studies have proved that same plant species growing in different ecological or environmental conditions show significant differences in the accumulation and production of primary and secondary metabolites. Long term acclimatization or local adaptation, seasonal alterations related to phenology, environmental changes in the biotic and abiotic factors, geographical differences involving different populations (genetic differences within a plant species), different environmental conditions of the individual species‟ growth location, especially when they have genetic homogeneity can be the main reasons for the variation in the levels of metabolites for individual plant species (Sampaio et al., 2016). Studies carried out in Artemisisa aucheri revealed that A. aucheri got adapted to the changes in altitude, soil type and texture, moisture level, temperature and radiation which occurred by the change in its antioxidant enzymes activity and production of secondary metabolites (Khajehzadeh et al., 2014). Due to the wide- distribution of P. zeylanica and great adaptive response capability to different environmental conditions and the ability to produce secondary metabolites, molecular approach to study the genetic diversity using molecular markers can be used to understand the relation between the environmental data and their response with respect to the secondary metabolite production. Genetic diversity assessment within and between plant populations can be studied using various markers such as morphological markers which are based on visually accessible traits such as flower colour, seed shape, growth habits and pigmentation; biochemical markers (allelic variants of enzymes called „isozymes‟ which are detected by electrophoresis and specific staining) and DNA– or molecular markers which are widely used. Molecular markers include a huge variety of markers which can be employed for genetic and molecular variation analysis. Molecular markers are broadly divided into three classes based on the method of their detection,

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Introduction viz., hybridization based (example, Restriction fragment length polymorphisms – RFLPs), polymerase chain reaction, PCR-based (example, Rapid amplified polymorphic DNAs – RAPDs), Amplified fragment length polymorphisms – AFLPs), and DNA sequence-based (example, Simple Sequence Repeat or Microsatellite – SSR, sequence- tagged site – STS, sequence-characterized amplified regions – SCAR, expressed sequence-tag based SSRs – EST-SSR, and Single Nucleotide Polymorphisms – SNPs) depends on the availability of short oligonucleotide repeats sequences in the genome of plants (Govindaraj et al., 2015). Genetic variability using the random amplified polymorphic DNA (RAPD) markers was studied in Cassia tora from different agro- climatic zones of Madhya Pradesh (Tilwari et al., 2016).

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