REVIEW OF LITERATURE Chapter 2 Review of Literature

“Study the past, if you would define the future.” -Confucius

The recent vogue on research has 2.1. Brief history of enlightened all over the world and a Mimosoids form a major group within plethora of evidences have been collected legumes. According to Cronquist (1981), to confirm the immense potentiality of Mimosoids have been usually recognized medicinal used in traditional either as the family Mimosaceae or as the healthcare systems. Several medicinal subfamily Mimosoideae of the family plants have been identified and studied (Leguminosae) under the order using modern scientific approaches for . Mimosoideae consist of about 80 their phytochemistry. Selected members of genera and 3,370 species of trees, shrubs, Mimosoideae have already been remarked and lianas found mainly in tropical, as valuable medicinal plants, which are subtropical, and warm temperate regions rigorously used in Ayurvedha and Unani of the world (Luckow et al., 2003). The systems of medicine. Previous members of Mimosoideae are usually phytochemical reports on some species of characterized by their bipinnately Mimosaceae have already revealed their compound, alternate leaves; spicate or probable role in medical care. capitate inflorescence; bisexual flowers, This chapter, besides representing a rarely unisexual, actinomorphic, in tight general overview, considers selected clusters with numerous stamens and members of Mimosoideae from the legume fruits. Besides, most of the species perspectives of Ethnobotany, under Mimosaceae exhibit an Parmacological activities, Antioxidant associationship with nitrogen fixing activities, Neuroprotective activity, bacteria. Phytochemistry, focusing also on 2.2. Ethnomedicinal studies Molecular documentation of selected taxa as well as on rhizobial diversity. ‘Ethnomedicine’ may be defined as the sum of knowledge of plants, skills and

REVIEW OF LITERATURE 10 practices based on the oral theories, beliefs, survey in Caprivi region of Namibia and experiences curing diseases and (Chinsembu and Hedimbi, 2010) reflected disorders by native people belonging to the beneficial effects 6 Mimosoids treating different culture (Tamuli and Saikia, 2004). different HIV/AIDS-related opportunistic Several plants are being used to treat infections. Saini et al. (2008) mentioned different ailments since time immemorial. the ethnomedicinal value of five acacias Utilization of different plant species of (Acacia nilotica, A. tortilis, A. senegal, A. different families like, Asteraceae, catechu and A. jacquemontii) in Rajasthan Poaceae, Malvaceae, Mimosaceae etc. of India which were regularly used to treat were mentioned in Indian traditional and asthma, toothache, stomach complaint, skin ayurvedic medical system to alleviate infections, caugh, leprosy, indigestion and dysentery, inflammation, burning diarrhea. Simultaneously, selected sensation, asthma, leucoderma, leprosy, members of Mimosaceae are also used by cholera, vaginal and uterine complaints, the indigenous people of Bargarh district of bile, bilious fevers, piles, jaundice, leprosy, Orissa, India for their local therapeutic bronchitis, cold and cough, fatigue, blood purposes (Sen and Behera, 2008). In diseases etc (Kirtikar and Basu, 2006). continuation, Saha et al. (2014a) described Selected members of Mimosaceae, the use of M. pudica (root and leaf especially, pudica, M. hamata, decoction) as a remedy of leucorrhea and Samanea saman, Prosopis cineraria, breast cancer whereas A. nilotica was Parkia biglandulosa, Albizia procera, found to have anti-diabetic property (Saha Acacia senegal, A. chundra etc. were et al., 2014c) in Malda district of West reported to be used as ethnomedicine by Bengal, India. local people of the Idar-Vadali forest area 2.3. Medicinal properties of selected of Sabarkantha district of India to treat Mimosoids rheumatoid arthritis, fever, headache, piles, fistula, swellings, diarrhea, diabetes, Plants have been exploited for the cataract, hydrocele erysipelas, ulcer, management of diseases for centuries leucorrhoea and also used as anti-venom because of their very limited adverse agent in case of scorpion sting (Patel and effects. Therefore, their scientific Jangid, 2013). An ethnomedicinal report evaluation is a logical way of searching from Araku valley Mandalam, new drugs. Moreover, 80% of world Visakhapatnam district, India claimed the population depends entirely on herbal effectiveness of Mimosaceae in local medicines prepared almost exclusively healthcare management (Padal and Sathya from plants. Numerous indigenous vathi, 2013). Besides, ethnomedicinal medicinal plants have been found to be

REVIEW OF LITERATURE 11 successfully used to control different (Solomon-Wisdom and Shittu, 2010). ailments, which can counter the high cost Antibacterial activity of A. concinna bark and poor availability of the current was also observed against P. aerogenosa synthetic drugs for many rural populations and S. aureus (Vergeese and Sivaraj, in developing countries like India. 2012). Furthermore, the antimicrobial Consequently, different medicinal activity of the A. nilotica against several members of Mimosoideae have already bacterial strains was also examined by been evaluated for their various Khan et al. (2009) while ethyl acetate pharmacological activities followed by extract of Albizzia lebbeck leaves isolation of active principles. In this confirmed successive antibacterial activity section, an overview of different against gram positive and gram negative pharmacological activities of selected bacteria (Rahul et al., 2010). Antibacterial Mimosoids is summarized below: activity and antifungal activity of Samanea saman leaves were also noticed against 2.3.1. Antimicrobial activity Pseudomonas aeruginosa, Fusarium Antimicrobials are such kind of substances solani and Trichophyton longifusus (Azhar that kill microorganisms or inhibit their et al., 2009). growth. Numerous plants are being used as 2.3.2. Anti-inflammatory activity antimicrobial agent since the beginning of human civilization. Likewise, plenty of Inflammation may be regarded as a plants under Mimosaceae were studied for complex pathophysiologic process and can their antimicrobial activity. Genest et al. be initiated in response to injury involving (2008) reported a comparative account of the accumulation of cells and exudates in antibacterial and antioxidant activities of the affected tissues (Markiewski and dichloromethane (DCM) and methanolic Lambris, 2007). Several plants were being extracts of stems of Mimosa rubicaulis and applied therapeutically for many years M. pudica exhibiting considerable leading to the production of major anti- encouraging activity against Bacillus inflammatory drugs. Therefore, natural subtilis, E. coli, Staphylococcus aureus products with anti-inflammatory activity and Pseudomonas aeruginosa. In addition, are a chief concern in the present potent antibacterial activity of M. pudica circumstance. Studies with M. pudica was also tested by Balakrishnan et al. leaves revealed significant (p<0.05) (2006). The ethanolic leaf extract of inhibitory activity in a dose-dependent Acacia nilotica effectively showed highest manner than the standard drugs, zone of inhibition at 70mg/ml extract indomethacin when it was tested on concentration against the bacteria carrageenan-induced paw oedema and Campylobacter coli, isolated from goats cotton pellet granuloma in rats (Goli et al.,

REVIEW OF LITERATURE 12

2011). In addition, similar type of healing activity of ointment containing experiment with ethanolic leaf extract of methanolic and aqueous extract of M. M. pudica was executed by Mistry et al. pudica root at 2% level in rat models. (2012) and the result was found to be Concurrently, similar type of result was significant against acute and chronic also found in bark extract of Albizia inflammation. Catechin, isolated from lebbeck (Gupta and Jain, 2010). Acacia catechu revealed the inhibition of 2.3.4. Antinociceptive activity Cyclooxygenase (COX) and 5- Pain, a subjective symptom, is affected by Lipoxygenase (LOX) enzyme activity psychological factors which could be showing its anti-inflammatory potentiality alleviated by different chemical agents by (Altavilla et al., 2009). Further, the means of central or peripheral butanolic fraction of Acacia pennata dried mechanisms. Different plant species have leaves exhibited significant protective been considered as natural pain-relievers effects against chemical stimuli (acetic in search of new potent drugs. The acid and formalin) as well as an inhibitory methanolic extract of A. catechu was effect in carrageenin-induced rat paw proved as a relieving agent from pain oedema in the late phase (Dongmo et al., when tested in acetic acid-induced gastric 2005). pain mice models (Rahmatullah et al., 2.3.3. Wound healing activity 2013). The aqueous extract of Mimosa Wound healing is an immune-mediated pudica exhibited significant (p<0.001) obscure process where the skin or other inhibition of writhing response in acetic soft tissue repairs itself after injury. The acid-induced animal model which might healing process begins with the clotting of be due to the inhibition or reduction of bloods as well as a set of biochemical proinflammatory mediators (Karthikeyan actions takes place to repair the damage and Deepa, 2010). On the other hand, (You and Han, 2014). Numerous plants analgesic activity of a Brazilian native were explored in the management and plant, Abarema cochliacarpos was treatment of wounds over the years as they evaluated in mice model by Silva et al. promote the repair mechanisms in natural (2009) exhibiting higher activity of bark way. The methanolic extract of aerial parts extracts than that of standard drug used. and roots of M. pudica was found to be a 2.3.5. Antidiabetic activity potent candidate revealing greater wound Diabetes, a metabolic disorder critically healing activity in Wistar Albino rats affecting the population, is rapidly compared to the standard drug Gentamicin emerging as a major public health (Kannan et al., 2009). In addition, Kokane challenge in developing countries. et al. (2009) reported noteworthy wound

REVIEW OF LITERATURE 13

Although, several drugs and interventions 2.3.6. Diuretic activity are available to manage diabetes, in most Diuretics are the drugs which increase the of cases these are either expensive or show rate of urine flow, sodium excretion and adverse effects like hypoglycemia. are used to adjust the volume and Therefore, there always remains a need to composition of body fluids. Traditionally, find an effective and safety drug for the several plants were claimed to be treatment of diabetes. Despite of having possessed diuretic properties, but lack decreased or nil adverse effects, natural proper clinical validation. However, a few products play a vital role in this regard. reports are available regarding the diuretic For example, the ethanolic leaf extract of activity of members of Mimosoideae. M. pudica revealed significant decrease of Sangma et al. (2010) revealed that the blood glucose level in alloxan induced aqueous extract of M. pudica leaves was diabetic Wistar rats as compared to significant (at 100mg/kg) showing diuretic standard, Metformin whereas pet ether activity in normally fed albino rats with extract did not show any significant increased electrolyte excretion (p<0.01 for decrease in serum glucose level up to 7 urine output, p<0.01 for Cl, p<0.05 for K+, days of treatment (Sutar et al., 2009). and p<0.01 for Na+). The methanolic Likewise, the methanolic extract of the extract of Albizia lebbeck was pods and tender leaves of Acacia nilotica administered to experimental rats at a dose was found to be very beneficial to treat of 200 and 400mg/kg for diuretic activity diabetes (Gilani et al., 1999). Besides, and obtained significant increase in the significant reduction of blood glucose volume of urine and urinary concentration level was observed in diabetic albino rats of Na+, K+ (Sivakumar et al., 2013). at the doses of 250 and 500mg/kg body 2.3.7. Antidiarrheal activity weight (p<0.001) by the application of ethyl acetate extract of Acacia catechu Diarrhea is a frequent digestive chaos (Ray et al., 2006). The bark extract of caused by enterotoxins of several bacteria. Albizia odoratissima also significantly (p< Indigenous systems of Indian therapeutic 0.01) reduced the levels of serum exhibited several numbers of medicinal cholesterol, triglycerides, serum glutamic- plants to be used as antidiarrheal activity. oxaloacetic transaminase, serum glutamic- For instance, the crude ethyl acetate pyruvic transaminase, alkaline phosphatase extract of A. catechu at a dose of 250mg/ as well as the extract decreased the level of kg was found to be highly significant total proteins in alloxan induced diabetic (p<0.001) against diarrhea in albino rats in mice suggesting their potent role as respect of latent period of onset of diarrhea antidiabetic agent (Kumar et al., 2011). (Ray et al., 2006) whereas the ethanolic

REVIEW OF LITERATURE 14 leaf extract of M. pudica inhibited castor young seedless pods of A. nilotica (Bansal oil induced diarrhea and PGE2 induced and Goel, 2012). enteropooling in Wistar albino rats and 2.3.9. Hypolipidemic activity found 200 and 400mg/kg was significant Hypolipidemic drugs are the substances (p<0.001) dose (Khalid et al., 2011). that reduce the level of lipids and Besides, the bark powder of A. nilotica lipoproteins in the blood. The plant species was found to be a potent inhibitor when namely, M. pudica exhibited potent castor oil and magnesium sulphate induced hypolipidemic activity (p<0.05) against diarrhea in Swiss albino mice was studied atherogenic diet in wistar albino rats by by Misar et al. (2006). Similar type of lowering the serum levels (Rajendran and result was also reported by Besra et al. Krishnakumar, 2010). (2002), when they tested the antidiarrhoeal activity of Albizzia lebbeck seeds 2.3.10. Hepatoprotective activity employing conventional rodent models. Hepatoprotection is such type of capability 2.3.8. Antiulcer activity to prevent the damage of liver. Natural products have been used traditionally for Ulcers are the lesions on the surface of the prevention and treatment of liver skin or a mucous membrane caused by disease. Scientific research on superficial loss of tissue. Ulcer healing is a hepatoprotective activity has been complex process involving the supported the claims of the medicinal combination of wound retraction and re- efficacy of several herbal compounds. epithelialization. Several medicinal plants Ethanolic (50%) extract of M. pudica within Mimosoideae were mentioned in exhibited significant hepatoprotection ayurvedic medicine for their antiulcer when the extract was examined on CCl - properties over the years. For example, 4 induced liver damage in Wistar albino rats Vinothapooshan and Sundar (2010) (Kumar and Kumar, 2010). Additionally, reported the antiulcer activity of different methanolic leaf extract of M. pudica was extracts of M. pudica in aspirin, alcohol also found to be hepatoprotective (p<0.05) and pyloric ligation induced models of by means of lowering of serum levels gastric ulcer in albino rats revealing ulcer (Sohil and Sundaram, 2009). damage suppression capability of all Subsequently, ethanolic extract of Acacia extracts (p<0.001). The aqueous extract of concinna pods exhibited significant A. catechu heartwood significantly hepatoprotective effect in CC1 induced inhibited the formation of ulcers in the 4 liver damage rat model (Maqdoom, 2016). pylorus ligated rat models (Patankar et al., Further, significant (p<0.001) 2011). Similar type of activity was also hepatoprotective activity was observed at a noticed in hydroethanolic extracts of

REVIEW OF LITERATURE 15 dose of 250 mg/kg p.o. of ethyl acetate swim endurance test and acetic acid extract of Acacia catechu on albino rats induced writhing test model (Chaudhary et after the administration of seven days (Ray al., 2012). Further, significant et al., 2006). immunological adjuvant activities of saponin extracts from the pods of A. 2.3.11. Immunomodulatory activity concinna was examined by An immunomodulatory agent is a drug or Kukhetpitakwong et al. (2006). inhibitor that may be used as an 2.4. Antioxidant activities of selected immunosuppressant or an immunostimulator based on its effect on Mimosoids the immune system. The logical design of Reactive Oxygen Species (ROS) or free novel drugs from traditional formulation radicals such as hydroxyl radical, singlet offers new prospects in modern healthcare oxygen, superoxide anion and hydrogen management. Ayurveda, one of the oldest peroxide play a chief role in the traditional medical systems in India, development of various diseases including reveals certain plants which strengthen the atherosclerosis, heart disease, ageing, host immune system. For instance, the hot immunosuppression, and others (Young water extract and butanol fraction of and Woodside, 2001). The free radicals in Albizzia lebbeck bark successfully proved human body are produced by means of immunostimulatory effect against aerobic respiration or from exogenous macrophage migration model as well as sources and react with various biological cell mediated arms of the murine immune molecules namely proteins, lipids and system (Barua et al., 2000). The aqueous deoxyribonucleic acids resulting in the extract of Acacia catechu stimulated the imbalance between oxidants and murine neutrophil adhesion and the antioxidants. Moreover, oxidative stress or phagocytic index. Additionally, the extract excessive oxidation of cellular substrates was found to be helpful in protection result in the formation of type II diabetes, against cyclophosphamide induced neurodegenerative diseases, or even some neutropenia in murine system which was types of cancer. The most effective evident through its immunoglobulin pathway to reduce the action of free production (Ismail and Asad, 2009). The radicals causing oxidative stress is ethanolic extract of Albizia lebbeck leaves antioxidative defense mechanism. have been reported to be exhibit strong Antioxidants may be defined as the immunomodulatory effect by increasing compounds inhibiting or delaying the the swimming or survival time (P<0.001) oxidation of other molecules by means of and also decreased the writhing produced reducing the initiation or propagation of by glacial acetic acid (P<.001) employing

REVIEW OF LITERATURE 16 oxidizing chain reactions. (Muthukumaran et al., 2011). In addition, reduced lipid peroxidation and superoxide Medicinal plants play a key role in treating dismutase (SOD) level was observed when various human ailments due to presence of leaf extract of M. pudica was tested in rat certain components of therapeutic value. model (Muthukumaran et al., 2010; The study done so far on medicinal plants Nazeema and Brindha, 2009). Kalaivani and vegetables strongly supports the idea and Mathew (2010) studied the antioxidant that plant constituents having antioxidant and free radical scavenging activities of A. activity are capable of exerting protective nilotica methanolic leaf extract, effects against oxidative stress in demonstrating greater potentiality of the biological systems (Cao et al., 1996). In extract to scavenge DPPH, hydroxyl fact, plant based drugs are an important radical, prevent lipid peroxidation and to resource of therapeutic agents with easier possess superior reducing power. Crude availability, relatively cheaper cost and 70% acetone and 50% ethanolic extracts non-toxic nature as compared to modern from the leaf and bark of Acacia nilotica medicine. Various methods are used to were tested for DPPH activity and found investigate the antioxidant property of that acetone leaf and bark extract was plant samples. In this section, merely more effective than the ethanolic extract compiled descriptions of selected (Gowri et al., 2011). Moreover, a Mimosoids are summarized. comparative evaluation of the antioxidant Zhang et al. (2011) demonstrated the activities of 3 acacia species (A. nilotica, antioxidant activities of the methanol A. seyal and A. laeta) demonstrated extracts of M. pudica through DPPH and superior bioactivities of A. nilotica (Abdel- FRAP assays as well as quantified the total Farid et al., 2014). The bark extract of phenolic and flavonoid contents. The Acacia catechu exhibited significant results demonstrated potent antioxidant inhibition of DPPH, H2O2 and reducing activities in the sequence of leaf> whole power activity in-vitro-cally followed by plant> seed> stem. Among the five significant increase level of superoxide flavonoids isolated, 5,7,3´,4´-tetrahydroxy- dismutase, catalase, glutathione-S- 6-C-[β-D-apiose-(1→4)]-β-D- transferase and reduced glutathione at the glycopyranosyl flavone revealed trolox dose of 100 and 200mg/kg bwt (Alam et equivalent antioxidant capacities. In a al., 2013). Antioxidant activity of A. further experiment, ethanolic leaf extract concinna pods was determined by three of M. pudica exhibited moderate hydrogen different assays DPPH, ABTS-radical peroxide and nitric oxide scavenging scavenging assay and linoleic acid activity with IC value of 449.60±2.55μg/ 50 peroxidation assay which reflected its ml and 78.1±1.75µg/ml respectively

REVIEW OF LITERATURE 17 potent inhibitory activity with lower IC50 factors for several pharmacological value (Poomanee et al., 2015). activities. A glimpse of phytochemicals Subsequently, bark extract of Albizia identified so far from the selected lebbeck was also proven as potent free members of Mimosoideae is enlisted in radical scavenger (Suruse et al., 2013; this section. Vasanthi et al., 2014). In addition, M. pudica, one of the most well studied antioxidant activity of three enzymes members of Mimosaceae, was accounted including superoxide dismutase (SOD), to have several bioactive components like catalase (CAT), glutathione peroxidase flavonoids, phenols, alkaloids, terpenoids, (GPX) and glutathione S transferase (GST) glycosides, quinines, tannins, saponins and were assessed in diabetic rat model with coumarin (Gandhiraja et al., 2009). A few decline level of damage after the other phytochemicals including mimosine, administration of A. lebbeck (Resmi et al., tyrosine 3,4-dihydroxypiridine, 2006). mimosinamine, mimosinic acid were 2.5. Phytochemistry reported in M. pudica by Johnson et al. (2014). Further, five compounds namely Plants are the remarkable sources of 5,7,3′,4′-tetrahydroxyl-6-C- β-D- chemical compounds having therapeutic glucopyranosyl flavone, 5,7,3′,4′- effect on the body, referred to as tetrahydroxyl-8-C- β-D-glucopyranosyl phytochemicals. Virtually, these flavone, succinic acid, β-sitosterol and phytochemicals in plants perform a variety stigmasterol were isolated and identified of physiological functions including from the same plant i.e. M. pudica (Yuan defense mechanism against pathogens like et al., 2006). The active metabolites such bacteria, fungi as well as insects. Every as gallic acid, protocatechuic acid, plant synthesizes different types of pyrocatechol, (+)- catechin, (-) phytochemicals which play an important epigallocatechin-7-gallate, (-) role in pharmaceutical, food and chemical epigallocatechin-5,7- digallate, (-) industries. In addition, these bioactive epicatechin, (+) dicatechin, quercetin, (+) phytocompounds serve as templates for leucocyanidin gallate, sucrose and (+) preparing several synthetic drugs. The catechin-5-gallate was reported to be phytochemicals have been divided into found in bark extracts of A. nilotica several groups such as, alkaloids, (Anonymous, 2001; Malviya et al., 2011; flavonoids, phenols, saponins, coumarins, Singh et al., 2009) followed by anthocyanins, essential oils etc. A plethora protocatechuic acid, ellagic acid, of evidences revealed that these different leucocyanidin, m-digallic dimer 3,4,5,7- types of phytochemicals are the main tetrahydroxy flavan-3-ol, oligomer 3,4,7-

REVIEW OF LITERATURE 18 trihydroxy flavan 3,4-diol, 3,4,5,7- A. chinensis leaves and their structures tetrahydroxy flavan-3-ol and (-) were explained as quercetin 3'-O-beta-D- epicatechol from gum of A. nilotica glucopyranosyl-3-O-rutinoside, (Malviya et al., 2011; Singh et al., 2009). kaempferol 3,7-di-O-beta-D- Another important Mimosoid namely, A. glucopyranoside, rutin, D-pinitol, luteolin catechu was reported to possesses 7-O-beta-D-glucopyranoside, (+)- epicatechin, epigallocatechin, epicatechin lyoniresinol 3alpha-O-beta-D- gallate, phloroglucin, protocatechuic acid, glucopyranoside, (-)-lyoniresinol 3-alpha- quercetin, poriferasterol glucosides, O-beta-D-glucopyranoside, syringin (Liu lupenone, procyanidin, L-arabinose, D- et al., 2009). Lupeol, epilupeol, lupenone, galactose, D-rhamnose, aldobiuronic acid, α-spinasterol and α-spinasterone were and taxifolin (Jain et al., 2007; Sharma et found to be present in Samanea saman al., 1997). Further, 12 compounds namely (Azhar et al., 2009; Ragasa et al., 2014). 4-hydroxybenzoic acid, kaempferol, A few main active phytoconstituents of quercetin, 3,4',7-trihydroxyl-3', 5- some members of Mimosaceae are dimethoxyflavone, catechin, epicatechin, summarized in Fig. 2.1. afzelechin, epiafzelechin, mesquitol, 2.6. Molecular diversity of different ophioglonin, aromadendrin, and phenol were isolated from A. catechu by Li et al. Mimosoids and Rhizobium (2010) whereas A. concinna was found to 2.6.1. Mimosoideae be own tartaric acid, oxalic acid, succinic acid, calycotomine, nicotine and rutin Genetic marker stands for genetic (Gupta and Nigam, 1970). The differences between individual organisms phytoconstituents reported in Albizzia or species that are located in close lebbeck bark are melacacidin, friedelin, D- proximity to genes. All genetic markers catechin, β-sitosterol, albiziahexoside, hold specific genomic locations within betulnic acid and echinocystic acid while chromosomes known as loci. Three types the leaves contains albigenic, albigenin, of genetic markers have been discovered kaempferol, quercetin; albizziahexoside, so far such as, morphological markers, tannins, proteins, carbohydrates, amino biochemical markers and DNA markers. acids and saponins. In addition the pods The morphological markers are typically contains 3’, 5 Dihydroxy 4’, 7 dimethoxy characterized by phenotypic characters flavone, and N- Benzoyl L phenyl alaninol including flower color, seed shape, growth (Rahul et al., 2010; Verma et al., 2013). habits or pigmentation (Sumarani et al., Further, eight bioactive compounds were 2004). Biochemical markers are the isolated from the 95% ethanolic extract of differences in enzymes that are

REVIEW OF LITERATURE 19

Mimosine Mimosinamine β-sitosterol

Stigmasterol Gallic acid (+)- catechin

Quercetin Protocatechuic acid Ellagic acid Fig. 2.1. Selected phytocompounds present in different Mimosoids. distinguished by electrophoresis and plant. Baring the use of DNA markers in specific staining (Pillai and Lekha, 2008). the construction of linkage maps, they The DNA markers are the most might be applied in plant breeding such as predominant markers arise from different assessing the level of genetic diversity classes of DNA mutations such as within germplasm and cultivar identity substitutions (point mutations), (Jahufer et al., 2002; Winter and Kahl, rearrangements (insertions or deletions) or 1995). errors in replication of tandemly repeated In essence, DNA markers might be DNA (Paterson, 1996). Besides, DNA broadly divided into three categories based markers are abundant in number and are on the method of detection: hybridization- not influenced by any environmental based; PCR based and DNA sequence factors and developmental stages of the based (Winter and Kahl, 1995). In

REVIEW OF LITERATURE 20 addition, DNA markers may expose on the specificity of restriction enzymes, genetic differences between individuals of which are extremely sequence-specific and the same or different species which can be cut the DNA only at their recognition sites visualized by means of gel electrophoresis (Heun et al., 1991). In addition, RFLP is and staining with ethidium bromide or virtually used in the identification of genes silver nitrate or detection with radioactive for genetic disorders, genome mapping, or colorimetric probes. A plethora of determination of risk of disease, and evidences suggested that DNA markers paternity testing. RFLP markers are also play a vital role in enhancing crop frequently used to distinguish between two improvement and global food production organisms or species. The DNA from by improving the efficiency of different ecotypes, various geographical conventional plant breeding programs isolates, and different inbred lines of a (Ortiz, 1998). species contain many RFLP.

DNA markers or DNA fingerprinting However, these techniques are restricted techniques can be employed to resolve only for genetic diversity, analysis of how closely related populations may be, as species, cultivars and varieties but not in well as to classify the individuals plant identification. Recently, a new providing better resolution of genetic modified molecular technique i.e. DNA relationships. Of the various DNA barcoding was developed offering a new fingerprinting techniques developed for dimension in the scientific community plant research, random amplified (Hebert and Gregory, 2005). In fact, DNA polymorphic DNA (RAPD), a PCR-based barcoding is a novel, modern and molecular technique, has become innovative technique which can be used to increasingly popular which are being used explore the evolution, identification and to evaluate the genetic relationship among genetic relatedness of unknown plants and species, cultivars and varieties (Williams animal species by using a short stretch of et al., 1990). A major advantage of RAPD DNA sequence. Recently, chloroplast and markers is that it requires no prior mitochondrial genes are being used to sequence information and knowledge study the sequence variation at generic and about any particular gene in a target taxon species level. The chloroplast genes such (Palumbi, 1996). Restriction fragment as matK and TrnL-F have been utilized by length polymorphism (RFLP) is another various workers to study the plant type of molecular technique in molecular evolutionary pattern as well as to solve the biology to differentiate minor nucleotide various anomalies in the taxonomic levels. sequence variations in homologous The matK gene exhibits three times high fragments of DNA. This technique relies rate of nucleotide substitution than that of

REVIEW OF LITERATURE 21 the large subunit of Rubisco (rbcL) and species of Acacia were obtained by Nanda found to be six fold higher at the amino et al. (2004) employing 253 of distinct acid substitution rate (Johnson and Soltis, bands through RAPD. The result reflected 1994; Olmstead and Palmer, 1994) that A. farnesiana and A. catechu were the elucidating advanced phylogenetic signal closest member sharing about 30% of for resolving evolutionary relationships similarity whereas A. auriculiformis shares among the plant species at all taxonomic about 28% similarity with A. farnesiana levels. and A. catechu. In addition, A. mollissima shares about 18% of similarity with A. Therefore, despite of rapid development arabica. Further, the genetic variability of and widespread application of molecular nine Acacia nilotica subspecies of various techniques, very little is known regarding origins was also analyzed using RAPD by genetic variability within and among the Ndoye-Ndir et al. (2008) exhibiting large taxa of Mimosoideae at the DNA level. differences between subspecies but no Subsequently, there has always been a correlation between geographic distances debate among the taxonomists regarding and genetic distances was established. the taxonomic position of several Genetic differentiation among the six populations within the order Fabales varieties of A. caven were examined by owing to similar types of morphological means of RAPD supporting similar kind of characters. Since, the morphological trend as found in taxonomic differentiation variation between species is difficult to (Pometti et al., 2010). Subsequently, distinguish; an appropriate knowledge of several RAPD and ISSR markers were molecular documentation would help to employed to study the genetic diversity in understand the genetic relationship among Kenyan populations of Acacia senegal the different genera in Mimosoideae. (Josiah et al., 2008). Besides, phylogenetic The genetic relationships among the three analysis chloroplast DNA of 22 species of species of Mimosa namely, M. pudica, M. Acacia was carried out using RFLP pigra and M. invisa were analyzed using indicating that A. nilotica and A. 30 RAPD primers. Out of which eleven farnesiana are sister species, while A. primers revealed 83% of polymorphism in nilotica is Afro-Asiatic and A. farnesiana all three species with a total of 92 bands. is America in origin (Bukhari et al., 1999). The polymorphism percentage at A few RAPD and ISSR markers were interspecific level varied from 25% to further utilized to analyze 172 individuals 100% as well as M. pudica was found to representing eight populations of Albizia be closely related to M. pigra and M. lebbeck in different geographical range invisa (Sulain et al., 2013). High degree of and the genetic diversity was found to be diversity (∼70%) within the six tree

REVIEW OF LITERATURE 22 ranged from 1.23 to 1.38 while the total heterogeneous group of gram negative, gene variability was 0.34 (Aparajita and aerobic, heterotrophic, non-spore forming Rout, 2009). During the DNA barcode microbe (Hirsch et al., 1993). It contains analysis of Acacia, Robinson and Harris, granules of poly-β-hydroxybutyrate which (2000) documented that the tribe Acacieae are refractile by phase contrast microscopy and genus Acacia are not only and produces an acidic reaction in mineral- monophyletic but also the subgenera salts medium containing mannitol or other Acacia and Aculeiferum are sister taxa and carbohydrates. Rhizobium includes the neither of them appeared closely related to largest number of species into the family. subgenus Phyllodineae. Barcoding However, the original genus Rhizobium analysis of acacias from three different has undergone several subsequent changes continents showed that all of three cpDNA in recent years giving rise to many other regions (rbcL, matK and trnH-psbA) taxa. distinguished and supported the newly In Bergey’s Manual of Systematic proposed genera of from Acacia Bacteriology, Rhizobium was included as well as discriminated sister species along with other three genera namely, within either genera and differentiated Bradyrhizobium, Agrobacterium, and biogeographical patterns among Phyllobacterium within the family populations from India, Africa and Rhizobiaceae and the separation of these Australia (Newmaster and Ragupathy, genera was predominantly based on the 2009). Nevill et al. (2013) demonstrated ability to stimulate the production of root the novel use of DNA barcoding for seed or leaf nodules in host plant species identification and demonstrated the (Jordan, 1984). The recent of practical potential of DNA barcoding for Rhizobiaceae, as well as of any other the growing discipline of restoration bacterial groups is mostly supported by the ecology of Acacia in the Midwest of phylogenetic analyses based on 16SrDNA Western Australia. sequences. A revision and dismemberment 2.6.2. Rhizobium and genetic diversity of the genus Rhizobium and its relatives of the class alphaproteobacteria was led by Rhizobium is a type genus of the family their phylogenetic studies. Rhizobiaceae of the order Rhizobiales in Characterization of the Rhizobium genome the class alphaproteobacteria. The genera at molecular level is the most Rhizobium consists of about 44 recognized discriminating method for assessing the species including some latest novel species variability among strains and isolates of like R. sphaerophysae, R. pusense, R. the bacteria (Demezas et al., 1991; Thies vallis and R. herbae (Qin et al., 2012). It et al., 2001). The workers used different can be characterized as rod shaped,

REVIEW OF LITERATURE 23 type of primers to obtain ‘PCR- and subjected to RAPD analysis revealing fingerprints’ to characterize rhizobial RAPD is a very discriminative and isolates at the strain level. The primers, efficient method for differentiating and designed to study the genetic diversity, are studying genetic diversity of Rhizobium RAPD, 16S ribosomal RNA genes, strains (Rajasundari et al., 2009). RAPD repetitive element sequences (REP, ERIC profiling was further found to be an and BOX), 16S–23S rRNA intergenic efficient discriminatory method when spacer regions or genes for nitrogen different root-nodule rhizobia were fixation and nodulation (De Bruijn, 1992; collected from chickpea and green pea Thies et al., 2001). roots for their genomic diversity (Qureshi et al., 2014). The genetic diversity of Amongst handful of work done so far, indigenous soybean rhizobia, isolated from Harrison et al. (1992) reported that the use different soil types in eastern Croatia, was of RAPD primers may produce varied studied by using different PCR amplification patterns from different fingerprinting methods such as 16S rDNA, Rhizobium isolates, especially directly PCR-RFLP, rep-PCR and RAPD analysis. from nodules providing this method as a Highly specific and reproducible patterns potential one for examining genetic were found that enabled accurate strain structures or strain differentiation as well differentiation (Sikora and Redzepovic, as relationships in Rhizobium populations. 2003). Apart from these, different Young and Cheng (1998) further molecular techniques such as, rep-PCR, suggested that RAPD technique is a ERIC-PCR, BOX-PCR and (GTG)5-PCR potential tool for the construction of were further utilized for genotyping of genetic maps and useful in identification of different bacterial strains of Rhizobium the genetics and systematics of different (Blazinkov et al., 2007; Menna et al., populations of rhizobia. Nine soil rhizobia 2009; Versalovic et al., 1991). were isolated from different field locations