1.0 INTRODUCTION

There is a global increase in utilizing drug from plant sources which projected to be sixty billion around the universe (Barnes et al., 2004). Medicines from plants are efficient in curing diseases. The traditional systems of medicine namely Ayurveda, Siddha and Unani are well established in India and are widely acknowledged to be effective and safe without any side effects (Farnsworth, 1998).

Recent years has seen the vast increase in medicinal plants because of its non chemical origin and exhibit remarkable efficacy in the treatment of various ailments (Siddhiqui et al., 1995). A vast number of plants are known to occupy various places in earth. Traditional medicine has been improved in developing countries as an alternative solution to health problems. There is a need for development of new drugs of plant origin against human diseases due to the resistance of pathogens against existing drugs (Govil, 2002).

The World Health Organization (WHO) has compiled a file of twenty thousand curative plants used around the globe. A large number of species have local uses within the country or spread over several countries in a region. The medicinal virtues of the raw materials including chemical contents and composition of the plants merited inclusion in national pharmacopoeias and official formularies in different countries (Amar jyothi Das, 2012).

Treatment of various diseases was carried out with the use of compounds obtained from medicinal plants (Akerele, 1988). The plants need more attention due to their important role in primary healthcare delivery system for improvement of people’s health (Hamayun, 2003). It is an essential component of human healthcare especially for the rural communities who solely rely on forest plants for food, shelter, energy and medicine. The medicinal values of plants lie in some chemical substances that produce a definite physiological action on human body (Chalchat, 1998).

Essential oils have potential application in medical procedures and in the cosmetic, food and pharmaceutical industries (Tongnuanchan and Benjakul, 2014). Essential oils are concentrated liquids of complex mixtures of bioactive compounds and can be extracted from several plant parts. Essential oil which contains several bioactive compounds was the good source (Thorpe, 2007). Inspite rapid fragmentation of natural habitats is greatly narrowing the distribution of the plant and increasing the risk of losing genetic diversity (George, 1984).

1.1 In vitro micropropagation

In vitro cell culture was developed by Gottlieb Haberlandt (1962) from various plant species like Laminum purpureum, Eicchornia crassiples and cultured in glucose enriched Knop’s salt solution. In vitro tissue culture is performed aseptically to produce plants that are similar to plants grown in environmental conditions. Plants are produced either from cells or from whole plant under in vitro conditions this technique is most effective in development of agricultural plant species (Thorpe, 2007). In vitro techniques have been successfully applied to solve the problems of conventional micropropagation in a large number of medicinal plants. In vitro propagation refers to true to type propagation of selected genotypes under laboratory conditions. New plants are generated by different explants such as single cells, protoplasts, pieces of leaves, roots and node on basal media with necessary nutrients. Conservation through vegetative propagation is time consuming however, tissue culture offers an alternative tool for rapid multiplication of disease free propagules in a short period (Muralidharan, 1997).

According to red list of threatened species, 113 endangered plant species, 44 plant species are critically endangered and 87 vulnerable species (IUCN, 2000). The development of plant tissue culture technology holds great promise for conservation and enhancement of valuable medicinal plants. It has many advantages over conventional methods of propagation, which suffers from several limitations (Jeyachandran, 2012).

Tissue culture technique is developed in the improvement of the growth conditions of the plants and the facilitation of international germplasm exchange, genetic transformation, production of cryopreservation and production of secondary metabolites (Alderete, 2006). The compounds obtained from in vitro propagated plants are varied from the wild variety. Instead the structural organization of the plants was not modified by micropropagation  and the  medicinal effects of plant materials typically result from the combinations of secondary metabolites such as Tannins, Alkaloids, Carbohydrates, Steroids, and Phenol compounds, Flavonoids, Resins, Fatty acids, Gums which are capable of producing definite physiological action on human body (Joshi, 2009).

To improve production of high quality plants, micropropagation is required. It is carried out using a selective media consisting of growth hormones that helps in development of morphological characters from naturally grown plant parts (Debergh and Read, 1991).

Preliminary phytochemical and Gas Chromatograph and Mass Spectrometry (GC-MS) analysis of secondary metabolites are advantage for the micropropagation of medicinal plants and it has been reported in many plant species (Singh and Chaturvedi, 2010).

In vitro micropropagation technique is advantageous than conventional methods since it does not depend on seasonal changes, production in mass quantities, characteristic specific clone production, preserving of endangered plant species (Saini and Jaiwal, 2000). Conservation planners have sought objectives and quantitative criteria for setting priorities among the elements of biodiversity to be protected (Williams, 2002).

The micropropagation is known to produce plants that are genetically identical to parent plant. Less variation was observed in the secondary metabolites content than their wild variety (Yamada et al., 1991).

The aim of producing exact replicas of the original plant selected for its desirable characters and considerable progress has been made in the recent years in micropropagation of many plant species (Wang and Hu 1982). Today there is an enormous resurgence of interest in all herbal products and a rediscovery of the traditional use of medicinal herbs, includes the discovery of new useful compounds produced by natural plant populations in very small quantities or compounds that may not be produced by the adult plants which are available in cultures and the biochemical characterization and identification of active compound (Banerjee and Shrivastava, 2006).

The main industrial goal of the plant tissue culture is to produce a large number of plants in a month instead of years (Chopra et al., 1956).                MS (Murashige and Skoog) medium is foremost medium for growth compared to B5 and SH medium. Cytokinins are growth hormones generally used in tissue culture to stimulate cell division and affects shoot and root morphogenesis. Reduction in growth of primary root and density of lateral root is caused by cytokinins (Laplaze, 2007).

The developed tissue culture technique will increase the scarce knowledge about the in vitro response of this native germplasm with potential relevance as an endangered plant species, it is a necessary step in many experiments like clonal propagation, formation of virus free plants and genetic transformation. The entries of virus in the wild variety the plant will be change their genetic nature and compound synthesis.  The large scale growth of plants in liquid culture with bioreactors for production of valuable compounds derived secondary metabolites and recombinant proteins used as biopharmaceuticals (Georgieva et al., 1996).

1.2 Molecular characterization

Genetic studies are fundamental for the management and conservation of valuable medicinal plants. The use of molecular markers is a powerful tool in the genetic study of populations. The molecular marker approach for identification of plant varieties of genotypes seems to be more effective than traditional morphological markers. Since it allows direct access to the hereditary material and makes it possible to understand the relationships connecting individuals (Williams, 1990). Identification of plant species, phylogenetic analysis, population studies and genetic linkage mapping is carried out by molecular markers based on Polymerase Chain Reaction (PCR) (Paterson et al., 1991). Several DNA markers have been successfully employed to assess the genomic stability in regenerated plants including those with no obvious phenotypic alternations (Rahman and Rajora, 2001).

1.2.1 Random amplified polymorphic DNA (RAPD)

Randomly Amplified Polymorphic DNA (RAPD) analysis has been known to detect genetic diversity, genetic profiling, plant species conservation and random locations of the genome along with the cost effectiveness, technical simplicity and does not require template DNA for prior sequence information (Yu and Nguyen, 1994).

RAPD markers were then used for the linkage analysis of some phenotypical characters in plants, such as fruit skin color and comparison of genetic stability of the conserved plants and the controls (Inoue et al., 2006). RAPD Polymerase Chain Reaction method is also faster and more sensitive than biochemical methods. The advantages of RAPD technique is to analyze number of samples economically, the DNA printing is very specific which forms independent ontogeny expression. Besides, all genomes can be analyzed using unlimited number of markers (Titin Purnaningsih, 2013).

RAPD is a simple, foremost, rapid and simple assay marker that requires low quantities of template DNA, and randomly spread throughout the genome. These markers provide good genome coverage (Perez et al., 1998).

1.2.2 Inter Simple Sequence Repeat (ISSR)

ISSR is a fast and highly reliable genotyping technique, amplification of primer requires single sequence repeat motif and is based on variation in the regions between adjacent inversely oriented microsatellites. The technique is exceptionally informative, extremely reproducible and exhibits tremendous detecting polymorphism level (Pathak and Dhawan, 2012).

ISSR markers make use of longer primers (15-30 mers) as compared to RAPD primers (10 mers), that allow the following use of high annealing temperature that leads to higher stringency. In several plant species, RAPD and ISSR markers have known to be efficient to detect genetic fidelity. They have been competent in naming phylogenetic bond between Bacopa monnieri (L.) (Martin et al, 2004).

1.3 Qualitative and Quantitative analysis

Discovery of useful drugs primarily involve phytochemicals. Pharmaceutical companies have found interest in producing new drugs with the aid of phytochemical analysis of the plants and are commercially important (Wadood et al., 2013).

The potential of higher plant species, limited percentage has been investigated of their phytochemical analysis and fraction submitted to biological and pharmacological screening is even smaller. Hence any phytochemical analysis of medicinal plant will reveal only a very narrow spectrum of it components. Therapeutic agents require pharmacological screening of either natural or synthetic compounds. Random screening as tool in discovering new biologically active molecules has been most productive in the area of antibiotics (Gerhartz et al., 1986). Phytochemical constituents of plants are Alkaloids, Steroids, Tannins, Glycosides, Volatile oils, Resins, Phenols Flavonoids, Tannins and Carbohydrates are deposited in their specific parts like leaves, bark, seeds, fruits and root. The beneficial therapeutic effects of plant materials occurs from the mutualism between these secondary products and plants and finds potential use in medicine and pharmacological applications (Tonthubthimthong et al., 2001).

The Gas Chromatography and Mass Spectroscopy is the universally used technique for the identification and quantification of the unknown organic compounds in a complex mixture that can be determined by interpretation and also by matching the spectra with reference spectra. The volatile compound of plants is composed of biogenically derived hydrocarbons and oxygenated materials (Ronald Hites, 1997).  Extracts from natural products, comprising of fruits, vegetables and medicinal herbs are known to be effective against various human diseases    (Wu et al., 2002). Radical scavenging is the process of reaction that takes place between phytochemical compounds and free radical, the gained electron gets delocalized over the phenolic antioxidant and the aromatic nucleus prevents the extension of the free radical chain reaction. Instead polyphenolic compounds inhibit oxidation through a variety of mechanisms (Cuvelier et al., 1992).Plant metabolism function depends on primary and secondary metabolites. Primary metabolites consist of Chlorophylls, Carbohydrates, Amino acids and Proteins, while secondary metabolites is composed of Saponins, Alkaloids, Flavonoids, Steroids and Tannins (Kumar et al., 2009).

1.4 Antioxidant

The antioxidant is used in industrial chemicals to prevent oxidation, and in foods that contain natural chemicals, body tissue that has beneficial health effects. Antioxidants molecules inhibit oxidation that can produce oxidative damage inducing free radicals, prevents oxidative stress, lipid free radical, cellular and tissue damage (Gulcin, 2009). In recent years, the importance of natural antioxidants from plants has significantly increased (Sarikurkar, 2011). Antioxidant compounds such as Phenolic acids, Polyphenols and Flavonoids are known to scavenge free radicals like peroxide, hydroperoxide or lipid peroxyl and thus prevent the oxidative mechanisms that lead to degenerative diseases (Valentao et al., 2002).

Medicinal plants have been extensively studied for their antioxidant activity. Traditionally herbs have been used in nutrition, medicine, flavoring, beverages and cosmetics. Intakes of antioxidant rich fresh fruit, vegetables and tea have been found to prevent cancer and cardiovascular diseases        (Willcox et al., 2004). In vitro experiments carried on antioxidant compounds in higher plants inhibited free radicals and reactive oxygen species and exhibited protection against oxidation damage. These compounds have related structures to synthetic antioxidants and thus can be inferred as potential antioxidants activity (Ali et al., 2008).

1.5 In vitro cytotoxicity assay

In 1952, A. E. Moore, L. Sabachewsky and Toolan established the Human Epithelial type-2 (HEp-2) cell line by injection with epidermoid carcinoma tissue from the larynx of 56 year old male that produced tumours in irradiated cortisonized weanling rats. It could support the growth of 10 of 14 arboviruses, measles virus, and it has been in use for experimental studies in rats for tumor production, hamsters, mice, embryonated eggs and terminal cancer patient volunteers (Toolan, 1954).

Cytotoxicity analysis offers a vital means of selecting compounds for consideration in drug discovery. The selection of drug using a specific viability or cytotoxicity assessment technology could be influenced by explicit research goals. For in vitro human cell culture methods, if a compound interferes with cellular integration, if it considerably modifies cellular morphology, alters cell growth rate or causes cell death, then the compound is termed as cytotoxic (George et al., 2012).

1.6 In vitro anti fungal activity

The increasing incidence and prevalence of fungal infection in developing countries is attributed to immune compromised state such as use of anticancer drugs, immunosuppressive agents, HIV-positivity and etc., most infections of skin and its appendages, the hair and nail are caused by a homogenous group of keratinophilic fungi called the dermatophytes        (Pfaller et al., 1994). Dermatophytes are a group of molds that are related morphologically and physiologically between them, causing infections in humans and animals (De Vroey, 1985).  At least one-fifth of the world populations suffers from mycoses infection. Dermatophyte infections, particularly those involving the skin and mucosal surfaces constitute a serious problems in developing countries and are directly connected with the skin (Tinea corporis) fungal infections such as Feet (Tinea pedis), Hair (Tinea capitis), Nails (Tinea unguium),  Groin (Tinea cruris) because of the prevailing moisture and temperature conditions (Martin and Kobayashi, 1993). Tinea corporis was the most common dermatophytes. Dermatophytes are known to spread by direct contact from people, animals and soil and indirectly from fomites (Barry L Hainer, 2003).

Dermatophytosis, also known as zoonosis, have created  health hazardous due to the close contact between human, particularly children, and animals such as dogs, cats, birds and small rodent and pocket pets. The clinical symptoms may not pose a serious threat, however effective treatment is costly and time consuming since the increasing resistance incidence in known fungal pathogens to the currently available antibiotics has become apparent (Alexander and Perfect, 1997).

There is an increase in the risk of infection by dermatophytes due to factors related to the trend of living in communities, animal contact, and the usage of antineo plastic and antibiotics drugs. Clotrimazole, Miconazole, Fluconazole, Itraconazole and Ketoconazole (Imidazole group) are very effective drugs but they could be harmful to human health (Shahi et al., 1999). Drug resistant strains have caused failures such as host toxicity of available polyenes and fungi static mode of action azoles in current antifungal therapy (Baker and David Rogers, 2006).

1.6.1 Resistant fungus

Treatments to fungal infections have failed due to development of resistant strains. There is need to develop antifungal drugs using in vitro methodology against dermatophytes that are resistant. Clinical and Laboratory Standards Institute (CLSI) have previously carried out micro assays, agar dilution, E test and colorimetric micro dilution, these methodologies could not be carried in many laboratories.

A recent study comparing colorimetric broth microdilution testing of fluconazole with reference broth macrodilution and broth microdilution methods demonstrated excellent agreement among the three methods but broth microdilution method performed according to NCCLS guidelines demonstrated good agreement compared with broth macrodilution method (Diamond, 1991). Antifungal susceptibility testing is becoming more important in the clinical laboratory because the frequency of serious fungal infections, especially yeast are known to cause mucosal infections and are also known to cause male and female genital infections in immunocompromised patients, has increased significantly in recent years (Banerjee et al., 1991)

Cleome is known to be the largest genus that comprises 180 to 200 herbaceous annual species and shrubs distributed widely in tropical and subtropical regions. It has major diversity of 150 species in tropical regions, (Raghavan, 2006). It is also known as spider flower and mountain bee plant. Medicinally used as leaf paste on headache, skin diseases and also as food for pregnant women in African countries. Preparation of black paint is done by boiling Cleome leaves (Sungwarl and Supanee, 2006).

In extreme conditions, C. gynandra are eaten as vegetable. Oil from Cleome chelidonii seeds has been used as insect repellent. Cleome arabica contains higher amount of flavonoids (Rukmini, 1978). The seeds of Cleome viscosa are reported to have nutritive value while the juice of leaves is applied to the skin as counter irritant and whole plants are used too many biological properties such as PRSV melon virus WMV-2, ZYMV melon viruses and skin disease (Berger, 2005).

The leaves and stems of Cleome rutidosperma, along with other nine commonly used medicinal plants of Nigeria and suggested that these medicinal plants could potentially be used as raw materials in drug formulation      (Edeoga et al., 2003). The leaves and flower of Cleome viscosa was used for antimicrobial and antifungal activity against bacterial and fungal organisms that are pathogenic. This plant has been used to raise the formation of blood during childbirth. They are useful in treating vomiting, diphtheria, and stomach disorders. The plant could be intercropped with cabbage to reduce diamondback moth as well as thrip attacks (Silue, 2009).

Hence, the focus of the present study is in vitro micropropagation of Cleome species, Molecular characterization of micropropagated plants with wild plants. Further carryout the pharmacological activities of                 Cleome rutidosperma and Cleome viscosa.

4.1 Collection, identification and authentication of Cleome rutidosperma

and Cleome viscosa

4.1.1 Description of Cleome rutidosperma DC.

Common Name : Fringed spider flower or Purple Cleome

Vernacular Name : Seru walai

Synonyms  : Cleome ciliate, Cleome burmannii, Cleome thyrsiflora,

  Cleome guineensis

Systematic classification

Kingdom  : Plantae

Subkingdom  : Tracheobionta

Infrakingdom  : Streptophyta

Superdivision : Embryophyta

Division  : Magnoliophyta

Subdivision  : Spermatophytina

Class   : Dicotyledonae

Order   : Brassicales

Family  : Cleomaceae

Genus   : Cleome

Species  : rutidosperma

4.1.1.1  Origin and Distribution

Cleome genus comprises 150 to 200 species, the majority of species found in tropical America, whereas about 55 are known from tropical Africa. Cleome rutidosperma is native to tropical Africa and has been introduced and become naturalized in tropical and subtropical regions of Asia, the Americas and the West Indies. Cleome rutidosperma is a low growing herb belonging to the family Cleomaceae, it is a common herb that grows as a weed in disturbed and rural habitats, principally in areas with humid and hot environmental conditions. It is often found as a weed of disturbed ground, roadsides, gardens, crops abandoned lands, and has also been found as an epiphyte on trees, stone walls and cliff faces. This species is included in the global compendium of weeds (Randall, 2012), where it is considered to have moderate economic impacts in a wide range of crops, due to its scrambling habit that smothers and stunts young crop plants. C. rutidosperma has been listed as invasive in China, Malaysia, India, Thailand, Vietnam, Australia, and the Domican republic (Waterhouse and Mitchell, 1998).

4.1.1.2  Taxonomical Description

C. rutidosperma is an annual herb, up to 70 cm tall, widely branched, erect or sometimes spreading. Alternate leaves 3 palmatisect, rhomboid-elliptic to lanceolate leaflets, generally asymmetric, the central 0.5-6 x 0.2-2.5 cm, the lateral smaller, acute to acuminate at the apex, cuneate at the base, ciliolate serrulate margins, conspicuous nerves specially prominent below, petiole up to 7cm. Inflorescence racemes, solitary to few flowered, lax and not clearly demarcated, very short or up to 20 cm. long; bracts usually similar in size to the leaves. Flowers in the axil of leaf-like trifoliate bracts,  pedicels in flower up to 2.5 cm, in fruit to 3.5 cm. Narrowly lanceolate sepals 4, 2-5 x ca 0.5 mm, sub glabrous to sparsely pubescent, often with glandular hairs. Petals 4, white, pink, lilac, violet or blue, narrowed into a basal claw, oblanceolate to elliptic lamina, apiculate. Stamens 6, filaments 5-9 mm, anthers 1-3 mm. Ovary linear, cylindrical, 3-10 mm long, with a gynophore 1.5 mm,  glabrous or with some short hairs and sessile glands,  very short style,  capitate or truncate stigma, papillose (Figure 4.1, 4.2 and 4.3). Capsule 2.5 -7.5 x 0.2-0.5 cm, on a gynophore to 13 mm long, linear-ellipsoid, sometimes slightly torulose, glabrous or glabrescent, beak 2-8 mm, valves with prominent longitudinal anastomosing nerves. Subglobose seeds, slightly laterally compressed, up to 2 mm in diameter, with longitudinal striations and prominent transverse ridges, glabrous, reddish-brown, dark brown or black, whitish elaiosome present  (Widespread, 1972).

4.1.1.3  Medicinal Importance

The whole plant of C. rutidosperma has medicinal value and is known to have a numerous pharmacological effects like antipyretic (prevent fever), antimicrobial, free radical scavenging activities and antiplasmodial activities (Bose et al., 2005). The plant is frequently used in traditional medicine      (Akah and Nwambie, 1993). Leaf sap is applied in Ghana, Gabon and DR Congo to cure earache and deafness. In Ghana a leaf extract is used to treat irritated skin and in Nigeria it is used to treat convulsions. Pollen of this species was found present in honey from Malaysia. In Malaysia planting of                 C. rutidosperma around field edges considered as part of an insect control programmed (Maishihah and Kiew, 1989).

4.1.2 Description of Cleome viscosa L.

Common Name : Asian spider flower

Vernacular Name : Naikkaduku

Synonyms  : Cleome acutifolia, Sinapistrum viscosum, Arivela

                                   viscosa.

Systematic classification

Kingdom  : Plantae

Subkingdom  : Tracheobionta

Infrakingdom  : Streptophyta

Superdivision : Embryophyta

Division  : Magnoliophyta

Subdivision  : Spermatophytina

Class   : Dicotyledonae

Order   : Brassicales

Family  : Cleomaceae

Genus   : Cleome

Species  : viscosa

4.1.2.1  Origin and Distribution

Cleome viscosa occurs in northern tropical Africa, from Cape Verde and Senegal to Egypt, it is absent in southern Africa, inspite present in Madagascar and other Indian Ocean islands. Cleome viscosa is widespread in peninsular Arabia, Asia, Australia and tropical America. Cleome viscosa occurs in marshland and found in both under seasonal dry and humid conditions. Cleome viscosa its autecology and provided information regarding its distribution and abundance which is helpful for its cultivation on a commercial scale       (Jansen, 2004).

4.1.2.2  Taxonomical Description

An annual, sticky herb with a strong penetrating odor and clothed with glandular and simple hairs. Leaves are digitately compound, with 3 to 5 leaflets. Leaflets are obovate, elliptic-oblong, very variable in size, often 2-4 cm long, and petiole up to 5 cm. Racemes elongated, up to 30 cm long, with corymbose flowers at the top and elongated mature fruits below bracteates. Flowers 10 – 15 mm across, yellowish, pedicels 6 – 20 mm long, bracts foliaceous. Sepals oblong lanceolate, 3 – 4 mm long, 1 – 2 mm wide and glandular pubescent. Petals 8 – 15 mm long, 2 – 4 mm broad, oblong –  obovate. Stamens 10 – 12, not exceeding the petals, gynophore absent (Figure 4.4, 4.5 and 4.6) (Vaidyaratnam, 2010).

4.1.2.3  Medicinal Importance

The C. viscosa leaves, flowers, seeds and roots of the plant are widely used in traditional and folkloric systems of medicine as an anti inflammatory, antimicrobial, antipyretic, anthelmintic, immunomodulatory and hepatoprotective activities. The leaves and root are particularly useful for treating skin disease (Mali, 2010). C. viscosa leaves and young shoots used as vegetables. The whole herb was used to treat fungal infections, cough, bronchitis and cardiac disorders (Kirtikar and Basu 1975). Seeds have served as raw materials for the extraction of Coumarinolignoids, a valuable chemical entity needed by pharmaceuticals industries for liver diseases and immunomodulation (Chattopadhyay et al., 2004). The analgesic, antipyretic and anti-diarrheal activities of the extract of C. viscosa and it was noted that the fresh leaves are used for jaundice, seeds and leaf are used to treat viral infections (Devi et al., 2003).

4.1.3 Collection of plants

Cleome rutidosperma DC. and Cleome viscosa L. were collected from natural habitat of Sirumailur village (The latitude 13.0938995 and longitude 80.292356 are the geocoordinate of the Sirumailur), Kanchipuram district, Tamil Nadu.

4.1.3.1  Identification plants

The plants was identified and authenticated by Botanical Survey of India, Coimbatore, India, and voucher specimens were deposited.

4.2. In vitro micropropagation of Cleome rutidosperma and Cleome viscosa

with different plant growth regulators of different concentration and

combination.

4.2.1 Source of explants

Node and shoot tips were used as explants from healthy wild plants of    C. rutidosperma and C. viscosa. The explants were cut into 5 mm in length and used for in vitro micropropagation studies.

4.2.2 Preparation of stock solutions

The plant tissue culture media formulation described as Toshio Murashige and Folke Skoog (1962) experiment referred as MS medium was selected as the optimal culture medium.

MS medium stock solutions prepared for plant tissue culture as follows;

Macronutrients – Ammonium nitrate 1650mg, Potassium nitrate 1900mg, Calcium chloride 440mg, Magnesium sulfate 370mg and Potassium phosphate 170mg were dissolved in one liter sterile distilled water and transfer in to sterile storage bottle (Table – 4.1).

Micronutrients – Potassium iodide 0.83mg, Boric acid 6.2mg, Manganous sulfate 22.3mg, Zinc sulphate 8.6mg, Sodium molybdate 0.25mg, Copper sulfate 0.025mg, Cobaltous chloride 0.025mg were dissolved in one liter sterile distilled water and transfer in to sterile storage bottle (Table – 4.2).

Iron source – 27.85mg of Ferrous Sulphate and 37.30mg of Disodium ethylenediaminetetraacetic acid dihydrate were heated separately and dissolved in one liter sterile distilled water later, and transfer in to sterile storage bottle (Table – 4.3).

The organic supplements – Myo – inositol 100mg, Nicotinic acid 50mg, Pyridoxine HCl 50mg, and Thiamine HCl 50mg, and Glycine 200mg were dissolved in one liter sterile distilled water and transfer in to sterile storage bottle (Table – 4.4).

Carbon source – 3% sucrose was added into the one liter solutions. The pH was adjusted to 5.6 – 5.8. The preparation was then gelled with 0.8% w/v agar (Table – 4.5).

These stock solutions were prepared and stored at 4⁰C until further use.

Table: Composition of revised Murashige and Skoog medium:

Table – 4.1: Major inorganic nutrients stock solution

Table – 4.2: Minor inorganic nutrients stock solution

Table – 4.3: Iron source stock solution

Table – 4.4: Organic supplements stock solution

Table – 4.5: Carbone source

4.2.3 Growth regulators

Plant growth regulators (PGRs) such as cytokinins 6-Benzyl amino purine (BAP), Kinetin (KIN) and auxins Indole-3-acetic acid (lAA), a-Naphthalene acetic acid (NAA),  2,4-Dichlorophenoxy acetic acid (2,4-D), Indole-3-butyric acid (IBA) at different concentrations and in combinations used for the study. Each hormone was dissolved in respective solvents and used for micropropagation (Table – 4.6).

4.2.4 Preparation of plant growth regulators

The Preparation of plant growth regulators stock solutions were as follows.

Cytokinins such as Kinetin 100mg was dissolved in 20ml of 0.1N NaOH and diluted with 980ml of distilled water to make up to 1.0mg/1.0ml concentration. The same concentration of                       6-benzyl amino purine (BAP) at 1.0mg/1.0ml concentration was prepared and stored at 4^ºC.

Auxins such as Indole-3-acedic acid (IAA) (100 mg), Indole-3-butyric acid (IBA) (100mg) and α-Naphthaline acedic acid (NAA) (100mg) were dissolved separately in 20ml of 0.1N NaOH and diluted with 980ml of distilled water to make up to 1.0mg/1.0ml concentration and stored at 4^ºC.

100mg of 2,4-Dichlorophenoxy acetic acid (2,4D) was dissolved separately in 20ml of 70% ethanol and diluted with 980ml of distilled water to make up to 1.0mg/1.0ml concentration and  stored at 4^ºC

All these hormones were added in to MS medium before the sterilization.

Table – 4.6: Solubility of plant growth regulators                             (cytokinins and auxins)