1. INTRODUCTION

Ocimum tenuiflorum is one of the most sacred herbs of India, and is an integral part of ancient Hindu traditions. According to Hindu mythology, Ocimum tenuiflorum originated as one of the 14 “Ratnas (gems or treasures)” from the ocean as the ultimate sacred plant to enhance health and remove diseases. Ocimum tenuiflorum is believed to be a manifestation of the Goddess Lakshmi, the wife of God Vishnu, and is worshiped for health, wealth, and happy married life (Raina et al., 2013). Necklaces made from Ocimum tenuiflorum stems or roots are auspicious to devotees who wear them around their neck or wrist to seek God’s blessings. The plant is considered a blessing from God, a religious symbol, and a magic herb as described in many ancient medicinal texts such as Ayurveda, Siddha and Unani (Engels and Brinckmann, 2013). Thus, Ocimum tenuiflorum has been a permanent fixture in Hindu homes, temples, and sacred shrines and its Sanskrit name “Tulsi” (a word for “the incomparable or matchless one") truly defines the significance of this legendary Ayurvedic plant used since time immemorial.

Traditional uses of Ocimum tenuiflorum as medicine usually include, but are not limited to the treatment of: abdominal issues, oral infections, cough, colds, tumors and cancer, digestive tract problems, respiratory inflammation, arthritis, asthma, ulcers, wounds, hypoglycemia, bronchitis, urinary issues, cardiovascular problems and many others (Das and Vasudevan, 2006; Mohan et al., 2011; Engels and Brinckmann, 2013; Kumar et al., 2013; Mondal et al., 2009; Singh and Verma, 2010). Although the whole plant is medicinally important, the leaves are generally the most common form of treatment, and are either used raw or steeped in hot water. The medicinal properties of Ocimum tenuiflorum leaves include: antimicrobial, anticancer, antistress, adaptogenic, stimulant, expectorant, nervine, antipyretic and antiperiodic (Engels and Brinckmann, 2013 These beneficial properties have led to Ocimum tenuiflorum being named “Queen of Herbs”, “Incomparable One” and “The Mother Medicine of Nature” and one of the most valued medicinal and religious herbs in India (Singh and Verma, 2010). The knowledge of this traditional medicinal herb is expanding to other cultures due to its unique therapeutic properties.

1.1 Botany

The genus Ocimum is a very important aromatic group of herbaceous plants comprising about 160 species of herbs and shrubs from the tropical and subtropical regions of Asia, Africa, and Central and South America. However, the major place of diversity appears to be in Africa. This genus is characterized by a great variability in its morphology and chemo types. The ease of its cross pollination contributes to a myriad of subspecies, varieties,

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and yielding various essential oil used by the industry and is considered as one of the largest genera of the Lamiaceae family (Sobti, 1977; Choudhuri, 1979). It is an annual, aromatic, branched herb, 30 to 150 cm high (Vina and Murillo, 2003). It is distributed in the tropical and temperate regions of the world.

Several species and varieties of plants of the genus Ocimum have been reported to yield oil of diverse nature commonly known as basilica oils and their numerous medicinal uses (Mshana et al., 2000; Matasyoh et al., 2007). This essential oil is rich in phenolic compounds and a wide array of other natural products including polyphenols such as flavonoids and anthocyanins (Sajjadi, 2006). In 1977 as a result of their intensive research work on ocimum species, Regional Research Laboratory, Jammu, has developed four new strains with interesting odour, viz., RRL-01, RRL-02, RRL-03 and RRL-04. In additional to the development of Methyl cinnamate-rich strain, they have also synthesized many new strains for high eugenol content. For example a hybrid strain of Ocimum gratissimum L. using recurrent selection technique of breeding and named it as “Clocimum” (Vaidya, 1977; Prakash and Gupta, 2005). There are many cultivars of basil, which vary in their leaf color (green or purple), flower color (white, red, purple) and aroma (Sajjadi, 2006). Ocimum sanctum L., known as ‘Tulsi’ in Hindi and ‘Holy Basil’ in English, is an erect softy hairy aromatic herb or under shrub found throughout India. Tulsi is commonly cultivated in gardens. Two types of Ocimum sanctum L. are met within cultivation: (i) Tulsi plants with green leaves known as Sri Tulsi and (ii) Tulsi plants with purple leaves are known as Krishna Tulsi. The inflorescence is a long spike with tiny purple flowers. Ocimum sanctum L. is held sacred by Hindus and is used as medicinal plants in day-today practice in Indian homes for various ailments (Prakash and Gupta, 2005). Different parts of Tulsi plant e.g. leaves, flowers, stem, root, seeds etc., are known to possess therapeutic potentials and have been used, by traditional medical practitioners, as expectorant, adaptogenic, analgesic, anticancer, anti-malaria, anti-diarrhea, dysentery, skin diseases, antiasthmatic, antiemetic, diaphoretic, antidiabetic, antifertility, painful eye diseases, hepatoprotective, hypotensive, hypolipidmic and antistress agents. Tulsi has also been used in treatment of fever, bronchitis, arthritis, convulsions etc. Several other pharmacological effects, such as antitumor, (oral & topical), anti-ulcer, antimicrobial, anti-hyperlipidemic, and anti-viral activities, have also been attributed to ursolic acid.The essential oils extracted from Tulsi leaves also possess anti-fungal and anti-viral activity (Prakash and Gupta,2005). Basil is a popular culinary herb, and its essential oils have been used extensively for many years in the flavoring of confectionary and baked goods, condiments (e.g., ketchups, tomato pastes, chili sauces, pickles, and vinegars), sausages and meats, salad dressings, nonalcoholic beverages, ice cream, and ices.

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Basil oil has also found a wide application in perfumery, as well as in dental and oral products (Suppakul et al., 2003).

Ocimum tenuiflorum is included in the family Lamiaceae, along with several other medicinal herbs such as salvia and mint (Gershenzon et al., 1992; Baran et al., 2010) and over 65 species of basil that are used in the food, pharmacology and perfume industry (Trevisan et al., 2006; Makri and Kintzios, 2008). The species is closely related to sweet basil (Ocimum basilicum), a commonly used herb in Europe and North America. Ocimum tenuiflorum has also been described as Ocimum tenuiflorum (basil with small flowers) or Ocimum gratissimum (very grateful basil). Of these, the species name O. sanctum is favoured due to the wide range of its uses in religious and cultural traditions (Engels and Brinckmann, 2013).

Three distinct forms of Ocimum tenuiflorum are commonly distributed throughout the Indian subcontinent: 1) Sri or Rama Tulsi with green leaves; 2) Krishna Tulsi with dark green to purple leaves; and 3) Vana Tulsi with green leaves but the plant grows in the wild (Engels and Brinckmann, 2013). Of these, the Rama and Krishna Ocimum tenuiflorum are most commonly grown in homes and commercial production for use in Ayurvedic preparations. The plant is found mainly in subtropical and tropical areas of Asia including India, China, Malaysia, Sri Lanka, and Thailand in addition to Australia and Africa, at altitudes of up to 1800m in India to sandy dry conditions in China (Engels and Brinckmann, 2013; Kumar et al., 2013).

1.2 Morphology

Ocimum tenuiflorum is an upright plant with many branches and can grow from 20 to 150 cm in length supported by a square stem that is hairy and lignified at the base (Mondal et al., 2011; Mohan et al., 2011; Gupta et al., 2002; Engels and Brinckmann, 2013; Das and Vasudevan, 2006; Jürges et al., 2009). The leaves of the plant are highly aromatic, similar to clove, and are arranged opposite, alternate, and found to be elliptic or ovate in shape, with hairs on both adaxial and abaxial sides and margins that are toothed, serrated or entire (Gupta et al., 2002; Jürges et al., 2009; Kumar et al., 2013). The inflorescences of the plant are long cylindrical racemes purple in colour. The flowers are in compact whorls with bell shaped petals, 60-100mm in length and produce small yellow-brown fruits (Kumar et al., 2013; Gupta et al., 2002; Jürges et al., 2009).

1.3 Medicinal Properties of Ocimum tenuiflorum

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Medicinal effect of Ocimum tenuiflorum is believed to be due to the complexity of the constitutes in the plant, leading to many positive influences in the human body. Innumerable medicinal benefits of Ocimum tenuiflorum have been recorded in many different regions and local languages of India. Recently, scientific evidence of therapeutic effect of Ocimum tenuiflorum has started to emerge in mainstream medical journals mostly from studies using in vitro bioassays and small clinical trials. A few recent examples of such studies on Ocimum tenuiflorum include: antimetastatic activity against Lewis Lung carcinoma cells (Magesh et al., 2009), cardioprotective inhibition of lipid peroxidation in rats induced with myocardial infarction, and high cholesterol or high cadmium diets (Sharma et al., 2001; Suanarunsawat et al., 2010; Ramesh and Satakopan, 2010), immunostimulation through elevation in levels of TNF-α, IFN-γ and IL-2 cytokines in rats infected with Salmonella typhimurium (Goel et al., 2010), neuroprotection and normalization of brain function through modulation of neurotransmitters (Yanpallewar et al., 2004; Ahmad et al., 2012; Muthuraman et al., 2008; Ravindran et al., 2005) and prevention of radiation mediated cell death in mice (Subramanian et al., 2005; Ganasoundari et al., 1997). Other examples of the medicinal properties of Ocimum tenuiflorum are antimicrobial benefits as a mouth rinse (Agarwal and Nagesh, 2011), inhibition of bacterial gonorrhoea (Shokeen et al., 2008), reduced acne (Viyoch et al., 2006), anthelmintic activity (Asha et al., 2001), wound healing (Shetty et al., 2008; Goel et al., 2010) and reduced levels of plasma glucose (Agrawal et al., 1996; Chattopadhyay, 1993; Vats et al., 2004, 2002), triglyceride and cholesterol (Rai et al., 1997). Ocimum tenuiflorum can also reduce diabetic symptoms and blood pressure (Kochhar et al., 2009) and stimulate insulin production in the pancreas (Hannan et al., 2006) as well as, subdue skin, breast, and gastric cancer because of the antioxidative properties (Baliga et al., 2013).

In numerous rat studies, the inclusion of Ocimum tenuiflorum improved the stress response through normalizing changes and associated oxidative damage induced by stresses such as noise, restraint and an increased swimming time (Archana and Namasivayam, 2000; Gupta et al., 2007; Maity et al., 2000; Samson et al., 2007; Tabassum et al., 2009; Bathala et al., 2012; Sood et al., 2006).

Ocimum tenuiflorum has been described to improve symptoms of disorders such as Alzheimer’s or dementia, anxiety (Bhattacharyya et al., 2008), depression and cerebral reperfusion (Yanpallewar et al., 2004) indicating its anti-stress influence on the central nervous system (Joshi et al., 2011; Pemminati et al., 2010; Yanpallewar et al., 2004; Khanna and Bhatia, 2003; Bhattacharyya et al., 2008).

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Although the most widespread use of Ocimum tenuiflorum remains to be medicinal, recent studies have shown that this plant is effective against a variety of biological pollutants present in water (Sundaramurthi and Dhandapani, 2012; Parag et al., 2010). Ocimum tenuiflorum essential oils have antifungal and antiaflatoxigenic effects which can be used for storage and improvement of shelf life of food products such as ‘Tofu’ (Kumar et al., 2010; Anbarasu and Vijayalakshmi, 2007). There is evidence that Ocimum tenuiflorum extracts can improve the shelf life of bananas and may also be a viable alternative to chemical fungicides for the management of crown rot disease (Sangeetha et al., 2010). Strong antimicrobial effects of the essential oil constituents against pathogenic fungi and both gram-positive and gram-negative bacteria have also been demonstrated (Pandey and Madhuri, 2010; Amber et al., 2010).

1.4 Tissue culture and micro propagation

In vitro micropropagation is an effective mean for rapid multiplication of species in which it is necessary to obtain a high progeny uniformity. Therefore, the interest in using these techniques for rapid and large-scale propagation of medicinal and aromatic plants has been significantly increased (Sahoo et al., 1997). Many in vitro studies have been conducted on Lamiaceae species, including the Ocimun genus, using different explants, like nodal segments (Ahuja et al., 1982; Shahzad and Siddiqui, 2000; Begun et al., 2000), leaf explants (Phippen and Simon, 2000), young inflorescence (Singh and Sehgal, 1999) and axillary buds (Begun et al., 2002).

2. REVIEW OF LITERATURE

Pattnaik and Chand (1996) have reported on in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/L for O. americanum and 1.0 mg/L for O.sanctum. Incorporation of 0.5 mg/L gibberellic acid (GA3) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoots of O. americanum were rooted

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on half strength MS supplemented with 1.0 mg/L IBA, whereas O. sanctum rooted best on medium with 1.0 mg/L NAA.

Sahoo et al. (1997) An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N6-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l−1, 4.4 µM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl−1, 1.1 µM). Gibberellic (GA3) at 0.4 mg·l−1 (1.2 µM) added to the medium along with BA (1.0 mg·l−1, 4.4 µM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l−1 (5.0 µM) indole-3-butyric acid (IBA).

Singh and Sehgal, (1999) In vitro micropropagation of holy basil (Ocimum sanctum L.), an Indian medicinal herb, has been accomplished on Murashige and Skoog (MS) medium utilizing young inflorescence explants. MS supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ) produced only non-morphogenetic callus. Direct multiple shoots differentiated within 2--3 weeks when explants were cultured on MS containing 6-benzyl aminopurine (BAP). Of the various levels of BAP tested, MS + BAP (1.0 mgl−1) produced the maximum number of shoots. Incorporation of indole-3-acetic acid (IAA) (0.05 mgl−1) along with BAP (1.0 mgl−1) in the culture medium showed a marked increase in the number of shoots. About 92% of the in vitro regenerated shoots rooted on MS hormone-free medium within 2--3 weeks of culture and 85% of the micro propagated plantlets could be successfully established in soil, where they grew normally.

Phippen and Simon (2000) successfully developed an efficient plant regeneration protocol for basil (Ocimum basilicum L.). Explants from 1 month old seedlings yielded the highest frequency of 85% regeneration with an average of 5.1 shoots per explant. The regeneration protocol was carried out on three basil varieties (Sweet Dani; methylcinnamate; Green Purple Ruffles). Callus and shoot induction was initiated on Murashige and Skoog basal medium supplemented with thidiazuron (16.8 μM) for approximately 30 days. Shoot induction and development were achieved by refreshing the induction medium after 14 days. The most morphogenetically responsive explants were from the first fully expanded true

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leaves of greenhouse-grown basil seedlings. Developing shoots were rooted in the dark on media with thidiazuron removed. Within 20 days, rooted plantlets were transferred and acclimatized under greenhouse conditions where they developed normal morphological characteristics.

Shahzad and Siddiqui (2000) Multiple shoot regeneration from nodal explant callus has been achieved in O. sanctum [O. tenuiflorum]. The explants cultured on Murashige-Skoog (MS) medium supplemented with growth regulators released phenolic exudate which adversely affected the culture response; the explants turned brown and ultimately perished. The addition of 50 mg/l ascorbic acid (AA) to MS medium checked the release of phenolic exudates. Treatment of the explant with AA when AA was omitted from the medium induced callus as well as caulogenesis and rhizogenesis from the callus and also directly from the explant. MS with 2,4-D at 2 mg/l proved best for the induction of organogenic callus; on subculturing the callus on MS with BA at 5 mg/l + NAA at 0.2 mg/l + glutamic acid at 50 mg/l multiple shoots differentiated. Early callus induction followed by profuse rhizogenesis was observed on MS medium with NAA at 5 mg/l + BA at 0.5 mg/l. Lateral bud break occurred on MS medium with adenine sulfate at 5 mg/l + IAA at 0.5 mg/l.

Begum et al. (2002) In vitro regenerated shoots of Ocimum basilicum L. by excising, sectioned inter nodal pieces and subcultured individually in the nutrient medium which produced in an average of eight multiple shoots per transfer. For rooting, in vitro grown shoots were excised from the culture flask and implanted individually on root induction medium containing half strength of MS salts and 0.1 - 1.0 mg/l NAA, IBA or IAA. The highest frequency and healthy rooting was observed on MS medium containing 1.0 mg/l NAA. In vitro regenerated plantlets were transferred on to specially made plastic tray containing coco-peet as potting mixand thereafter successfully established under ex vitro condition. The survival percentage of transplanted plantlets was 75.

Dode et al. (2003) described the procedure for micropropagation of O. basilicum using cotiledonary leafs from in vitro geminated plants. Cotyledons from in vitro germinated seeds were used as initial explants, put in MS (Murashige and Skoog, 1962) medium with 0.2mg.L-1 NAA (1-Naphthalene acetic acid) in combination with 0–5mg.L-1 BAP(6-Benzyl aminopurine) and kept at 28 ± 1°C, 16-h light photoperiod and 48μmol.m-2.s-1 luminous density flow, for 45 days. The highest efficiency of shoot formation after 45 days occurred in the medium containing 5mg.L-1 BAP and 0, 2mg.L–1 NAA. The presence of NAA inhibited root formation, when combined with different concentrations of cytokinin (BAP, 1 to 5mg.L-

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1). Higher BAP concentrations induced an increase in the number of explants with shoots and a higher number of shoots/cotyledon.

Lee and Yi (2003) used Basil (Ocimum basilicum L., Lamiaceae) leaf (5mm´5mm) and internode (10 mm) were excised from two weeks grown seedling and cultured on MS medium supplemented with various concentrations of NAA, IAA, BA, Kinetin, 2iP, and TDZ. There is no difference between leaf and internode explants for callus induction. But the combinations and concentrations of plant growth regulators were shown to be critical factors for callus growth and plant regeneration. Callus induction and multiplication was highest when explants were cultured on MS medium containing 1 mg L-1 NAA + 1 mg L-1 BA. Plant regeneration was highest when callus was transferred on MS medium containing 0.5 mg L-1 IAA + 0.5 mg L-1 2iP and 1.0 mg L-1 IAA + 0.5 mgL-1 2iP. According to combination of plant growth regulators, callus was showed various colors (e.g., green, purple, and yellow). These appearances suggest that plant growth regulators affected different second metabolic pathway. These calluses were subcultured on MS medium supplemented with various plant growth regulators. But the callus colors were not shown to be critical factors for callus multiplication and plant regeneration of basil.

Bodhipadma, et al. (2005) aseptically germinated Seeds of the plant were in vitro. Leaves from seedlings were cut and used for callus induction on MS basal medium containing 0, 0.5, 1 and 2 mg/l 2, 4-D under light condition for 8 weeks. Besides, nodes from these seedlings were cultured on MS hormone-free medium for nodal plantlet regeneration. The leaves from these new plantlets were also excised for callus induction using the same procedure. It was found that callus from both leaves of seed-germinated plantlets and leaves of nodal plantlets developed maximally on MS basal medium supplemented with 0.5 mg/l 2, 4-D.

Kumar et al., (2005) found profuse callus initiation from internodes and leaf explants of Ocimum basilicum inoculated on MS medium supplemented with 1 mg/ L 2,4 - D. Indirect organogenesis was observed from the leaf and internode inoculated on MS medium supplemented with 0.3 – 0.5 mg/L IAA and 3 – 5 mg/L BA. Multiple shoots were initiated from the shoot tip and nodal explants inoculated on MS medium fortified with BA or Kin alone or in combination. Maximum multiple shoots were obtained in MS medium supplemented with 5 mg/l BA and 10 mg/l Kin. The shoots were obtained via direct and indirect organogenesis were further sub cultured on MS medium supplemented with 4mg/l BA and 1mg/l GA3 Numerous shoots were obtained after repeated subcultures. Rooting was observed from the shoot in the regeneration medium. The plantlets on hardening showed 85% survival rate.

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Gopi et al. (2006) developed a rapid system for regeneration of the important medicinal plant, Ocimum gratissimum L, from nodal explant. Single node explants were inoculated on basal MS (Murashige and Skoog, 1962) medium containing 3% (w/v) sucrose, supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for direct plant regeneration. Maximum numbers of shoot (14.3±1.5) were observed on the medium containing 0.5 mg/l BAP and 0.25 mg/l IAA after four weeks of culture. Regenerated shoots were separated and rooted on same half strength MS medium supplemented with 0.5 mg/l of IAA alone for three weeks. Well-developed complete plantlets were transferred on to specially made plastic cup containing soilrite.

Banu and Bari (2007) used Shoot tip and leaf explants of Ocimum sanctum Linn. to culture in different concentrations and combinations of growth regulators (BAP, Kin, 2, 4-D, IAA and IBA) in MS medium to observe shoot multiplication, callus induction, callus regeneration and root induction. Among the different concentrations and combinations of growth regulators, the highest percentage of shoot formation and highest average number of shoots were observed 90 and 5.88%, respectively in 0.2 mg L-1 BAP from shoot tip explants. Callus induction was obtained within 12-15 days of culture from leaf explants. The highest frequency (90.00%) of organic callus induction was observed in MS medium containing 1.0 mg L-1 NAA. Shoot regeneration occurred when the calli were sub cultured in Ms medium supplemented in BAP formulation. The highest percentage of shoot regeneration was obtained 90.00 in 0.2 mg L-1 BAP. In vitro grown shoots rooted best on MS medium containing 0.1 mg L-1 NAA.

Mohanpriya (2007) In vitro cultures were taken up to standardize the protocol for micropropagation and callus mediated regeneration. Among the different concentrations of cytokinins tried for micropropagation, the maximum numbers of shoots (5-6) were obtained in MS medium supplemented with BAP 2.0 mg/L. Among the various concentration of cytokinins tried, media supplemented with 1.0 mg/L Kinetin (3.65cm) was found to be the optimal media for elongation of microshoots. Elongated microshoots were transferred to root induction media. MS + 0.1 mg/L IBA + 1.5 mg/L NAA was found to be optimal media combination for root induction with a response of 11.60 ± 1.05 roots/plant. Among different concentrations of 2,4-D tried for callus induction in MS medium, maximum callus induction (100%) was obtained in media supplemented with 0.5mg/L 2,4-D. The callus was subcultured for organogenesis in MS media supplemented with BAP (0.1- 0.8 mg/L). One hundred percent somatic embryogenesis and maximum regeneration of callus was obtained in MS medium supplemented with 0.8 mg/L BAP.

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Siddique and Anis (2007) established an efficient protocol for rapid micropropagation of Ocimum basilicum. Multiple shoots were induced by culturing shoot tip explants excised from mature plants on a liquid Murashige and Skoog (MS) medium supplemented with 5–100 µM of thidiazuron (TDZ) for different treatment duration (4, 8, 12 and 16 d). The optimal level of TDZ supplementation to the culture medium was 50 µM for 8 d induction period followed by subculturing in MS medium devoid of TDZ as it produced maximum regeneration frequency (78 %), mean number of shoots (11.6 ± 1.16) and shoot length (4.8 ± 0.43 cm) per explant. A culture period longer than 8 d with TDZ resulted in the formation of fasciated or distorted shoots. The regenerated shoots rooted best on MS medium containing 1.0 µM indole-3-butyric acid (IBA). The micropropagated shoots with well developed roots were successfully established in pots containing garden soil and grown in greenhouse with 95 % survival rate.

Chandramohan and Sivakumari (2009) selected leaf and shoot explants for micropropagation of Ocimum basilium L. in MS media supplemented with different concentration and combination of plant hormones like IAA, NAA, 2,4-D and BAP. High frequency of multiple shooting was obtained from medium containing 2mg/l BAP. Nodal explants were introduced on MS medium supplemented with different concentration of BAP for multiple Shooting. Shoots were initiated within 7 days. The results showed that all Concentration of BAP (0.5-5 mg/l) had high frequency of multiple shooting.

Zi Xiong Lim et al. (2009) carried out induction of callus from leaf explants by incubating leaf explants on Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid (2,4-D), picloram, and indole-butyric acid (IBA) at 0, 1, 3, and 5 mg/L as well as the combination of 3 mg/L picloram with different concentrations (0, 0.5, 1.0, 1.5, and 2.0 m g/L) of 6!benzylaminopurine (BAP) or kinetin. The studies revealed that all the leaf explants incubated on phytohormone supplemented medium formed callus. Leaf explants grown on 3 mg/L picloram formed callus after 8±1 days of culture, and degree of callus formation w as found to be the highest (++++) among all the single auxin treatments. In contrast, the degree of callus formed from the leaf explants cultured on MS medium supplemented with combination of auxin and cytokinins were evidently lower than those in the single auxin treatments. Leaf explants cultured on kinetin-supplemented MS medium showed a higher degree of callus formation (++++) as compared to BAP-supplemented MS medium.

Saha et al. (2010) developed an efficient plant regeneration protocol using nodal explants of Ocimum kilimandscharicum Guerke, a medicinally important herbaceous plant

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species belonging to the family Lamiaceae. Axillary shoot bud proliferation was initiated from nodal explants cultured on MS medium supplemented with various concentrations of 6-benzyladenine (BA) (0.5–3.0 mg/l), kinetin (KN) (0.5–3.0 mg/l) and 2-isoPentenyladenine (2-iP) (0.5–3.0 mg/l). The maximum number of shoots (6.09±0.05), with average length 3.83±0.11 cm, was achieved with medium containing 1.0 mg/l BA. Shoot culture was established by repeated subculturing of the original nodal explants on shoot multiplication medium after each harvest of newly formed shoots. 20–30 shoots were obtained from a single nodal explant after 5 months. Rooting of shoots was achieved on half-strength MS medium supplemented with 1.5 mg/1 Indole-3-butyric acid (IBA) and 2% sucrose. Well-developed plantlets transferred to plastic pots containing soil and vermiculite (1:1) showed 81.13% survival.

Gogoi and Kumaria (2011) evolved an efficient method of organogenesis and plantlet regeneration from callus for large-scale propagation of Ocimum tenuiflorum within a short span of time. Callus developed from axillary buds cultured in Murashige and Skoog, (MS) medium supplemented with growth regulators viz. indole-3 acetic acid (IAA), α- naphthalene acetic acid (NAA), 6-benzyl aminopurine (BAP) and kinetin (Kn) either singly or in combination. Explants treated with different combinations of growth regulators showed 100% response of nodal explants in all the combinations of NAA and Kn but maximum number of shoot buds was recorded in medium supplemented with 13.42μM NAA and 2.32μM Kn in combinations. The developed shoots showed best rooting response when cultured in MS+26.85μM NAA+02.32μM Kn. The compost mixture comprising soil and cow dung (2:1) was found to be the most suitable substratum with the plantlet survivability of 82.85%.

Hakkim (2011) initiated callus from leaf explant on Murashige and Skoog's (MS) medium supplemented with 2,4 dichlorophenoxyacetic acid (2,4-D) 1 mg/L and kinetin (KIN) 0.1 mg/L. Suspensions were established by transferring friable callus to MS liquid medium supplemented with growth regulators such as 2,4-D (1 mg/L) + KIN (0.1-0.5 mg/L), 2,4-D (0.5–2.5 mg/L), NAA (0.5-2.5 mg/L), and IAA (0.5-2.5 mg/L) individually.

Mathew and Deepa (2011) used Cotyledonary leaves of Ocimum species viz., Ocimum basilicum L., Ocimum sanctum L. & Ocimum gratissimum L. for comparative studies on somatic embryogenesis. Murashige and Skoog (MS) medium with 2,4-Dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) was used to initiate callus. MS with 1.0 mg l-1 2,4-D + 0.5 mg l-1 BA was found suitable for the development of callus with maximum weight and lesser days to induction for O. basilicum and O. sanctum whereas MS

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with 0.5 mg l-1 2,4-D + 0.5 mg l-1 BA initiated callus of maximum weight with high % of response and lesser days to induction for O. gratissimum. High % of response to callus induction was found in MS with1.5 mg l-1 2,4-D + 0.5 mg l-1 BA for O. basilicum and O. sanctum. Differentiation into globular stage of somatic embryos was observed in all cultures but with variation in duration, % response and embryo colour, on transfer of sub-cultured callus to MS media containing different concentrations and combinations of BA, Kinetin (KIN) & Indole Acetic Acid (IAA). Maximum differentiation into globular shaped somatic embryos was observed in all concentration ranges of KIN with or without IAA and in BA (2.0, 3.0 mg l-1) which had coconut water (CW) as an additional supplement.

Livadariu (2011) experimentally studied to illustrate the influence that exogenous cytokinins (BAP and TDZ), and auxins (NAA or IBA) have, added in the composition of the artificial nutritive medium, for modeling the in vitro propagation through direct somatic embryogenesis in two cultivars of basil (Ocimum basilicum L. var. Marseille and Ocimum basilicum L. var. Red Rubin), starting from different types of explants (leaf, cotyledon, epicotyl, hipocotyl and radicle). The best embryogenic response was recorded for experimental variant consisting in TDZ cytokinin and IBA auxin in quantities of 1 mg/l respectively 0,5 mg/l, and cotyledon type explant.

Asghari et al. (2012) carried out, two successive experiments: first, the effects of explants source on MS medium supplemented with four different concentrations of BAP were studied in order to investigate the morphogenic responses; and second, the effects of different levels of two growth regulators (BAP and IAA) either individually or in combination on multiple shoot induction from nodal segments were evaluated. Maximum percent of regeneration (96.67±0.33) and average number of shoot (5.6±1.15) were observed on the medium containing 11 μM BAP + 0 μM IAA. Regenerated shoots were separated and rooted on the same half strength MS medium supplemented with 3.42 μM IAA alone for two weeks. Similarly in the second experiment, increasing BAP concentration led to decreased rooting. Moreover, a positive correlation between increasing the BAP level in culture media and vitrification of regenerated shoots was observed. The lowest and the highest vitrification values were achieved in the media containing 0 and 33 μm BAP, respectively.

Daniel et al. (2012) micropropogated Ocimum basilicum L. using axillary explants on Murashige & Skoog’s medium. Nodal explants produced proliferation of multiple shoots on the medium containing 0.5 mgl-1 BAP with 0.5 mgl-1 IAA. The elongated shoots were separated and cultured for root induction. Rooting of in vitro raised shoots were best induced on ½ strength MS medium supplemented with 1.5 mgl-1 IBA with highest percentage of

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shoot regenerating roots (89 %). The well rooted plantlets were acclimatized and successfully established in the natural condition with 90% survival.

Janarthanam and Sumathi (2012) developed protocol for multiple shoot induction and plant regeneration from nodal explants of Ocimum citriodorum. Nodal explants inoculated on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l Benzyl adenine (BAP) and 0.025 mg/l indole -3- acetic acid (IAA) showed better growth response (80%) and produced 15.2 ± 1.28 shoots per explant with an average length of 6.17 ± 0.29 cm after 35 days. Roots were induced after transfer to half strength MS medium supplemented with 0.5 mg/l Indole -3- butyric acid (IBA) produced 6.0 ± 1.0 roots with an average height of 4.9 ± 0.26 cm after 30 days.

Shahzad et al. (2012) developed an efficient in vitro micropropagation system for direct shoot growth of Ocimum basilicum, using nodal explants. The excised nodes were cultured on Murashige and Skoog (MS) medium containing two plant growth regulators (6-benzyladenine and 2- isopentanyl adenine) with various combinations and concentrations for the study of shoot induction. Addition of L-glutamine was essential to induce sprouting of axillary buds. The best condition for shoot growth was with 6-benzyladenine (BA) 10.0 μM + L-glutamine 30 mg/L in MS medium. The optimum shoot formation frequency was 100% and about 13.4 ± 1.80 shoots were obtained from each explant after 8 weeks of culture. Shoots (more than 4 cm) were rooted most effectively in 5.0 μM indole-3-butyric acid (IBA) supplemented with half-strength MS medium.

Kalakoti (2013) used different concentrations of growth harmones namely, NAA (100 μl – 500 μl and BAP ( 100 μl – 200 μl) were used and their effect on the in vitro development of Ocimum kilimandscharicum was determined. Ocimum kilimandscharicum Gueke, was cultured on MS media. The maximum number of shoots, with average length 10-11 cm, was achieved with medium containing 100 μl NAA and 300 μl BAP . Shoot culture was established by repeated subculturing of the original explants. In this way, 20-30 shoots were obtained from single apical explants after 4 month. Rooting of shoots was achieved on MS media supplemented with 100 μl BAP and 400 μl NAA. The callus was achieved on MS media supplemented with 200 μl NAA and 500 μl BAP. Well developed plantlets transferred to pots containing soil showed 68% survival.

Kibler (2014) developed a micropropagation protocol and characterize the medicinal plant Ocimum sanctum L. An efficient system was established for in vitro multiplication of shoots (2.5 shoots/explant) using BA (1.1 μM) and GA3 (0.3 μM). The addition of AIP, a phenolic pathway inhibitor, at 2 μM along with AC (0.6%) improved the formation of shoot

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(6.3 shoots/ explant) and also alleviated the problem of liquification of culture medium. Microshoots, rooted in a medium containing 0.5 μM IBA with AC (0.6%), had a high survival rate (83%) when transplanted into the greenhouse.

Sheelu et al. (2014) used different concentrations of BAP in MS media in different explants of O. gratissinum. The nodal and shoot tip explants were taken and sterilized using bavistin, tween 20 and HgCl2. The explants were introduced into MS media containing various concentrations of BAP. 0.5 mg/l BAP showed the best performance of proliferation by inducing shoots in 95% cultured nodal explants. The explants produced the highest number (12.0+0.5) of shoots per culture on the medium with 2.3+0.29cm average length of shoots per culture. On the same medium, shoot tip explants, produced shoots in 75% of the culture. Explants produced the highest number 6.9+0.23 of shoots per culture, their average length being 1.8+0.29cm.

Deepak et al. (2014) formulated an efficient method for rapid propagation using young shoot of Ocimum sanctum. Young shoot cultured in Murashige and Skoog, (MS) medium containing different growth regulatory components like as indole-3 acetic acid (IAA), α-naphtholene acetic acid (NAA), 6-benzyl aminopurine (BAP), and sucrose. The callus was further elongated by transferring it in fresh media after a specific time interval. The maximum number of shoots was achieved with medium containing BAP. Rooting of shoot was achieved by using MS medium supplemented with 2.0 mg/l IAA and 3% sucrose. Well developed plantlets transferred in polycup containing sterile soil with compost material and finally well established in the field with 60-70% survival rate.

Leelavathi et al. (2014) developed a repeatable protocol for rapid clonal multiplication using in vitro apical bud of Ocimum basilicum for commercial purpose and mass cultivation of diseases free plants. Plant regeneration from cultured in vitro apical bud explants of Ocimum basilicum was obtained by direct shoot development on Murashige and Skoog’s basal medium supplemented with BAP (8.88 µM) and Kinetin (9.28 µM). MS + BAP (8.88 µM) was found to be the most suitable medium for initiation and multiplication of shoots from apical bud. Regenerated shoots were grown on the same medium for further development, 74 days old culture when sub cultured on the same medium exhibited large number of healthy multiple shoots of 6-8 cms in height. These 15-20 healthy multiple shoots developed roots on the same media thus avoiding an additional step of in vitro rooting. Complete plants thus obtained were transferred to sterilized soil in plastic pots for 4-6 weeks and then to field.

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Ekmekci and Aasim (2014) provided a reliable and reaptable in vitro plant regeneration protocol of cultivated sweet basil of Turkey. Basil seeds were surface sterilized with 2.5% NaOCl. Epicotyl, hypocotyl and shoot tip explants were isolated from 12-14 days old in vitro grown seedlings. The explants were cultured on MS medium containing 0.80-2.40 mg/L TDZ with or without 0.10 mg/L IBA alongwith 1.0 mg/L PVP and 3.0 g/L activated charcoal. Cent percent callus induction were recorded on all explants on all culture mediums. Shoot regeneration frequency of epicotyl, hypocotyl and shoot tip explant ranged 75.0-100, 25.0-83.33 and 66.67-100% respectively. Maximum number of shoots from epicotyl (3.22) and shoot tip (3.58) were scored on MS medium containing 2.40 mg/l TDZ-0.10 mg/l IBA. Whereas, hypocotyl explant induced maximum number of (5.17) shoots per explant on MS medium with 2.0 mg/l TDZ.

Kőszeghi (2014) applied meta-Topolin (mT) (N6 -(2-hydroxybenzyl) adenine-9-riboside) and aromatic cytokinin as Benzyladenine (BAP) in the micro propagation of sweet basil (Ocimum basilicum L.) was tested for the first time and plant growth parameters assessed to determine the optimum level of these cytokinins. Additionally, the rate of root-growth inhibition due to these two cytokinins was also assessed. Our results show that 1 mg/l (4.43 µM) BAP and 0.5 mg/l (2.07 µM) mT produced the most favourable effects on new shoot developments. Meta-Topolin was shown to increase the quality of the plants and in comparison with BAP fewer distortions were observed. No significant differences in root-growth inhibition between the mT and BAP were detected.

Gopal et al. (2014) in vitro propagated Ocimum basilicum Linn. var. pilosum (Willd.) Benth. Shoot buds were used as source of explants on MS media supplemented with different concentrations of growth regulators for callus growth, induction of multiple shoots and roots respectively. MS media with 1.5 mg/L of kinetin and 0.5 mg/L of NAA showed 95.5% shooting, maximum number of shoots (7.33) and relatively better shoot lengths (4.15 cm). Excised shoots were carefully transferred to half-strength MS medium supplemented with 1.0 mg/L indole-3-butyric acid (IBA) for root induction and it yields 86.6% rooting. Whereas, average root length and number of roots observed were 1.73 cm and 3.31 respectively per explants.

Anamika et al. (2014) established a protocol rapid micropropagation of Ocimum citriodorum Vis., an endangered medicinal herb. The cotyledons were excised from the in vitro germinating seedlings and used as explants for the present study. The explants yielded the highest frequency of 87.49% shoot regeneration with an average shoot length of 4.98 cm on Murashige and Skoog (MS) medium supplemented with 1 mg l-1 6- benzylamino

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purine (BAP) + 0.1 mg l-1 napthalene acetic acid (NAA) + 500 mg l-1 casein hydrolysate (CH) + 25 mg l-1 adenine sulphate (AS). Alteration from the optimal concentration of BAP resulted in the formation of callus. Regenerated microshoots were separated and rooted on MS medium containing NAA (0.5 mg l-1).

3. MATERIALS AND METHODS

3.1. Collection of explants

For the present study the small plantlets of Ocimum tenuiflorum were obtained from Botanical garden of MMASC, Sirsi which is located in Western Ghat region. Explants used in the experiment were auxillary buds and leaves.

3.2. Preparation of media

Nutrient media used for the present study was MS medium (Murashige and Skoog, 1962). All the stock solution was prepared in sterilized well stopper bottle and maintained at 4 – 10 0 C. Glycine and Myoinositol were freshly added at the time of media preparation.

(a) Auxins and their preparation:

Auxins like Indole- 3- acetic acid (IAA), Indole-3 butyric acid (IBA), 1- Naphthyl acetic acid (NAA) and 2, 4 - Dichloro phenoxy acetic acid (2,4-D) were used in these experiments. A stock solution containing 1 mg/ml of auxins was prepared in sterile distilled water, after dissolving it in 0.1 N NaOH.

(b) Cytokinins and their preparation:

6-Benzyl amino purine (BAP) and Kinetin -6- furfuryl amino purine (Kin) were used for the present study. A stock solution containing 1 mg/ml of cytokinin was prepared in sterile distilled water, after dissolving it in 0.1 N HCL.

3.3. Preparation of Media

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The MS nutrient media (Murashige and Skoog, 1962) was used for micro propagation, callus induction, somatic embryogenesis and regeneration studies. The composition of the media is given in Appendix-I; Distilled water was used for the preparation of culture media. The stock solutions were mixed in required proportion for each medium. Growth hormones were added and the pH was adjusted to 5.8 using 0.1 N NaOH or 0.1 N HCL. Gelling agent (agar-agar) at a concentration of 0.8 % was added and steamed to melt and poured into the medium. It was then screw capped bottles (25 ml per bottle) and then autoclaved at 121oC temperature, 15 PSI pressure for 15 minutes. The autoclaved media then removed and allowed to attain room temperature. The bottles then maintained at 4 to 10o C.

3.4. Surface sterilization of explants

Explant used: a) Leaves and b) Nodes

a) Leaves

Procedure of sterilization:

1. New and fresh leaves are collected in conical flask from our campus medicinal garden.

2. Leaves are rinsed thoroughly with tap water

5 to 10 minutes

3. Leaves are washed with tween 80 by adding 5 drops of tween 80 in 100ml of distilled water

5 to 10 minutes

4. Leaves are rinsed with Distilled water until the lather is washed off properly.

5. Leaves are kept drowned in 100ml distilled water containing 5ml of sodium hypochlorite

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15 minutes

6. Leaves are rinsed with Distilled water twice to remove the traces of sodium hypochlorite.

7. Leaves are now treated with 1% Bavistin in distilled water

30 minutes

8. Leaves are rinsed with Distilled water until all the traces of bavistin are completely removed.

9. Inside LAF, leaves are treated with 0.1gm mercuric chloride in 100ml water

3 minutes

10. Leaves are rinsed with sterile water thrice to remove the traces of mercuric chloride.

b) Nodes

Procedure of sterilization:

1. New and fresh nodes are collected in conical flask from our campus medicinal garden.

2. Nodes are rinsed thoroughly with tap water

5 to 10 minutes

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3 . Nodes are washed with tween 80 by adding 10 drops of tween 80 in 100ml of Distilled water

15 minutes

4. Nodes are rinsed with Distilled water until the lather is washed off properly.

5. Nodes are kept drowned in 100ml distilled water containing 5ml of sodium hypochlorite

20 minutes

6. Nodes are rinsed with Distilled water twice to remove the traces of sodium hypochlorite.

7. Nodes are now treated with 2gms of Bavistin in 100ml distilled water

30 minutes.

8. Nodes are rinsed with Distilled water until all the traces of bavistin are completely removed.

9. Inside LAF, nodes are treated with 0.1gm mercuric chloride in 100ml water for

5 minutes.

10. Nodes are rinsed with sterile water thrice to remove the traces of mercuric chloride.

3.5. Inoculation of explants

The working table of the laminar airflow chamber was first surface sterilized with 70 % ethanol. Sterile petri dishes and tools (forceps, scalpels, sterile cotton and sterile paper towels) that were used for inoculation were kept in the laminar airflow chamber. The ultra

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violet light was switched on for 20 minutes prior to inoculation then switched off. Then the hands were sterilized with 70% alcohol. The forceps and scaples were dipped in 70% ethanol and flamed, cooled and used for inoculation. Then the culture flasks were inoculated with the sterilized explants in the laminar airflow chamber for raising aseptic cultures.

3.6. Culture conditions

The cultured bottles were incubated at 25±2o C under cool white inflorescent light with a photoperiod of 16 hours light and 8 hours of darkness

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4. RESULTS AND DISCUSSION

In general, for the conservation of valuable medicinal plant resources, micro propagation through axillary bud proliferation has been proven to be a handy tool (Pierik, 1978; Chu, 1992). Studies by Castillo, (1998) and Rui and Pulli, (2004) has revealed that the type of plant growth hormone and its dosage influenced the frequency of shoot formation. Aswath and Choudhary, (2002) used cytokinins in the media to stimulate axillary or adventitious shoot development. He found that the type and concentration of cytokinin had profound effects on shoot multiplication.

Table 1: Effect of various combinations of cytokinins and auxins on shoot induction of Ocimum plantlets

BAP(mg/l) Kn(mg/l) NAA(mg/l) %s urvival No. of ShootsShoots length

0.0 2.5

0.2

70 2 5

0.5 2 70 3 6

1.0 2.5 80 6 6

1.5 1 80 7 6

2.0 0.5 80 7 3

2.5 0 90 7 3

3.0 0 70 7 3

The tried combinations were BAP (0 to 3.0 mg/l), Kn (0 to 2.5 mg/l) keeping NAA (0.2 mg/l) constant (Table 1). BAP was In order to get maximum number of shoots, and Kn was used to elongation of shoot length. The maximum number of shoots (7) and shoot length (6 cm) were obtained from combination of 1.5 mg/l BAP, 1 mg/l Kn and 0.2 mg/l NAA. The combination showed 80% survival. The combination is in correlation with studies of Gopi et al. (2006); Gogoi and Kumaria (2011). The increase in BAP concentration caused decrease in shoot length.

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Table 2: Effect of different combinations of growth regulators on callus induction

2,4-D NAA(mg/l) BAP(mg/l) % survival Callus(mass)

0.0 1.0

0.5

30 ++

0.2 0.8 45 ++++

0.4 0.6 55 +++

0.6 0.4 25 ++

0.8 0.2 30 ++

1.0 0.0 40 ++++

Various combination of 2,4-D (0 to 1 mg/l), NAA (0 to 1 mg/l) keeping BAP (0.5 mg/l) constant (Table 2). The combination of 0.4 mg/l 2.4-D, 0.6 mg/l NAA mg/l and BAP 0.5 mg/l showed maximum callus mass with 55% survival. The combination of 1 mg/l 2, 4-D showed maximum callus was with 40% survival. It is similar to that of Chandramohan and Sivakumari (2009); Zi Xiong Lim et al. (2009); Bodipadma et al. (2005). Increased 2,4 D concentration caused drying of callus upon subculturing. BAP was used in this combination in order to avoid shock to the callus while it is subcultured.

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5. CONCLUSION

Ocimum tenuiflorum is one of the most sacred herbs of India, and is an integral part of ancient Hindu traditions. The genus Ocimum is a very important aromatic group of herbaceous plants comprising about 160 species of herbs and shrubs from the tropical and subtropical regions of Asia, Africa, and Central and South America. Several species and varieties of plants of the genus Ocimum have been reported to yield oil of diverse nature commonly known as basilica oils and their numerous medicinal uses. This essential oil is rich in phenolic compounds and a wide array of other natural products including polyphenols such as flavonoids and anthocyanins.

An efficient protocol for callus initiation and shoot initiation of Ocimum tenuiflorum using leaves and auxillary buds as explants has been established in this work. The tried combinations were BAP (0 to 3.0 mg/l), Kn (0 to 2.5 mg/l) keeping NAA (0.2 mg/l) constant for auxillary bud explants. Various combination of 2,4-D (0 to 1 mg/l), NAA (0 to 1 mg/l) keeping BAP (0.5 mg/l) constant.

The maximum number of shoots (7) and shoot length (6 cm) were obtained from combination of 1.5 mg/l BAP, 1 mg/l Kn and 0.2 mg/l NAA. The combination showed 80% survival. The combination of 0.4 mg/l 2.4-D, 0.6 mg/l NAA mg/l and BAP 0.5 mg/l showed maximum callus mass with 55% survival. The combination of 1 mg/l 2, 4-D showed maximum callus was with 40% survival.

From the above results obtained it can be concluded that the present study offers a protocol for rapid propagation of Ocimum sanctum using axillary buds and inflorescence explants. This could be useful for large-scale multiplication as well as extraction of secondary

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metabolites in higher amounts. Thus the use of investigated volatile oils and phenolic compounds have tremendous value in herbal product market could allow the rapid development of a new industry.

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APPENDIX – ICOMPOSITION OF MS MEDIUM

(Murashige and Shoog, 1962)

Components Composition (mg/L) Stock solution (W/V) g

Macro Nutrients (10 X)

NH4NO3 1650 16.50

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KNO3 1900 19.00

MgSO4.7H2O 370 3.70

KH2PO4 170 1.70

CaCl2.2H2O 440 4.4

Micro Nutrients (1000X)

Na2MoO4.2H2 0.25 0.025

CuSO4.5H2 O 0.025 0.0025

C0Cl2.6H2O 0.025 0.0025

MnSO4. 4H2O 22.3 2.23

ZnSO4.7H2O 8.6 0.86

H3BO3 6.2 0.62

KI 0.83 0.083

Iron EDTA (1000X)

FeSO4.7H2O 27.8 2.78

Na2EDTA.2H2O 37.3 3.73

Vitamins (1000X)

Nicotinic acid 0.5 0.05

Pyridoxine HCl 0.5 0.05

Thiamine 0.5 0.05

Glycine 2 0.2

Myoinositol 100 10

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