ocimum tenuiflorum callus and shoot induction

1. 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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2. 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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3. 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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4. 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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5. 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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6. 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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7. 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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8. 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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9. 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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10. 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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11. 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
11 | P a g e

12. 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
12 | P a g e

13. 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
13 | P a g e

14. (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.
14 | P a g e

15. 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
15 | P a g e

16. 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
16 | P a g e

17. 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 121o
C 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
17 | P a g e

18. 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
18 | P a g e

19. 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
19 | P a g e

20. 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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21. 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 Shoots
Shoots
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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22. 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.
22 | P a g e

23. 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
23 | P a g e

24. 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 – I
COMPOSITION 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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36. 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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