Biosci. Biotech. Res. Comm. 11(2): 256-262 (2018)
An ef cient protocol for
regeneration of
an important medicinal plant
Amit Kumar*, Mu da Fayaz, Musadiq Hussain Bhat and Ashok Kumar Jain
School of Studies in Botany, Jiwaji University, Gwalior (M.P.)-474011, India
An ef cient in vitro protocol has been established for propagation of elite plant of Vitex negundo L. (Verbenaceae)
commonly known as Nirgundi. It is a large woody aromatic and multipurpose medicinal shrub. It is used medicinally
throughout the greater part of India. This species is widely used in Chinese herbal medicine and is the second most
important for treatment of chronic bronchitis. Leaf extract of this plant possess antibacterial and antitumor activity.
In the present study, nodal segments of Vitex negundo were taken as source of explants and grown on MS media
with 3% Sucrose and 0.8% agar-agar, supplemented with different concentrations of BAP, KIN (0.5 – 3.5 mgl
) and
TDZ (0.5-2.0), with various auxins (NAA, IBA, TIBA), incubated under a photoperiod of 16h illumination of light
and 8h dark at 25±2
C. MS + 1 mgl
BAP was found to be the best concentration for shoot regeneration (90%). The
regenerated shoots were sub-cultured for rooting, using different concentrations of IBA and NAA. Present optimized
micropropagation protocol offers the possibility of germplasm conservation and mass cultivation of this important
medicinal plant.
*Corresponding Author:
Received 29
March, 2018
Accepted after revision 26
June, 2018
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007 CODEN: USA BBRCBA
Thomson Reuters ISI ESC / Clarivate Analytics USA and
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© A Society of Science and Nature Publication, Bhopal India
2018. All rights reserved.
Online Contents Available at: http//
DOI: 10.21786/bbrc/11.1/10
Medicinal plants have been the subject of curiosity since
times immemorial (Constable, 1990). Almost every civi-
lization has a history of medicinal plant uses. About
80% of the people living in developing countries depend
on indigenous medicines to meet their primary health
care needs. About 85% of these traditional medicines
involve the consumption of plant extracts. Out of 250
species of the genus Vitex, near about 14 species have
been found to occur in India. Vitex negundo L. (Verben-
aceae) is a perennial aromatic, large woody shrub, tri or
Amit Kumar et al.
penta-foliate leaves with purple color  ower in branched
tomentose cymes. It is commonly called as Chaste tree,
Nirgundi (Hindi) and Monk’s pepper. It is an important
agro-forestry tree (200–300 cm high) found throughout
the greater part of India and has been included in the list
of valuable plant species due to its wide use in the Indian
system of medicine (Kapur et al., 1994). It posseses vari-
ous medicinal properties (Muthuswamy et al., 2012; Basri
et al., 2014; Bano et al, 2015; Lad et al., 2016).
The plant possesses anti-arthritic, hepatoprotective,
anti-in ammatory, anti-allergic, insecticidal, antioxi-
dant, antibacterial, immunomodulatory, antifungal as
well as mosquito repellant activities (Islam et al., 2013;
Zheng et al., 2014; Singh et al., 2015; Lad et al., 2015;
Lad et al., 2016). Leaves are aromatic, used as an antifer-
tility drug (Bhargava, 1986) and possess snake neutral-
izing activities, (Minu et al., 2012) (Muthuswamy et al.,
2012; Durairaj et al., 2014) Dharmadasa et al., (2016)
also reported the anti-snake venom properties. Leaves
are antiparasitic and used as alternative vermifuge and
anodyne. They are also very effective to reduce in am-
matory swellings of joints in rheumatism and relieve
catarrh and headache. Root is used as tonic, diuretic
and expectorant. It regulates hormones, enhances breast
milk production and possesses progesterogenic proper-
ties as well (Au et al., 2008; Arora et al., 2011; Basri
et al., 2014; Haider et al; 2017).
Betulinic acid, ursolic acid and -sitosterol are some
of its active constituents, isolated from its leaves which
have been found to possess anti-cancer, anti-HIV and
angiogenic properties, respectively (Basri et al., 2014). In
nature the species propagates through stem cutting and
seeds. Based on our preliminary investigations propaga-
tion with vegetative cuttings is very slow and the sur-
vival rate is very limited. Propagation through seeds is
hindered due to poor germination. Thus conventional
propagation through seeds and vegetative cutting is
not an adequate solution to meet the demand for this
rare medicinal plant. Hence this study was carried out
to develop an ef cient protocol for its mass cultivation.
Nodal explants were excised from elite plants of Vitex
negundo growing in medicinal plants garden, School of
Studies in Botany, Jiwaji University, Gwalior (M.P). The
excised nodal explants of V. negundo were washed for
10 min under continuous stream of running tap water.
Surface sterilization was done by treating the explants
with 4% (v/v) Tween-20 (detergent; SRL, Pvt. Ltd, Mum-
bai, India) and rinsed with distilled water. These explants
were then treated with 2% (w/v) bavistin solution (Sys-
temic fungicide; BASP India Ltd., Mumbai India) for 5
min and followed by treatment with freshly prepared
0.1% HgCl
(SRL, Mumbai, India) for 3 min with con-
tinuous shaking under a laminar  ow cabinet. These
explants were  nally washed 2-3 times by sterile dis-
tilled water prior to implantation in semisolid media.
The MS (Murashige and Skoog, 1962) basal medium
was supplemented with 6-Benzylaminopurine (BAP),
6-Furfuryl-aminopurine (KIN), Thidiazuron (TDZ), Indole-
3-butyric acid (IBA), 2,3,5-triiodobenzoic acid (TIBA),
-naphthalene acetic acid (NAA), at various concentra-
tions and in various combinations for rhizogenesis. Full
and half strength MS basal medium with IBA and NAA
at different concentration was employed. All the plant
growth regulators were procured from SRL and Himedia-
Qualigens, SRL, Glaxo, CDH, Titan biotech and Himedia.
3% (w/v) sucrose (SRL, Mumbai, India) was used as Car-
bon source, solidi ed with 0.8% agar-agar and pH was
adjusted to 5.75 using 0.1 N NaOH or 0.1 N HCl. 20 ml
media (aprox.) was dispended in each 150×25 cm test
tube (Borosil, India), tightly covered with air tight plastic
test tube caps and sterilized by autoclaving at 1.06 kgcm
at 121°C for 15 min. The explants were cultured in verti-
cal orientation in test tubes containing semisolid medium.
Cultures were maintained at 25±2 °C temperature with a
relative humidity of 55±5 % under regular cycle of light
(450-460 μW cm
) by cool day light emitted from  uo-
rescent incandescent tubes (40 W, Philips &Finolex, India)
of 16 hr light followed by 8 hr dark period.
After root formation, healthy plantlets with well
developed root system were removed from medium and
washed under running tap water to remove the medium.
These are then transferred to plastic pots (5 cm diam-
eter) containing autoclaved mixture of soil, sand and
vermicompost (1:1:1). Subsequently acclimatization was
achieved by covering the plastic pots with polythene
bags to maintain humidity. Plants were irrigated with
of major salts of MS media. After 1 week, 3-5
holes are made in the poly bags. Plants were irrigated
after every 5 days. The potted plants were maintained in
the culture room. After 30 days the plantlets were potted
in earthen pots with garden soil.
The shoot response of explants was evaluated after
35 days of culture in terms of percentage of explants
producing shoots, average number of shoots per explant
and average shoot length per explant. For root response,
percentage of shoot producing roots, average number of
roots per explant and average root length was recorded.
All the values have been reported as mean value along
with standard error (Mean ± SE).
An ever increasing demand of uniform medicinal plants
based medicines warrants their mass propagation through
plant tissue culture strategy. Tissue culture technology is
Amit Kumar et al.
potent and has opened extensive areas of research for
biodiversity conservation. Tissue culture protocols have
been developed for a wide range of medicinal plants,
which includes endangered, rare and threatened plant
species. (Sharma et al., 2010). Conventional propaga-
tion methods are unable to meet the demand of the
pharmaceutical industries and drug research. Therefore,
it is necessary to develop a non-conventional method
for propagation to ful ll the demands of the drug mar-
ket (Rathore et al. 2008). In vitro propagation methods
offer a powerful tool for conservation of germplasm and
mass-multiplication of threatened plant species (Murch
et al. 2000). It helps in micropropagation of large num-
ber of plant in shorter time period, irrespective of season
and serves as an alternative source of plant propaga-
tion (Yadav and Singh, 2012; Yadav et al., 2013; Groach
et al., 2014). This method can be employed in multiply-
ing important endangered plant species which are dif-
cult to propagate by conventional means and saves the
plant from the extinction.
In order to establish an ef cient in vitro micropro-
pagation protocol for commercial exploitation of this
plant, nodal explants of V. negundo were inoculated on
MS medium supplemented with varied concentration (0,
0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 mgl
) of cytokinins
(BAP, KIN) and TDZ (0.5-2.0 mgl
). The nodal segments
cultured on growth regulator free MS medium showed
minimum signs of bud break even after 15 days of inoc-
ulation. The average number of shoots induced on MS
basal medium was 10% with an average shoot length of
0.72 ± 0.01 cm after 35 days of culture (Table 1).
However, addition of cytokinin was essential for dif-
ferentiation of multiple shoots from the nodal explants.
Of the three cytokinins tried, BAP was most effective over
the other two for induction of multiple shoots. Similar
effect have already been reported in various taxa like Cas-
sia angustifolia (Agrawal et al., 2002), Spilanthes acmella
(Pandey and Agrawal 2009), Aegle marmelos (Yadav
and Singh 2011), Tylophora indica (Faisal et al., 2007),
Achyranthes aspera (Ishwarya et al., 2018), Vitex trifolia
(Ahmad and Anis, 2014).The nodal segments responded
by initial enlargement of dormant axilary buds followed
by bud break within a week and multiple shoot induc-
tion and proliferation within 15 days of culture on BAP
containing media. 1 mgl
BAP was optimum in inducing
90% morphogenic culture with an average of 4.29±0.07
shoots per explant having an average shoot length of
3.28±0.31 cm after 35 days of culture (Table 1, Fig.1A).
BAP at 3.5 mgl
displayed poor morphogenic response
both in terms of average number of shoots and average
Table 1. Effect of growth regulators on shooting during in-vitro
culture of Vitex negundo L. on MS media.
% of shoot
Average number
of shoots per
(Mean ± SE)
Shoot Length (cm)
(Mean ± SE)
Control 0 10 0.12 ± 0.01 0.72 ± 0.01
BAP 0.5 80 3.48 ± 0.34 2.64 ± 0.59
1.0 90 4.29 ± 0.07 3.28 ± 0.31
1.5 80 2.59 ± 0.37 2.01 ± 0.27
2.0 70 3.11 ± 0.82 1. 81 ± 0.14
2.5 50 2.40 ± 0.30 1.78 ± 0.07
3.0 40 2.10 ± 0.15 1. 62 ± 0.14
3.5 30 2.00 ± 0.03 1.50 ± 0.18
KIN 0.5 70 2.43 ± 0.03 1.86 ± 0.18
1.0 60 2.87 ± 0.24 3.02 ± 0.24
1.5 80 3.47 ± 0.14 3.33 ±0.08
2.0 70 2.8 ± 0.2 2.17 ± 0.20
2.5 60 2.62 ± 0.18 1.81 ± 0.34
3.0 50 1.92 ± 0.20 1.61 ± 0.16
3.5 40 1.64 ± 0.07 1.63 ± 0.16
TDZ 0.5 60 2.16 ± 0.16 1.63 ± 0.19
1.0 50 2.4 ±0.28 1.46 ± 0.06
1.5 30 1.75 ± 0.25 1.7 ± 0.09
2.0 20 2.33 ± 0.33 1.43 ± 0.12
Amit Kumar et al.
FIGURE 1. In vitro plant regeneration through nodal segment and estab-
lishment of Vitex negundo L. (A) Cultures showing shoots on MS medium
with BAP (1.0 mgl
), (B & C) Multiple shoot development from nodal
explants on MS with BAP + IBA (1+0.50 mgl
) (D) Formation of light
creamy callus on TDZ (E) In vitro rooted shootlet with half MS medium
+ 0.75 ml
IBA (F) Acclimatized plantlets, 35 days old (G) Plants in pots.
Table 2. Effect of BAP 1 mgl1 and 1.5 mgl1 KIN in combination with different concentrations
of auxins on shoot bud regeneration from nodal explants of Vitex negundo
Concentration of
growth regulators
in mgl1
IBA TIBA Percentage
of explants
shoots (%)
Number of shoots
per explant
(Mean ± SE)
Shoot Length (cm)
(Mean ± SE)
0.25 60
2.66 ± 0.33 3.23 ± 0.09
90 6.12 ± 0.63 3.85 ± 0.21
0.75 70
3.14 ± 0.40 3.78 ± 0.39
0.25 50
2.8 ± 0.37 3.02 ± 0.17
0.50 70
3.5 ± 0.42 3.53 ± 0.08
0.75 60
2.5 ± 0.34 3.28 ± 0.10
KIN 1.5
0.25 70
4.37 ± 0.41 2.83 ± 0.26
0.50 80
5.88 ± 0.38 4.13 ± 0.43
0.75 60
4.16 ± 0.60 3.18 ± 0.17
0.25 60
4.33 ± 0.42 3.21 ± 0.20
0.50 80
5.2 ± 0.53 3.71 ± 0.15
0.75 70
4.38 ± 0.47 3.32 ± 0.12
Results were recorded after 35 days and are presented as Mean ± Std. Error.
Amit Kumar et al.
shoot length (Table 1). On increasing the concentration of
BAP, induction of multiple shoots was comparatively low
and average shoot length too decreased (Table 1). Except
BAP, all the tried concentrations of KIN and TDZ showed
poor morphogenic response in term of average number of
induced shoots and shoot length (Table 1). Considerable
callusing at the basal cut end of nodal segment along
with formation of multiple shoots was also reported in the
present study which agrees with the study on Azadirachta
indica (Arora et al., 2010) which showed similar results
(Fig.1 D). The formation of callusing at the basal cut ends
of nodal segment due to the action of accumulated auxins
at the basal cut proliferation, especially in the presence
of cytokinins (Marks and Simpson, 1994). The present
study also revealed the synergistic effect of BAP in com-
bination of auxin for effect shoot regeneration which has
also been reported in studies of Celastrus paniculatus (Lal
et al., 2010).
The highest number of shoots (6.12±0.63) developed
was observed in MS with BAP 1 + IBA 0.50 mgl
2 Fig.1 B & C). The highest proliferation rate (90%) was
also found at the same combination of plant growth
regulators in the medium.
The best results were observed on a medium contain-
ing BAP and IBA which is supported by earlier stud-
ies in Chonemorpha grandi ora (Nishitha et al., 2006),
Vitex negundo (Ahmad and Anis, 2011), Launaea cor-
nuta (Ambajo and Matheka, 2016). Mimosa pudica
(Bianchetti et al., 2017) Tylophora indica (Najar et al.,
2018), Ceropegia juncea (Binish, 2018), In these stud-
ies also synergitic effects were observed when Cytokinin
was used in combination with auxin. Among the two
different types of auxins employed for root induction on
in vitro excised shoots of V. negundo, IBA was found to
be most effective. A maximum of 90% shoots induced
an average of 12.12±0.83 roots with an average root
length of 4.98±2.50cm after 3 weeks on half strength
MS medium augmented with 0.75 mgl
IBA (Table 3).
The roots were induced directly from the shoot base
without callus formation at this concentration. (Table 3,
Fig.1 E). Similar responses have been already reported in
Spilanthes acmella (Pandey and Agrawal 2009, Yadav
and Singh 2010), (Reddy et al., 2014), Ceropegia juncea
(Binish, 2018),.However, at higher concentration of IBA,
the number of roots and root length showed decline.
Compared to IBA, poor rooting response was observed
at the concentration of IBA + full MS and NAA + full
and half MS. The tissue culture derived plantlets (Fig.1 F
& G) were acclimatized in the  eld condition with 90%
survival. Such micropropagated plants were found to be
morphologically similar to the mother plant.
An ef cient protocol has been developed for regen-
eration of Vitex negundo which offers a great potential
to cater the needs of different pharmaceutical industries.
In the present study, enhanced in vitro regeneration of
plants with combination of plant growth regulators such
as BAP, KIN, TDZ, IBA and TIBA was observed. This
will be helpful in understanding the callogenesis and
organogenesis through the nodal explants and to facili-
tate the mass propagation of Vitex negundo.
Table 3. Effect of growth regulators on rooting pattern of Vitex
negundo L. during in-vitro culture (full and ½ strength of MS
of explants
roots (%)
number of
roots per
root length
MS Full
0.50 70 5.71 ± 0.71 1.88 ± 1.20
0.75 80 6.75 ± 0.52 2.33 ± 2.21
1.0 60 4.33 ± 0.42 1.86 ± 2.10
MS half
0.50 80 7.57 ± 0.92 3.25 ± 3.76
0.75 90 12.12 ±0.83 4.98 ± 2.50
1.0 70 8.9 ± 0.89 4.53 ±3.92
MS full
0.50 70 4.85 ± 0.76 1.32 ± 0.77
0.75 80 6.25 ± 0.59 2.23 ± 2.62
1.0 60 4.33 ± 0.42 1.71 ± 3.37
MS half
0.50 70 6.8 ± 0.86 1.74 ± 0.07
0.75 90 8.42 ± 0.89 2.66 ± 0.30
1.0 60 7.33 ± 0.66 1.95 ± 0.13
Amit Kumar et al.
The authors are thankful to Director, Institute of Ethno-
biology and Head, School of Studies in Botany, Jiwaji
University Gwalior (M.P.) India, for providing necessary
facilities to carry out this work.
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