Int. J. Med. Arom.

Plants, ISSN 2249 4340

RESEARCH ARTICLE

Vol. 4, No. 1, pp. 48-55, March 2014

Efficient protocol for direct shoot organogenesis from in vitro raised

nodal explants of Solanum viarum (Dunal) - An important anticancer
medicinal plant
M.D. MAHADEV, CHANDRA SEKHAR PANATHULA, C.V. NAIDU*
Department of Biotechnology, Dravidian University, Kuppam-517426, A. P., India
*Corresponding author: Phone: +91-8772260386; Fax: +91-8570278209
Article History: Received 12th March 2014, Revised 25th March 2014, Accepted 27th March 2014.
Abstract: In the present study an attempt has been made to standardize a protocol for rapid direct plant regeneration from
in vitro derived nodal explants of S. viarum, which promoted shoot bud regeneration and resulted in maximum number of
multiple shoot induction in the medium supplemented with different concentrations of plant growth regulators such as
cytokinins like BAP, Kn alone or in combination with auxins like NAA/IBA/IAA. Among the combinations tested BAP
(2.0 mg l-1) and IBA (0.5 mg/l-1) produced maximum number of shoots (22.30.41) per explant. The maximum shoot
length (8.20.14 cm) was observed with Kn (1.0 mg l-1) and IBA (0.5 mg l-1) combination. All the in vitro raised shoots
with a length of 3.0-5.0 cm were transferred to rooting medium supplemented with different concentrations of auxins
such as NAA, IBA and IAA (0.2 1.0 mg l-1). The best rooting response was observed on IBA (0.6 mg l-1). The well
rooted plantlets were transferred to polycups containing soil and vermiculite in 1: 1 ratio for hardening. Finally the hardened plantlets were transferred to field conditions for maximum survivability.
Keywords: Multiple shoot induction; Nodal explants; Plant regeneration; Solanm viarum.
Abbreviations: BAP- Benzyl aminopurine; IAA-Indole-3-acetic acid; IBA-Indole-3-butyric acid; Kn- Kinetin; NAA- Naphthalene acetic acid.

Introduction
Multiplication of plants in vitro through
plant tissue culture methods have emerged in
late 1980s as a valuable approach to increase
plant productivity (Gupta et al. 1993). In recent
years, there has been an increased interest in in
vitro culture techniques which offer a viable
tool for mass multiplication and germplasm
conservation of rare, endangered, aromatic and
medicinal plants (Arora and Bhojwani 1989;
Sudha and Seeni 1994). Solanaceae family
comprises a number of plants widely known for
the presence of variety of natural products of
medicinal significance mainly steroidal lactones, glycosides, alkaloids and flavonoids. Solanum viarum (D.) is an ample branched, prickly shrub 1-2 meters tall at maturity. It is a member of economically significant family
Solanaceae. The plant grown in India as a richest source of steroids (Mullahey et al. 1987),
which yields an economically vital source of
glycoalkaloide solasodine which is present in

the fruit is a pioneer for the steroid disogenin

(Budhavari 1989). This is used in the production
of steroid hormone for treating cancer, rheumatic arthritis, addisons disease and for providing
contraceptives (Srinivasan 2005). Since the high
economical and pharmological values of secondary metabolites, industries are intensely attracted in utilizing the plant tissue culture technology for large scale production of these substances (Misawa 1994.). The present investigation was performed to determine the role of different plant growth regulators on direct shoot
regeneration from in vitro derived nodal explants of Solanum viarum.
Materials and methods
Plant material and surface sterilization
Nodes of axillary branches were excised
from the in vitro regenerated shoots of S. viarum
were used as explants as a prerequisite for experiments. Therefore for the explant source the

*Corresponding author: (E-mail) challagundlav <@> yahoo.co.in

2014 Copyright by the Authors, licensee Open Access Science Research Publisher.

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Int. J. Med. Arom. Plants

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In vitro direct shoot organogenesis from nodal explants of Solanum viarum

seeds were collected from mature and dried

fruits of healthy plants of S. viarum that are cultured in the herbal garden, Dravidian University,
Kuppam, A. P., India. Matured seeds were first
washed under running tap water for 30 mins. to
take away any adherent fruit tissues and dried
juice which might serve as an agent for fungal
contagion. Then the seeds were rigorously
washed with 0.4% bavistin (w/v) a fungicide
under sterile conditions for an hour and washed
thrice with sterile distilled water. This was followed by surface sterilization with 5% Tween20 (v/v) for 15 mins. and the seeds were sterilized in 70% alcohol (v/v) for 2 mins. Then the
seeds were washed with 0.1% HgCl2 (w/v) for 2
mins. Finally the seeds were washed thrice with
sterile distilled water to remove the traces of
HgCl2.
Culture medium and culture conditions
The seeds were inoculated on MS medium
(Murashige and Skoog, 1962) containing 0.8%
agar (w/v) supplemented with various concentrations of cytokinins BAP and Kn and auxins
(NAA, IAA and IBA) previous to autoclave, the
medium was adjusted to the pH of 5.8 and sterilized for 20 mins. at 121C for 15 lbs pressure.
The culture room conditions maintained for in
vitro cultures were 26C 2C and 60-70% relative humidity. Light intensity was 3000 lux with
a photoperiod of 18 hrs day light and 6 hrs in
dark. The primary shoots formed in vitro were
separated aseptically and cultured on MS medium supplemented with BAP (2.0 mg l-1).
Sub culturing
Axillary bud explants (1.0 2.0 cm) were
excised from the in vitro regenerated shoots of
S. viarum were inoculated on MS medium containing 3.0% sucrose and gelled with 0.8% agar
supplemented with various concentrations of
cytokinins like BAP, Kn alone or in combination with auxins such as NAA, IAA and IBA
were used for shoot proliferation. The pH of the
medium was adjusted to 5.8 before gelling with
agar and autoclaved for 20 mins. at 1210C for 15
lbs pressure. The cultures were maintained by
regular intervals of 21 days on fresh MS medium.
Mahadev et al.

Data analysis
Visual observations were recorded on the
frequency in terms of number of cultures responding for axillary shoot proliferation, shoot
development, number of shoots per explant, average length of the regenerated shoots, number
of roots per shoot and average root length.
Statistical analysis
All the experiments were conducted with a
minimum of 20 explants. All assays were repeated at least three times. The experimental
data were statistically analyzed by one-way
ANOVA using the DMRT (Duncans Multiple
Range Test) (P < 0.05) and were presented as
the mean standard error (SE).
Results and discussion
Effect of cytokinins alone on direct shoot regeneration from in vitro derived axillary bud explants of S. viarum
Effect of two different cytokinins BAP and
Kn on direct shoot regeneration was examined
separately. After two weeks of inoculation shoot
bud primordial was emerged along the cut portions of nodal explants, later they were developed as shoots. In BAP (0.5-3.0 mg l-1) alone
supplemented MS medium shoot initiation was
observed and the maximum mean number of
shoots (10.30.31) was observed at (2.0 mg l-1)
(Figure 1a) and mean shoot length ranged from
(2-4 cm) respectively. The optimum concentration of BAP was found to be (2.0 mg l-1). The
number of multiple shoots were decreased with
further increase in BAP concentration (> 3.0 mg
l-1). In Kn (0.5-3.0 mg l-1) alone supplemented
MS medium shoot bud initiation was observed
and the mean number of shoots ranged from
(4.0-8.0) with mean shoot length (3.0-5.0 cm).
The number of shoots were less compare to
BAP supplemented medium (Table 1). Direct
regeneration from nodal explant is another substitute stepfor clonal propagation and
germplasm conservation is a well-established
factor. In vitro regeneration of plants depends
on the interaction between endogenous growth
substances and the artificial growth regulators
which may be added to the medium.The stimuhttp://www.openaccessscience.com
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50
In vitro direct shoot organogenesis from nodal explants of Solanum viarum

lation of direct shoot regeneration depends on

the nature of the plant organ from which the explant was derived and is extremely dependent
on plant (George 1993a). Nodes contain axillary
meristem which can generate direct proliferation
of shoots. Preexisting meristems are the base of
the two pathways: direct shoot organogenesis
and direct shoot proliferation. Meristems are

highly reactive cells and plants regenerated from

them generally could show no variation in genotypic and phenotypic characters (Saeed et al.
1997). In the present study the cytokinin alone
supplemented medium multiple shoots become
visible after 2-3 weeks of inoculation this was
well reported in Celastrus paniculatus (Lakshmi
and Seeni 2001).

Table 1: Direct shoot organogenesis from nodal explants of in vitro raised S. viarum micro shoots.

BAP
0.5
1.0
2.0
3.0
1.0
2.0
3.0
1.0
2.0
3.0
1.0
2.0
-

Plant growth regulators

(mg l-1)
Kn
NAA
IBA
0.5
1.0
2.0
3.0
0.5
0.5
0.5
1.0
0.5
2.0
0.5
3.0
0.5
0.5
0.5
0.5
1.0
0.5
2.0
0.5
3.0
0.5
1.0
2.0
-

IAA
0.5
0.5
0.5
0.5

Regeneration
frequency
(%)
75
80
85
80
78
80
83
80
82
90
86
78
83
80
88
98
92
87
90
85
83
77
92
90

Mean no. of
shoots/
explant
6.50.13c
9.20.24i
10.3 0.31j
7.50.22de
4.20.51a
7.30.37d
8.60.23gh
6.80.42c
8.70.20h
12.20.31k
9.30.28i
7.20.14d
10.60.36j
5.80.43b
14.00.26m
22.30.41q
16.10.51p
10.50.17j
13.40.61l
7.80.28ef
10.30.18j
8.20.21fg
11.80.53k
7.90.36ef

Mean
shoot length
(cm)
4.50.15fg
3.60.32bc
2.40.23a
3.90.14cde
5.20.26i
4.70.19gh
4.00.25cde
3.70.52cd
5.30.41i
3.20.28b
4.60.35gh
4.10.15def
3.20.13b
5.00.12hi
5.40.22ij
3.80.25cd
4.70.52gh
8.20.14m
6.30.31k
7.20.26l
4.30.24efg
5.80.42j
4.50.28fg
6.30.51k

Data represent treatment means SE followed by different letter (s) within column indicate significant differences according to ANOVA and DMRT test (P < 0.05).
Effect of BAP in combination with auxin on direct shoot regeneration from in vitro derived
axillary bud explants
BAP, Kn in combination with auxins (NAA
or IBA or IAA) on direct shoot regeneration was
examined. BAP (0.5-3.0 mg l-1) in combination
with NAA, IBA and IAA (0.5 mg l-1) were used
to produce direct shoot regeneration from the
axillary bud explants of in vitro regenerated S.
viarum micro shoots. After two weeks of inoculation shoot bud primordial was emerged along
the cut portions of nodular explants, later they
were developed as shoots.In BAP and NAA
supplemented medium the mean number of
Mahadev et al.

shoots were ranged from (8.7-12.2) was obtained with shoot length (3.2-5.3 cm) respectively (Figure 1b). Whereas the lower concentration
of cytokinin BAP (1.0 mg l-1) and auxin IAA
(0.5 mg l-1) favored the mean shoot number
(10.30.18). With further increase in concentration of BAP the shoot number decreased to
(8.20.21). The highest number of shoots was
obtained in the combination of BAP (2.0 mg l-1)
and IBA (0.5 mg l-1) with (22.30.41) shoots
(Figure 1c) with a shoot length (3.80.25 cm)
(Table 1) but maximum shoot length was obtained on Kn (1.0 mg l-1) in combination with
IBA (0.5 mg l-1). The shoot number were decreased with further increase in the BAP conhttp://www.openaccessscience.com
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Int. J. Med. Arom. Plants

51
In vitro direct shoot organogenesis from nodal explants of Solanum viarum

centration (> 2.0 mg l-1). BAP preferred for

multiple shoot induction and proliferation from
nodal explants than Kn in the present study. In
general, BAP is the most effective growth regulator for stimulation and shoot proliferation.
BAP mimics as an inhibitor agent and drives
beside apical dominance of shoot induction and

shoot bud formation (Wang and Charle 1991).

Advantage of BAP over any cytokinin in induction and proliferation of shoots has been very
well standardized in Quercus rober (Puddephat
et al. 1997) and Solanum nigrum (Sridhar and
Naidu 2011).

Table 2: Root organogenesis of in vitro derived shoot lets of S. viarum supplemented with various
concentrations of IAA, NAA and IBA using half strength MS medium.
Plant growth regulators (mg l-1)
Regeneration
Mean number of
Mean root length
frequency
(%)
roots/shoot
(cm)
NAA
IBA
IAA
b
0.2
83
7.20.27
4.50.26g
d
0.4
85
9.30.34
4.80.41g
0.6
78
11.20.42f
3.70.52ef
h
0.8
65
14.50.35
2.40.27a
c
1.0
60
8.30.31
2.60.13ab
0.2
87
10.40.22e
7.00.12h
g
0.4
92
13.30.36
4.60.32g
0.6
98
19.20.22j
2.30.27a
h
0.8
90
14.10.35
3.50.15de
1.0
85
10.30.21e
3.20.18d
a
0.2
75
6.50.42
3.60.28def
0.4
80
9.70.43d
3.30.25de
g
0.6
85
13.20.30
4.00.16f
0.8
78
15.60.26i
3.00.29bc
f
1.0
70
11.60.51
2.40.18a
Data represent treatment means SE followed by different letter (s) within column indicate significant differences according to ANOVA and DMRT test (P < 0.05).
Effect of Kn in combination with auxin on direct
shoot regeneration from in vitro derived axillary
bud explants
Direct shoot induction from the in vitro derived axillary bud explants of S. viarum was also observed on Kn alone and in combination
with auxins like NAA, IBA and IAA but the
number of shoot formation was less when compared with BAP. The optimal concentration for
direct shoot regeneration using Kinetin was (2.0
mg l-1) in combination with IBA (0.5 mg l-1)
produced (13.40.61) mean number of shoots.
The maximum shoot length (8.20.14 cm) was
observed at Kn (1.0 mg l-1) and IBA (0.5 mg l-1)
(Table 1). In Kn and NAA supplemented medium the maximum shoot number (10.60.36)
was obtained with a mean shoot length
(3.20.13 cm) at Kn (2.0 mg l-1) and NAA (0.5

Mahadev et al.

mg l-1). Whereas in Kn and IAA supplemented

medium the maximum shoot number
(11.80.53) was obtained with a mean shoot
length (4.50.28 cm) at Kn (1.0 mg l-1) and IAA
(0.5 mg l-1). With further increase in the concentration of kinetin number of shoots were decreased.
The combination of cytokinin and auxin
seems to have a collaboration effect, as
cytokinins enhance cell division, stimulate axillary bud and adventitious shoot proliferation and
auxins regulate cell elongation, tissue swelling
and shoot expansion. The effect of auxins and
cytokinins on enhancing shoot regeneration has
been reported in several spices, such as Echinacea purpurea (Koroch et al. 2002) and Catalpa
ovate (Lisowska and Wysokinska 2000).

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In vitro direct shoot organogenesis from nodal explants of Solanum viarum

Figure 1: Direct regeneration of shoots from nodal explants of in vitro raised microshoots of S. viarum.
a) Shoot bud initiation from nodal explant after 7 days of inoculation on MS medium + BAP (2.0 mg l-1)
b) Initiation of multiple shoots from nodal explant on MS medium + BAP (2.0 mg l-1) + NAA (0.5 mg l1
)
c) Multiplication of shoots from nodal explant on MS medium + BA (2.0 mg l-1) + IBA (0.5 mg l-1)
d) Elongated multiple shoots regenerated from nodal explant on MS medium + BAP (3.0 mg l-1) + IBA
(0.5 mg l-1)
e) Initiation of roots from the regenerated shoots in vitro on MS medium + (IBA 0.6 mg l-1)
f) Plantlet showing elongated root system
g) Hardened plantlet in polycups containing soil and vermiculite in 1:1 ratio
h) Plantlet in field conditions.
Mahadev et al.

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53
In vitro direct shoot organogenesis from nodal explants of Solanum viarum

In the present study in vitro derived nodal

explants promoted shoot bud regeneration in
presence of the cytokinin BAP, Kn alone or in
combination with auxins like NAA/IBA/IAA
and resulted in maximum number of multiple
shoot induction. Among the auxins tested
(NAA, IBA and IAA) in combination with
BAP, natural auxin IBA proved to be the better
for providing maximum number of shoots.
Similar response was studied earlier in Platanus
acerifolia wild (Liu and Bao 2003). The next
best auxin which performed well was NAA in
combination with BAP. The effect of BAP and
NAA on direct shoot regeneration was reported
in Gysophila paniculata (Zukar et al. 1997) and
Spilanthes acmella (Saritha and Naidu 2008).
IAA in combination with Kn also have showed
the effect on direct shoot regeneration. These
results are in line with the observations of Stevia
rebaudiana (Preethi et al. 2011) and Cajanus
cajan (Misra 2002).
In vitro rooting
The in vitro developed shoots were excised
from clump of shoots and transferred on MS
medium supplemented with different concentration of auxins such as NAA, IBA and IAA (0.21.0 mg l-1). The best rooting response was observed on IBA (0.6 mg l-1) supplemented medium, (Figure 1e) where maximum number of
roots (19.20.22) were obtained followed by
IAA (0.8 mg l-1), NAA (0.8 mg l-1) supplemented medium where (15.60.26), (14.50.35)
number of roots were induced respectively. The
number of roots decreased with further increase
in auxin concentration (>0.8 mg l-1) (Table 2).
The excised shoots cultured on the MS medium
containing different auxin concentrations for
rooting in which the IBA performed well in
producing the maximum number of roots/shoot
with higher regeneration frequency these reports
were similar with the study on Mentha piperita
(Sujana and Naidu 2011). Rooted shoots
showed the maximum percentage of survival.
Among the plant growth regulators tested, in
most of the combinations, the comeback of direct regeneration from nodal explants were resulted in prompting shoot regeneration directly
from the nodal explants without forming
Mahadev et al.

callus. The excised nodes from in vitro derived

shoots were used as explants for further regeneration as this is also true in Cuminum cyminum
(Esmaeil Ebrahimie et al. 2007).
Acclimatization and hardening
Well-developed rooted plants were carefully
removed from culture tubes and washed to remove the leftovers of agar (Figure 1f). These
healthy shoots were transferred to tray containing autoclaved soil and vermiculate in 1:1 ratio
for acclimatization (Figure 1g). For a period of
8-10 days the plants were kept in polythene
membrane. After that the surviving plants were
transferred to pots and allowed to grow under
nursery shade conditions (Figure 1h). Finally
these acclimatized plants were planted in field
conditions with maximum survivability.
Acclimatization of regenerated plants to the
outside environment is the last stage in plant
tissue culture and its achievement depends on
different factors as proposed by various researchers (George and Sherrington 1984b). Prior
other investigation workers studied acclimatization of micropropagated plants in the green
house under field environments (Preece and Sutter 1991).
Conclusion
In the present study, regeneration of S.
viarum was greatly influenced by different plant
growth regulators supplemented in the media. It
can be concluded that based on readily available
nodal explants of S. viarum reproducible in vitro
direct shoot regeneration and proliferation, has
been described. The results obtained in the present study could be of enormous significance,
which contribute to the development in the
micropropagation of this economically important medicinal plant on commercial scale to
light the current day demand.
Acknowledgements: The authors wish to express their gratitude to the UGC BSR (NonSAP) New Delhi, India, for supporting this research work by providing financial assistance in
the form of fellowship.
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In vitro direct shoot organogenesis from nodal explants of Solanum viarum

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