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Ethnobotanical
Leaflets 12:
Micropropagation Studies on Vitex negundo L.
– A Medicinally Important Plant
Johnson, M.*1, Sonali Das, Yasmin,
N. and Rajasekara Pandian,
M.
Centre for Biotechnology
Research Department of Biotechnology
Muthayammal College of Arts and Science, Rasipuram
1 Present Address: Research Department
of Plant Biology and Plant Biotechnology
St. Xavier’s
College (Autonomous)
Palayamkottai,
Tamil Nadu, India – 627 002
*Author for correspondence: biojohnson@fastmail.fm
Tel: + 91 97 86 92 43 34
Issued 25 July 2008
Abstract
Vitex negundo Linn. belonging
to a gamopetalous family is a medicinally important
plant and has been micropropagated successfully by
culturing nodal segments. MS basal medium with 3% sucrose and augmented with
6 benzyl amino purine at 4.44 mm was showed the maximum number of shoot
proliferation from the nodal segments. Maximum percentage of shoot
proliferation was achieved on Murashige and Skoog’s basal medium with 3% and fortified with 6 Benzyl amino purine 4.44mm.
The in vitro raised shootlets transferred to half strength Murashige and Skoog’s medium
supplemented with auxin for rooting. Maximum
number (7.5+0.23) of rootlets were observed on MS medium
augmented with IBA (4.92mM). The in vitro raised plants were hardened then transferred to field
for re-establishment.
Key Words:
Vitex negundo, in vitro shoot proliferation, Clonal propagation.
Introduction
Vitex negundo
Linn., a member of the gamopetalous
family Verbenaceae,
is a medicinally important plant distributed throughout India.
The plant is bitter, acrid, thermogenic,
expectorant, carminative, digestive, stomachic, anodyne, anti-inflammatory,
antiseptic, cephalic, alterant, antipyretic,
diuretic, emmenagogue, depurative, rejuvenating,
ophthalmic, vulnerary and tonic. The roots used in vitiated conditions of vata, kapha, jajvara, cephalalgia, sprains, orchitis,
gout, splenohepatomegaly, otorrhoea,
inflammations ulcers, cephalagia, otalgia, arthritis, inflammation, dyspepsia, colic, verminosis, flatulence, dysentery, uropathy,
wounds, bronchitis, cough, malarial fever, haemorrhoids,
dysmenorrhoea, leprosy, dermatopathy,
ophthalmopathy and general debility. The bark is
used in vitiated conditions of vata, odontalgia, verminosis and ophthalmopathy. The flowers used in diarrhoea,
cholera, fever, haemorrhages, hepatopathy
and cardic disorders1 The whole plant is
having great demand on the market due to its medicinal value. The
conventional methods were applied for multiplication of this medicinal plant
the results are not satisfactory, in vitro propagation is an
alternative tool for large scale multiplication and a number of reports are
available for in vitro multiplication. The present study was initiated
to produce an in vitro multiplication procedure for rapid clonal propagation and field establishment of this
species.
Materials and Methods
Plants of vitex negundo L.
collected from Kollimalai, salem were
established in the green house and herbal garden. Young shoots were washed
with running tap water and surface sterilized in 0.1 (w/v) HgCl2
solutions for 60 sec. After rinsing 3-4 times with sterile distilled water,
leaves, stem nodes, internodes were cut into smaller segments (1cm) used as
the explants. The explants were placed horizontally as well as vertically on
solid basal Murashige and Skoog
(1962)2 (MS)
medium supplemented with 3% sucrose, 0.6%(w/v) agar (HIMEDIA, Mumbai) and
different concentration and combination of BAP,
Kin, NAA, IAA and 2, 4-D for direct and indirect organogenesis. The pH of the
medium was adjusted to 5.8 before autoclaving at 121oC for 15min.
The cultures were incubated at 25+2oC under cool
fluorescent light (2000 lux 14 hr photoperiod).
Four rooting plantlets were removed from culture, washed thoroughly with tap
water planted in small polycups filled with sterile
garden soil (3:1), covered by unperforated polybags, and hardened for 4 weeks in a mist chamber
before transfer to field.
Results and Discussions
MS medium augmented
with different concentrations of BAP was
used for multiple shoots emergence from the nodal segments. After 7 days of
inoculation, the buds were started for shoot proliferation (Fig. 1 A). The effect of BAP
on shoot multiplication from nodal explants is shown in table 1. MS medium supplmented with 6.66mM
of produced maximum number of shoots (8.2+0.79) with maximum
percentage (75.3+0.03) of multiple shootlets
formation (Fig. 1 B, C, D, E and F). MS medium augmented with BAP
induced multiple shoot formation was reported in Baliospermum axillare3, solanum surattense
4and Baliospermum montanum5. The present
study observations were directly coincided with the previous observations 3,
4 & 5. When the concentration of BAP
increased there is reduction in number of multiple shoots (Table 1).
The in vitro raised shootlets were transferred to half strength MS medium
with different concentrations and combinations of IBA,
IAA and NAA for rooting (table
2). Maximum number (7.5+0.23) of rootlets were observed on MS medium
augmented with IBA (4.92mM) (Fig. 1 G & H). The present study
report was directly coincide with previous workers observations Johnson and
Manickam5 in baliospermum montanum; sivasubramanian
et al.,6 in plectranthus vetiveroides and Johnson et al., 7 in Adenia hondala and Justicia jendurussa they got maximum number of rootlets on the
very same medium and hormone (1/2 MS+IBA
4.92mM) and their results supported
the present observation.
After 30 days of rooting, in vitro raised
plantlets were hardened in polycups containing a
mixture of sterile garden soil: sand (3:1), covered with polypropylene bags
and irrigated with 10 x diluted MS liquid medium. The plants were kept in a
culture room for 15 days. 90% of plants were successfully established in polycups (Fig. 1 I). After 15 days the polycups hardened plants, were transferred to pots and
kept in green house. 90% of plants were well established in the green house
condition. After one month, the plants were transferred to the field. About
80% of plants were established in the field. In the present study, we
successfully developed the protocol for in vitro propagation of the
medicinally important plant Vitex negundo (L).
REFERENCES
1. Warrier P.K., Nambiar V.P.K.,
and Raman Kutty . (1995) Vitex negundo L. In Indian medicinal plants Vol IV, Orient
Longmen Ltd., Madras
India.
2. Murashige, T. and Skoog
F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue
culture, Physiologia Pl. 15: 473-497.
3. Singh, K.and Sudarshana,
M. S. (2003) In vitro micropropagation of Baliospermum axillare
Blume. Indian Journal of Plant Physiology 8,
125-128.
4. Pawar, P. K., Pawar,
C.S., Narkhede, B.A., Teli,
N.P., Bhalsing, S.R. and Meshwari,
V.L. (2002) A Technique for rapid micropropagation
of Solanum surattense
Brum. f. Indian
Journal of Biotechnology 1,
201-204.
5. Johnson, M. and Manickam, V.S. (2003) In
vitro micropropagation of Baliospermum
montanum (Willd.) Muell-Arg- A Medicinal Plant. Indian Journal of Experimental
Biology 41,
1349-1351.
6. Sivasubramanian, S., Vallinayagam,
S., Raja D. Patric and Manickam,
V.S. (2002) Micropropagation of Plectranthus
ventiveroides (Jacob) Singh and Sharma – A
medicinal plant, Phytomorphology (52)
1, 55-59.
7. Johnson M., Manickam V. S., Nikhat Y., Sonali D., and Andal N. (2004) In
vitro multiplication of two economically important and endangered
medicinal plants – Justicia gebdarussa Brum and Adenia hondala (Gaertn) De Wilde Malaysian Journal of science 23 ( 2), 49 - 53 .
Table-1: influence
of plant growth regulators on the multiple shoot lets formation from the
nodal segments of vitex negundo L. cultured on MS medium.
|
S.No
|
MS medium with plant growth regulators (mm)
|
% of shoot lets formation +
S.E.
|
Mean length of shootslets + S.E.
|
No.of shootlets
/ Explant + S.E.
|
Callus formation
|
|
BAP
|
KIN
|
NAA
|
|
1
|
0.0
|
0.0
|
0.0
|
0
|
0
|
0
|
-
|
|
2
|
2.22
|
0.0
|
0.0
|
65.3+0.20
|
3.4+0.42
|
4.0+0.78
|
-
|
|
3
|
4.44
|
0.0
|
0.0
|
71.4+0.10
|
4.5
+0.50
|
7.0+0.87
|
-
|
|
4
|
6.66
|
0.0
|
0.0
|
75.3+0.03
|
6.0+0.68
|
8.2+0.79
|
-
|
|
5
|
8.88
|
0.0
|
0.0
|
70.8+0.20
|
7.1+0.73
|
6.6+0.56
|
-
|
|
6
|
11.1
|
0.0
|
0.0
|
68.1+0.01
|
5.0+0.64
|
5.0+0.02
|
+++
|
|
7
|
0.0
|
4.65
|
2.69
|
-
|
-
|
-
|
+++++
|
+ sign indicates degree of callus formation.
Table-2: influence
of plant growth regulators on the multiple root lets formation from in
vitro raised shoot lets of vitex negundo L
on half strength MS medium.
|
S.
No
|
½ MS medium with plant growth regulators (mM)
|
% of root lets formation + S.E
|
Mean length of root lets after 15 days + S.E.
|
Mean No. of rootlets / shoot + S.E.
|
Callus formation
|
|
IBA
|
IAA
|
NAA
|
|
1
|
0.0
|
0.0
|
0.0
|
0
|
0
|
0
|
0
|
|
2
|
4.92
|
0.0
|
0.0
|
85.3+0.03
|
8.0+0.5
|
7.5+0.2
|
-
|
|
3
|
9.84
|
0.0
|
0.0
|
74.4+0.1
|
6.0+0.2
|
5.5+0.1
|
-
|
|
4
|
0.0
|
5.71
|
2.69
|
77.3+0.1
|
7.2+0.1
|
6.1+0.3
|
+++
|
+ sign indicates
degree of callus formation
Fig. 1. Shoot proliferation in Vitex negundo L.
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