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Indian Journal of Biotechnology
Vol 5, April 2006, pp 246-248
In vitro propagation of Justicia gendarussa
Burm. f.–A medicinal plant
P Agastian, Lincy Williams and S Ignacimuthu•
Department of Plant Biology & Biotechnology and
*Entomology Research Institute
Loyola College (Autonomous), Chennai 600 034, India
Received 1 October 2004; revised 1 June 2005;
accepted 30 July 2005
An efficient protocol for in vitro propagation of Justicia
gendarussa Burm. f. has been developed. MS medium
supplemented with NAA induced prolific callus in both leaf and
nodal explants. Organogenic and chlorophyllous calli were
produced at lower concentrations of NAA (1.0 mg L-1) and BAP
(0.1 mg L-1). Thick and long roots with numerous root hairs were
produced with NAA (1.0 mg L-1) and BAP (0.1 mg L-1). Long
shoots were also formed. Of the in vitro grown 120 plantlets
transferred to the field 94% survived after 2 months of
transplantation to natural environment.
Keywords:
in vitro propagation,
gendarussa
organogenesis,
Justicia
IPC Code: Int. Cl.8 A01H4/00
Justicia gendarussa Burm. f. belonging to the family
Acanthaceae, is well-known for many of its medicinal
properties. It has anodyne, diaphoretic, diuretic,
antiphlogistic, antispasmodic, carminative, emetic,
febrifuge and laxative properties. The roots and leaves
are acrid, bitter, thermogenic, emmenagogue,
antiperiodic and insecticidal. They are useful in the
treatment of chronic rheumatism, cephalagia,
hemiplegia, facial paralysis, ostalgia, hemicrania,
cough,
bronchitis,
arthritis,
dysmenorrhoea,
amenorrhoea, internal haemorrhages, intermittent
fevers, ascites and debility1. Fresh leaves are used
topically to treat oedema of beriberi and rheumatism2.
Dried leaves are used as insecticide.
J. gendarussa grows along the beds of streams in
Himalayas. It is an erect, branched, smooth,
undershrub with long leaves having acute tips; small
flowers on long terminal pinkish spikes with purple
spots. The reported chemical constituents of Justicia
spp. are 2-(2′-amino benzylamino)-benzyl alcohol, 2_______________
•
Author for correspondence:
Tel & Fax: 0448174644;
E-mail: [email protected]
(2’-amino benzylamine)-benzyl alcohol-o-methyl
ether, 2-amino benzyl alcohol, 2-amino benzyl
alcohol o-methyl ether and β-sitosterol. Some spp.
exhibited varied responses like antimicrobial, antiinflammatory and antiplatelet aggregation effects3.
Moreover, cytotoxic and anticancer activities of
lignans isolated from Justicia spp. have also been
reported4.
The plant is propagated by seeds, whose viability is
only for a very short period and hence, it is important
to look for alternative methods to conserve it.
Moreover, owing to increasing exploitation of natural
population for its wide uses in traditional medicine,
the plant is also getting depleted. Hence an attempt
has been made for in vitro propagation of this
medicinal plant and this protocol offers an alternative
method for rapid multiplication of desirable clones.
Mature plants of Justicia gendarussa were
collected at a relatively warm (25 to 38°C) and humid
(70 to 95%) climate with about 20 cm rainfall during
the monsoon months (September-November) and
were grown in the garden. Tender leaves and nodal
regions of stem were used as explants. The explants
were soaked in 10% tween-20 for ten min. Then they
were washed thrice with sterile distilled water
followed by treatment with 0.03% bavistin,
(a fungicide) for 20 min and again washed thrice in
sterile distilled water. Explants were further surface
sterilized with 0.1% mercuric chloride for 4 min,
washed 5-7 times with sterile distilled water in a
complete aseptic ambience. Explants were trimmed
and placed on full strength MS medium5
supplemented with 8 g L-1 agar, 30 g L-1 sucrose and
appropriate concentrations of phytohormones.
Naphthalene
acetic
acid
(NAA)
and
6benzylaminopurine (BAP) were used in a 4 by 4 latin
square with concentrations 0, 0.1, 0.2, 0.5, 1.0, 1.5,
2.0 and 2.5 mg L-1. In all the cases, pH was
maintained between 5.6 and 5.8. Each treatment
consisted of fifteen replicates and the experiments
were repeated three times. A complete randomized
block design was used in all experiments and analysis
of variance and mean separation were carried out
using Duncan’s Multiple Range Test (DMRT) and
significance was assessed at 5% level6. Explants were
grown in a culture environment of either 25±2°C or
SHORT COMMUNICATIONS
20°C in continuous darkness, or a 16 h light18 h dark
cycle with a light intensity of 27 pE m-2 s-' or a
continuous low light regime of 0.5 pE m-2 S-I.
When the medium was supplemented with NAA,
prolific callus induction occurred in leaf and nodal
explants (Fig. 1; Table 1). To maximize callus
proliferation, a solid medium supplemented with 2 mg
L-I NAA was used. In this medium the callus doubled
in size every 15 d with routine subcultures. Callus
was found to be big, friable and white-pale green in
colour. When nodal explants were grown in medium
supplemented with low concentrations of NAA (1.0
mg L-') and BAP (0.1 mg L-'), they produced compact
chlorophyllous calli with shoots (Fig. 2). When leaf
explants were grown in medium supplemented with
BAP (2.0 mg L-I), hard callus was formed after longer
incubation time. This may be attributed to differential
physiological status and endogenous concentration of
growth regulators as revealed by thebstudies on
Quercus spp7.
The roots, developed from the callus after 15-20 d
of culturing in rooting medium, exhibited varied
morphological features. Lower concentrations of
NAA (0.1 to 1.0 mg L-I) without BAP gave maximum
number of tender roots (81nodal explant) also
recorded earlier in Hybarttltus e~zrzaespernzus8and
Datura nzeta?. Thick and long (25 cm) roots were
produced in large numbers in medium supplemented
with NAA (1 mg L-I) and BAP (0.1 mg L-') (Fig. 3).
Long chlorophyllous roots were formed in medium
supplemented with NAA (0.2-2.0 mg L-'). Very
minute, dense root hairs were produced from the thick
roots.
The inclusion of cytokinins and auxins to the
culture medium stimulated in vitro multiplication and
growth of shoots in several plant specieslO. In the
present study, shoots were produced from loth day
Table I-Effect
Medium
MS
MS + NAA
onwards and final observations were made on 20Ih
day. All combinations of NAA and BAP produced
long shoots (7.6k0.7 cm) with two to three shoots per
callus. However, 3 to 4 medium sized shoots appeared
in medium containing 0.5 mg L-I NAA and 0.1 mg L-I
BAP (Fig. 4). Only short shoots appeared at higher
concentrations of NAA and BAP. Nodal explants
produced calli with long shoots (10.71 cm) and
profuse rooting occurred at 0.1 mg L-' NAA and 1.0
mg L-' BAP. Maximum number (4.33) of shoots was
obtained on MS medium supplemented with 3 mg L-I
Figs I-%Plant regeneration In J. gerldani.s.va Burm. f.: 1, Callus
induction in nodal explant; 2, Shoot induction in callus using
nodal explant with single axillary bud; 3, Induction of shoot and
root from nodal explant; 4, Multiple shoot induction from in virro
nodal explant; & 5, Transfer of plantlet for hardening.
of NAA and BAP on callus induction and organogenesis from nodal explants of J. gendr~russa
Optimal hormonal
concentrations
(mi2 L-'1
Callus
Shoot
induction
formation
-
2.0
MS + BAP
0.2
MS + NAA
1.O
+ BAP
0.1
( n = 15; values in parenthesis are SD)
LSD at 5% level (* = 0.90) (# = 0.66)
-
0.2
3.0
0.5
0.1
Percentage
of response
Number of
shoots
Length of
shoots
Nature of Callus
50.34 (2.71)*
96.66 (2.50)*
75.34 (2.01)*
80.41 (1.60)*
1.00 #
2.1 1(0.70)*
4.33(1.03)*
3.20(1.24)*
4.8(0.52)*
5.4(0.61)*
6.3 (0.68)*
7.6 (0.70)*
Compact, very slow growth
Friable, bulky. organogenic
Compact, slow growth with thick roots
Friable, bulky, organogenic with
lengthy shoot and roots
INDIAN J BIOTECHNOL, APRIL 2006
248
of BAP. Similar findings have been reported in
Dictyospermum ovalifolium11. 120 rooted plantlets
were transferred directly to sand supplemented with
Hoagland solution (Fig. 5), and after acclimatization,
they were transferred to the field. 94% survival rate
was recorded after 2 months.
In the present study, both BAP and NAA induced
organogenesis and it appears that BAP is very
essential to induce organogenesis. Similar results have
been reported for legumes12 and Foxtail millet13.
Higher concentrations (>1.5 mg L-1) of NAA and
BAP induced thick and compact callus with no shoot
formation. These results presumably indicate a
threshold level of endogenous hormones in the
explants14. In conclusion, the present work
demonstrates a simple procedure for callus induction,
shoot and root formation of J. gendarussa using leaf
and nodal explants.
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