International Journal of Applied Research and Studies (iJARS)

ISSN: 2278-9480 Volume 3, Issue 3 (Mar - 2014)

www.ijars.in

Research Article

In Vitro Propagation of Limnophila aromatica (Lam.) Merr., An

Ornamental and Medicinal Aquatic Plant

Authors:
1 2
Sheeja George E *, Alphi Korath, 3 Aney kutty Joseph

Address For correspondence:

1, 2
Dept. of Marine Biology, Cochin University of Science and Technology, Kochi-682016, India
3
School of Management & Entrepreneurship, Kerala University of Fisheries and Ocean Studies
Kochi-682506, India

Abstract Limnophila aromatica (Lam.) Merr., belonging to the
family Scrophulariaceae, is a popular ornamental aquatic plant having
great demands in the aquarium industry. The plant is appreciated for
its medicinal importance too. In spite of its growing demands, the
plant is least available in India. Therefore tissue culture techniques
can serve as a powerful tool for the commercial production of this
plant. An efficient protocol for the micropropagation of L. aromatica
through multiple shoot proliferation from the nodal explants was
developed successfully. Treating the nodal explants with 15% (v/v)
commercial bleach (Robin liquid bleach, Reckitt Benckiser, India) for
10 minutes followed by a quick dip in 70% ethanol proved to be the
best sterilization procedure to obtain axenic cultures. The aseptic
nodal segments produced shoots within a week when cultured in a
medium containing full strength Murashige and Skoog (MS)
inorganic salts, 100 mg/l myo-inositol, 0.1 mg/l thiamine-HCl, 3%
sucrose, 0.8% agar as gelling agent and supplemented with 0.5 to 2.5
with mgl-1 6- Benzyl aminopurine (BAP) alone or in combination
with 0.1 or 0.2 mgl-1 - Naphthalene acetic acid (NAA). Maximum
number of shoots (5.13 shoots per explant, p 0.05) was formed on
MS medium supplemented with a combination of 2.0 mgl-1 BAP and
0.1 mgl-1 NAA. Maximum shoot length (2.69 cm, p 0.05) and
maximum number of roots were obtained on MS medium containing
0.5 mgl-1 BAP and 0.1 mgl-1 NAA. Rooted plantlets were
successfully acclimatized and transferred to aquarium
Keywords- Limnophila aromatica, ornamental, medicinal, aquatic
plant, in vitro propagation, nodal explants
I. INTRDUCTION
Limnophila aromatica, also called rice paddy herb, is a
tropical flowering plant belonging to the family
Scrophulariaceae. It is a popular aquarium plant, known for its
distinctive vibrant colored leaves, stalky stem and a large root
system. L. aromatica is also known for its rosemary herb, and
citrus aroma and is used in Vietnamese cruises to flavor soup
broths, sauces, and other foods [1]. Moreover the plant is used
in Ayurvedic and Folk-lore medicines too. In Ayurveda, the
plant is used in vitiated conditions of pitta, ulcers, galactic
impurities, anorexia, dyspepsia, helminthiasis, constipation,
inflammations etc. and plant juice is used as a cooling
medicine for fever and pharyngitis [2]. The leaf, stem and root
extracts of L.aromatica have antioxidant [1-5] and anti-
inflammatory [6] properties.
To the best of our knowledge, there is no report
available on the micropropagation of L. aromatica. Present
study was carried out to investigate the possibility of micro
propagating L. Aromatica for mass production to meet the
demands of the aquarium industry, as well as for the
production of in vitro plantlets that can be subsequently used
as plant source for the production of therapeutic compounds.
II. MATERIALS AND METHODS
A. Preparation and surface sterilization of the explants
Plants were collected from a local dealer in Ernakulam,
Kerala, India and where then grown in fresh water tanks.
Excised shoot tips, inter nodes and nodal segments (all with a
length of 1.5-2.0 cm) were washed under running tap water for
30 minutes and treated with 15% (v/v) commercial bleach
(Robin Liquid Bleach, Reckit Benckiser, India) for 10 minutes.
The explants were then immersed in 70% (v/v) ethanol for one
minute and washed several times with sterile distilled water.
Before inoculating into shoot formation media, they were again
trimmed to 1.0 cm.

Manuscript Id: iJARS/795 1

International Journal of Applied Research and Studies (iJARS)
ISSN: 2278-9480 Volume 3, Issue 3 (Mar - 2014)
www.ijars.in
B. Regeneration of shoots and roots from the explant
The shoot formation media consisted of MS mineral
salts [7], 1mgl-1 thiamine-HCl, 1mgl-1 pyridoxine-HCl, 1mgl-1
nicotinic acid, 2mgl-1 glycine, 100 mgl-1 myo-inositol, 3%
(w/v) sucrose, 0.8% (w/v) agar (Agar-agar, Sigma Chem. Co.,
St. Louis, MO) and supplemented with 0.5 to 2.5 mgl-1 6-
Benzyl aminopurine (BAP) alone or in combination with
0.1mgl-1 - Naphthalene acetic acid (NAA) for axillary shoot
multiplication. Since roots were developed spontaneously in
the above said shoot formation media, no other media were
tried for root formation. The pH of the medium was adjusted
to 5.8 and the medium was dispensed in 25150 mm culture
tubes before autoclaving at 1210 C for 15 minutes. Cultures
were maintained in 16-h day length at 2520 C in a culture
room.
Experiments were conducted separately to test the
effect of various concentrations of BAP alone and in
combination with 0.1mgl-1NAA on culture initiation, shoot
multiplication, shoot elongation and root formation.
Experiments were set up in Completely Randomized Design
(CRD) with six treatments for BAP alone (0, 0.5, 1.0, 1.5, 2.0,
2.5 mgl-1) and six treatments for BAP (0 2.5 mgl-1) in
combination with 0.1mgl-1NAA. Each treatment had ten
replications. Data were subjected to square root transformation
and analyzed by Univariate Analysis of Variance using SPSS
version 20.0. Significant differences between the means were
compared using Tukey Honestly Significant Difference (HSD)
[8].
III. RESULTS
In the MS basal medium, shoot buds regenerated in
100% of the shoot tip and nodal explants, but no direct shoot
regeneration was observed in the intermodal segments. Nodal
segments showed the best response (p 0.05) in the MS basal
medium with early bud initiation (Fig. 1) (5.98 days after
inoculation), and shoot multiplication (3.8327 shoots per
explant without callus formation); whereas it took 13.46 days
with the shoot tip explants for sprouting and the maximum
shoot number was 1.89 only. Therefore, further experiments
were conducted to test the effect of Plant Growth Regulators
(PGRs) on the in vitro response of nodal segment explants of
L.aromatica.
The morphogenic differentiation of explants towards
axillary bud proliferation was markedly influenced by the
concentration of BAP in the MS medium. In the absence of
hormones in the MS basal medium, the mean number of
shoots produced by the nodal segments was 3.8 (Table 1), and
BAP treatment significantly increased the number of shoots
that proliferated (p 0.05). Enhancement of shoot proliferation
by BAP in the MS media was also reported in Avicennia
marina [9] and Piper longum [10]. Efficiency of BAP for
shoot culture initiation and multiplication was also reported in
Bacopa monnieri [11-13]. In the present study, addition of
0.1mgl-1 NAA to the BAP- containing medium further
Manuscript Id: iJARS/795
increased the number of shoots per explant. While the
maximum number of shoots produced by BAP alone in the
medium was 4.59 at a concentration of 2.0 mgl -1 (Table 1),
media containing 2.0 mgl-1 BAP in the presence of 0.1 mgl-1
NAA induced 5.12 shoots per explant ( Table 2; Fig. 2). This
result shows that the optimum concentration of BAP in the MS
medium for maximum shoot proliferation from the nodal
explants of L .aromatica is 2.0mgl-1 (p 0.05), and addition of
0.1 mgl-1 NAA to the medium containing 2.0 mgl-1 BAP
interacts with BAP and induced maximum proliferation (p
0.05) of multiple shoots. This result is contradicted by the
opinion that exogenous auxin does not promote axillary shoot
proliferation [14] and supported by the reports on shoot
multiplication from the nodal segments of a closely related
species, Limnophila sessiliflora [15]. The present result is also
supported by the reports on positive interaction of NAA with
BAP in the MS medium on multiple shoot proliferation in
Hyacinthus orientalis [16] Cryptocoryne lucens [17],
C.tonkinensis [18] and C. wendtii [19].
The regenerated shoots elongated considerably in the
MS basal media as well as in media supplemented with
combinations of BAP and NAA. This observation is
contradictory to the reports on pronounced inhibition in
elongation and growth of shoots in woody plants [20], Pinus
strobus [21] and Ludwigia repens [22] on media containing
any concentrations of cytokinins. However the shoot length
showed an inverse relationship with the concentration of BAP
(Table 1 and Table 2) in the medium and shoot length
decreased with increase in the concentration of BAP in the MS
medium. Shoots elongated considerably in the medium
containing both BAP and NAA than in media containing BAP
alone. This result is in support to the opinion that high
cytokinin concentration in the medium has a suppressive effect
on axillary shoot elongation and addition of low
concentrations of auxins nullifies it [14, 21]. Maximum shoot
elongation (2.69 cm, p0.05) was obtained on MS medium
supplemented with 0.5mgl-1 BAP in combination with 0.1mgl-
1
NAA (Table 2, Fig. 3).
In the present study, it was observed that BAP in the
regeneration media do not inhibit root formation; which is
contradictory to the belief that rooting is difficult because of a
carry over effect from cytokinins in the shoot proliferation
medium [22]. Inhibition of root development by cytokinins in
the shoot regeneration media was reported in apple cultivars
[23], Eastern redbud [24] and silver maple [25], whereas root
regeneration from shoots regenerated on media containing
cytokinin was reported in B. monniera [13] and L. repens [22].
The root formation observed in the shoot regeneration media
in the present study may be because of the interaction of
endogenous and exogenous factors [14]. Maximum number of
sheejajebi@yahoo.co.in *Corresponding Author Email-Id
2
International Journal of Applied Research and Studies (iJARS)
ISSN: 2278-9480 Volume 3, Issue 3 (Mar - 2014)
www.ijars.in

roots (4.05 roots, p0.05) was observed in MS Means SD of 10 observations. Means with the same superscript in the same column belongs to same
homogenous subgroup.
medium containing 0.5mgl-1 BAP and 0.1mgl-1 NAA (Table
2).
Hardening was done by transferring the rooted
plantlets from culture tubes into plastic cups containing
sterilized Neopete, which were kept inside the culture room
with controlled conditions of 250 C and 16 h/day illumination
with cool fluorescent light for two weeks. The humidity was
adjusted at 80% for two weeks. The acclimatized plantlets
were then transferred to tanks (Fig 4), and 98% of them were
established successfully with similar phenotype of the mother
plants.
Figure 1. Culture initiation from the nodal segments of L.aromatica
IV. CONCLUSION
The present study was established for reproducible .
and higher efficiency of shoot regeneration in L.aromatica.
The procedure described here provides a rapid
micropropagation system that may also be applicable to other
species belonging to Limnophila.

TABLE I. IN VITRO RESPONSE SHOWN BY THE NODAL EXPLANTS OF Figure 2. Shoot multiplication in MS media containing 2 mgl-1 BAP
L.AROMATICA TO VARIOUS CONCENTRATIONS OF BAP
BAP Average
Average number Average Length
(mgl-1) number of
of Shoots/explant of Shoots
roots/explant
0.0 3.8327 0.106a
2.3983 0.093e 2.96290.153bc
bcd
0.5 4.4704 0.130
2.4379 0.086e 3.8042 0.177d Figure 1.
bc
1.0 4.3562 0.161
2.1583 0.113d 3.1591 0.149c Figure 2.
cd
1.5 4.5368 0.139 c c
1.9728 0.093 3.1386 0.233 Figure 3. Shoot multiplication MS media containing 2 mgl-1 BAP + 0.1 mgl-1
2.0 4.5989 0.235 d NAA
1.7883 0.045b 2.7188 0.094a
b
4.2982 0.168
2.5 1.4643 0.080a 2.7689 0.191ab
Means SD of 10 observations. Means with the same superscript in the same column belongs to same
homogenous subgroup.

TABLE II. IN VITRO RESPONSE SHOWN BY THE NODAL EXPLANTS OF

L.AROMATICA TO VARIOUS CONCENTRATIONS OF BAP IN COMBINATION WITH
0.1 MGL-1 NAA
BAP Average Figure 4. Plantlets after acclimatization
Average number Average Length
(mgl-1) number of
of Shoots/explant of Shoots
roots/explant
0.5 4.6249 0.105b
2.6994 0.595d 4.0590 0.166e
ACKNOWLEDGMENT
1.0 5.0779 0.128de d bcd
2.6451 0.061 3.31390.149 This forms a part of the PhD research work of the first author.
1.5 4.7820 0.189 c The authors are thankful to the Dept. Of Marine Biology,
2.3010 0.076c 3.5270 0.258d Microbiology & Biochemistry, School of Marine Sciences,
2.0 5.1276 0.091e Cochin University of Science and Technology, Cochin,
2.0903 0.033b 3.4501 0.327d
Kerala, India for providing the necessary facilities to carry out
a
2.5
4.2986 0.157
1.6530 0.091a 3.0649 0.838ab
this work.

Manuscript Id: iJARS/795 3

International Journal of Applied Research and Studies (iJARS)
ISSN: 2278-9480 Volume 3, Issue 3 (Mar - 2014)
www.ijars.in

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