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International Journal of Applied Research and Studies (iJARS)

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

Research Article

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

Ornamental and Medicinal Aquatic Plant

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
School of Management & Entrepreneurship, Kerala University of Fisheries and Ocean Studies
Kochi-682506, India

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

Manuscript Id: iJARS/795 1

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

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

Manuscript Id: iJARS/795 2

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

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
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
Figure 1. Culture initiation from the nodal segments of L.aromatica
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
BAP Average
Average number Average Length
(mgl-1) number of
of Shoots/explant of Shoots
0.0 3.8327 0.106a
2.3983 0.093e 2.96290.153bc
0.5 4.4704 0.130
2.4379 0.086e 3.8042 0.177d Figure 1.
1.0 4.3562 0.161
2.1583 0.113d 3.1591 0.149c Figure 2.
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
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.


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
0.5 4.6249 0.105b
2.6994 0.595d 4.0590 0.166e
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
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)

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