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info BANGLADESH JOURNAL

OF SCIENTIFIC AND
Bangladesh J. Sci. Ind. Res. 43(2), 215-222, 2008 INDUSTRIAL RESEARCH

BCSIR E-mail: bjsir07gmail.com

Micropropagation of Eclipta alba (Linn.) Hassk- a Valuable

Medicinal Herb
A.K. M. Sayeed Hassan,* Farhana Afroz, Laila Shamroze Bari, John Liton
Munshi, Miskat Ara Akhter Jahan and Rahima Khatun

Biological Research Division, BCSIR Laboratories, Dhaka-1205, Bangladesh.

Abstract

A protocol was established for mass propagation of a valuable medicinal herb, Eclipta alba (Linn.)
Hassk (Asteraceae) through in vitro culture. Apical and axillary buds of young sprouts from selected
plants were used as explants. Best shoot induction was observed on MS basal medium supplemented
with 0.5 mgl-1 BAP + 0.1 mgl-1 NAA, in which 94% of the explants produced 18 shoots per culture.
Repeated subcultures in the same medium, resulted rapid shoot multiplication with 26 shoots per cul-
ture. In vitro raised shoots rooted on half strength MS medium with 1.0 mgl-1 IBA +1.0 mgl-1 NAA.
For acclimatization and transplantation, the plantlets in the rooting culture tubes were kept in normal
room temperature for 7 days before transplanting in pots where plantlets were reared for three weeks.
The survival rate of regenerated plantlets was 80%.

Key words: Eclipta alba, Medicinal plant, Shoot proliferation, Micropropagation, Acclimatization

Introduction

Eclipta alba (Linn.) Hassk. (Fam. ity against Ranikhet disease virus. Eclipta
Asteraceae), a common herbaceous multi- alba is widely used in India as a cholagogne
branched annual weed of moist places with and deobstruent in hepatic enlargement, for
long lanceolate leaves, hirsute stem and jaundice and other ailments of the liver and
white flowers in axillary heads, grows all gall bladder. The roots have emetic and
over the Bangladesh (Ghani 1998). It is used purgative properties and it is applied exter-
as a tonic and diuretic in hepatic and spleen nally as an antiseptic to ulcer and wounds in
enlargement. It is also used in catarrhal jaun- cattle. The shoot extract shows anti micro-
dice and for skin diseases. The alcoholic bial activity against Staphylococcus aureus
extract of the plant has shown antiviral activ- and Escherichia coli (Anonymous 1952).

* Corresponding author
216 Micropropagation of Eclipta alba 43(2) 2008

Decoction of the plant is used to invigorate However, in Bangladesh, there is no report

the liver, to prevent premature graying of the on the establishment of a micropropagation
hair and to staunch bleeding, especially from protocol for Eclipta alba. The present study
the uterus (Chevallier and Andrew 1996). was therefore undertaken to develop a proto-
From the whole plant of Eclipta alba, a new col for mass clonal propagation of this
triterpene saponin, namely eclalbatin, important medicinal herb through in vitro
together with alpha-amyrin, ursolic acid and culture.
oleanolic acid have been isolated (Upadhyay
et al. 2001). In Ayurveda a large number of Materials and Methods
indigenous drugs have been mentioned pos-
The experiment was conducted at Biological
sessing analgesic properties. The total
Research Division in Bangladesh Council of
ethanol extract of Eclipta alba have been
Scientific and Industrial Research (BCSIR),
shown to posses analgesic properties
Dhaka. Healthy and profusely growing vine
(Sawant et al. 2004).
of Eclipta alba was collected from Bank
In recent years, there has been an increased Colony at Savar, Dhaka and BCSIR Campus,
interest in in vitro culture techniques which Dhaka and used as source of explants. Shoot
offer a viable tool for mass multiplication tips and stem nodes with a single axillary
and germplasm conservation of rare, endan- bud were used for this purpose. The explants
gered and threatened medicinal plants were washed thoroughly under running tap
(Ajithkumar and Seeni 1998; Prakash et al. water, pre-soaked in liquid detergent for
1999). Commercial exploitation and elimi- about 30 min, wiped with cotton and dipped
nation of natural habits consequent to urban- in 70% (v/v) ethanol for 1 min. They were
ization has led to gradual extinction of sever- then surface-sterilized with 0.1% (w/v) mer-
al medicinal plants. Micropropagation is an curic chloride for 5 min, followed by five
effective approach to conserve such rinses with sterile distilled water in front of a
germplasm. Further, genetic improvement is laminar air flow cabinet. The surface-steril-
another approach to augment drug-yielding ized explants were sized to 1-1.5 cm length
capacity of the plant (Tejavathi and Shailaja containing a single node with an axillary bud
1999). Therefore it is important to develop or a shoot tip with an apical bud. The
an efficient micropropagation technique for explants were placed vertically on the cul-
Eclipta alba to rapidly disseminate superior ture medium. The new shoots induced from
clones once they are identified. There have the in vitro cultures were further used as an
been few reports to date on micropropaga- explants for adventitious shoot regeneration.
tion of Eclipta alba using nodal explants
MS (Murashige and Skoog 1962) basal
(Franca et al. 1995; Borthakur et al. 2000).
medium was used for shoot proliferation and
 Hassan, Afroz, Bari, Munshi, Jahan and Khatun 217

adventitious shoot regeneration and half Established plants were transplanted in

strength MS was used for in vitro rooting. earthen pots under natural conditions and the
All media were supplemented with 30 gl-1 survival rate was recorded.
sucrose, 7 gl-1 agar (Difco) and dispensed
into 15x150 mm culture tubes and 250 ml Results and Discussion
conical flasks. The pH of the media was
Shoot tips and nodal explants of Eclipta alba
adjusted to 5.8 before autoclaving at 121OC
were cultured on MS media supplemented
for 20 min. The cultures were incubated for
with various concentration of BAP alone and
a 16 h photoperiod at 24 ± 2OC under a fluo-
BAP with NAA or IAA for shoot regenera-
rescent light.
tion. The explants were found to be swollen
Shoot proliferation from shoot tips and nodal and they produced four to five shoots within
explants was obtained in two separate sets of three-four weeks after inoculation (Fig 1a)
experiments. In the first experiment 0-2.5 on MS containing BAP alone but the number
mgl-1 BAP and 0-2.5 mgl-1 Kn were incorpo- of shoots increased up to 18 when the
rated into MS to select the best cytokinin for explants were cultured in MS with 0.5 mgl-1
the response of shoot induction. In the sec- BAP + 0.1 mgl-1 NAA (Table I, Fig 1b). Both
ond set, combination of BAP-NAA (0-2.5 the explants responded in the same medium
mgl-1) and BAP-IAA (0-2.5 mgl-1) were but highest numbers of micro shoots were
assessed for shoot multiplication. Number of observed to be induced from nodal explants.
new shoot proliferation of each culture was Combinations of BAP with IAA were not
recorded after every week of inoculation. found suitable for shoot induction (Table I).
Newly initiated shoots were separated and
For in vitro rooting, individual shoots (3-5 sub cultured repeatedly in fresh MS with 0.5
cm) were excised from the proliferated shoot mgl-1 BAP + 0.1 mgl-1 NAA, where the
cultures and implanted onto half strength MS number of shoots increased up to 26 per cul-
with different concentrations and combina- ture (Fig 1c). Dhaka and Kothari (2005)
tions of NAA, IBA and IAA. reported that maximum shoot proliferation in
Eclipta alba occurred when the explants
The rooted plants were taken out from the were cultured on MS medium supplemented
culture tubes, washed to remove agar gel with 1.0 mgl-1 benzylaminopurine (BAP)
adhered to the roots and transplanted to plas- and the shoot buds were further multiplied
tic pots with soil and compost (1: 1) for hard- and maintained on medium containing 0.5
ening. The plantlets were kept in a poly- mgl-1 BAP and 0.5 mgl-1 GA3. A similar phe-
chamber at 80% relative humidity, 32 ± 2°C nomenon was observed in Eclipta alba by
under a 12 h photoperiod for acclimation. other researchers (Gawde and Paratkar 2004;
218 Micropropagation of Eclipta alba 43(2) 2008

Table I. Effect of growth regulators in MS on morphogenic response of Eclipta alba shoot tips
and nodal segments.

Growth regulators(mgl-1) Shoot tips Nodal segments

BAP NAA IAA Explants form- Mean No. of Explants form- Mean No. of
ing shoots (%) shoot/explant ing shoots (%) shoot/explant
0.0 0.0 0.0 - - - -
0.5 0.0 0.0 73(0.8) 04(0.1) 76(2.3) 05(0.1)
0.5 0.0 0.1 83(0.8) 16(0.8) 88(1.8) 18(1.4)
1.0 0.0 0.2 68(6.6) 06(1.4) 72(0.8) 08(0.5)
1.5 0.0 0.5 71(1.4) 14(0.5) 73(0.1) 16(0.8)
2.0 0.0 0.5 76(0.8) 09(1.0) 79(0.5) 13(0.3)
0.5 0.1 0.0 94(0.5) 23(0.3) 96(0.7) 26(1.4)
1.0 0.2 0.0 81(1.0) 08(1.4) 84(0.1) 12(0.1)
1.5 0.5 0.0 78(1.8) 19(0.8) 81(1.4) 22(1.0)
2.0 0.5 0.0 72(1.4) 14(0.1) 76(0.8) 16(0.8)
Results are mean ± SE of three experiments with 15 replications.

Baskaran and Jayabalan 2005; Husain and medium to the pot under natural conditions.
Anis 2006; Han et al. 2007). About 80 percent of the transplanted plants
of Eclipta alba survived if the plants in the
Ninety five percent regenerated shoots root- rooting culture tubes were kept in normal
ed (Fig 1d) when cultured individually on room temperature for seven days before
root induction medium consisted of half- transplantation in pots and reared for three
strength MS salts with 1.0 mgl-1 IBA + 1.0 weeks. The plantlets were reared under semi-
mgl-1 NAA (Table II). Use of auxins singly controlled temperature (30±2OC) and light
or in combination for rooting was also (2000 lux) in a chamber with 80 percent
reported by different authors (Sahoo and humidity. During this period of acclimation
Chand 1998; Ajithkumar and Seeni 1998; shoots elongated, leaves expanded and
Rai 2002; Baskaran and Jayabalan 2005; turned deep green and healthier (Fig 1e ).
Sivakumar and Krishnamurthy 2000; Hassan
and Roy 2005; Rahman et al. 2006; Baksha After three weeks, plants were transferred to
et al. 2007). an open place and gradually acclimated to
outdoor conditions, where 80 percent plants
After four weeks the rooted shoots were survived. The technique described here
transferred to pots. None of the plantlets sur- appears to be readily adaptable for large
vived when directly transferred from rooting scale clonal propagation and plantation for
 Hassan, Afroz, Bari, Munshi, Jahan and Khatun 219

a b

c d e

Fig. 1. In vitro regeneration of Eclipta alba from shoot tip and nodal explants.

(a) Induction of shoots in four weeks of culture on MS + 0.5 mgl-1 BAP + 0.1 mg1-1 NAA
(b) Development and multiplication of shoots on MS + 0.5 mgl-1 BAP + 0.1 mgl-1 NAA after eight
weeks of culture.
(c) Development and multiplication of shoots on MS + 0.5 mgl-1 BAP + 0.1 mgl-1 NAA after
twelve weeks of culture.
(d) Rooting of in vitro regenerated shoots cultured on half strength MS + 1.0 mgl-1 IBA + 1.0
mgl-1 NAA in third weeks.
(e) Acclimatized regenerated plants of two months old.
220 Micropropagation of Eclipta alba 43(2) 2008

Table II. Effect of auxin(s) on root induction in regenerated shoots of Eclipta alba on half
strength MS.
Auxins(mgl-1) Shoots rooted(±SE) Days required for root induction
(%) (±SE)
IBA 0.5 82(0.8) 26(0.1)
IBA 0.75 73(0.1) 20(0.3)
IBA 1.0 66(1.0) 24(0.8)
NAA 0.5 81(0.3) 24(0.5)
NAA 0.75 76(1.4) 25(1.0)
NAA 1.0 73(0.5) 21(0.1)
IBA 1.0 + NAA 0.5 82(0.8) 23(1.4)
IBA 0.5 + IAA 0.5 71(0.1) 21(0.7)
IBA 0.5+ NAA 0.5+ IAA 0.5 68(0.7) 19(0.3)
IBA 1.0+ NAA 1.0 92(0.3) 18(0.8)
IBA 1.0+ IAA 1.0 76(0.8) 25(0.3)
IBA 1.0+ NAA 1.0 + IAA 1.0 62(0.5) 23(0.5)
Data were recorded after four weeks of culture. Results are mean ± SE of 15 replications.

sustainable use in the industry. Moreover, by Director, BCSIR Laboratories, Dhaka for his
standardizing the protocols for clonal propa- kind help during the study.
gation of selected elite plants, it is possible to
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Received : November 28, 2007;

Accepted : February 11, 2008