RESEARCH NOTE

An improved micropropagation of Eclipta alba by in vitropriming with chlorocholine chloride

Avik Ray Æ Sabita Bhattacharya

Received: 26 December 2006 / Accepted: 3 December 2007 / Published online: 18 December 2007

� Springer Science+Business Media B.V. 2007

Abstract An efficient method of micropropagation

for Eclipta alba from young nodal axils of shoot tip

explants has been developed by giving special

attention to ‘priming’ in vitro plantlets in view of

increasing their hardening ability after transplantation

ex vitro. Among 3 cytokinins—BAP, kinetin and

TDZ, BAP was found most effective in inducing and

proliferating adventitious shoots. The highest fre-

quency of responding explants (100%) and maximum

number of shoots (23.0) per explant were obtained

after 60 days culture on MS medium containing

8.8 lM BAP. Cent percent shoots developed roots

directly from shoot base when transferred to growth

regulator-free MS medium. For priming E. alba

microshoots, 6.3 lM of chlorocholine chloride

(CCC) was found most effective. The major changes

observed in 30 days old treated shoots were, produc-

tion of increased number of root, elevation of

chlorophyll level in leaves and increase in plant

biomass. Furthermore, arrested undesirable shoot

elongation made the plants sturdier and more suitable

for acclimatization. The primed micropropagated

E. alba plants were healthy and survived by higher

frequency (100%) in soil in comparison to the non-

treated plants (84% survival).

Keywords Eclipta alba � Micropropagation �Shoot multiplication � Cytokinin � Priming �Growth retardant

Abbreviations

BAP 6-Benzylaminopurine

TDZ Thiadiazuron

CCC Chlorocholine chloride

In India, Eclipta alba is traditionally used for nourish-

ment of hair and for curing a number of diseases

(Baskaran and Jayabalan 2005). The herb, due to its

hepato-protective property (Tyagarajan 1982; Singh

et al. 2001) is used commercially in preparing several

health tonics and liver supporting herbal drugs (Anon

2005). Conventionally the collection of Eclipta alba

raw materials are made from the waste plants of

cultivated crop fields where these are often routinely

treated with anti-weed phytotoxic chemicals (Oudhia

2005) found to penetrate into the plant body through

roots and foliages (Wehtje et al. 2005). In order to stop

using contaminated pharmaceutical raw materials, it is

necessary to develop an alternative source of healthy

plants propagated by a suitable agrotechnology.

Micropropagation is an efficient tool in this respect,

provided the in vitro-raised plantlets are strong enough

to withstand the transplantation stress. Priming of

micropropagated propagules has recently been recom-

mended for obtaining better acclimatized plants

(Nowak and Shulaev 2003; Hazarika 2003).

A. Ray (&) � S. Bhattacharya

Department of Botany, Bose Institute, 93/1, A.P.C. Road,

Kolkata 700 009, West Bengal, India

e-mail: avikray19@rediffmail.com;

avikray@hotmail.com

123

Plant Cell Tiss Organ Cult (2008) 92:315–319

DOI 10.1007/s11240-007-9328-y

The aim of this work was establishment of a

simple and efficient method for fast in vitro propa-

gation of Eclipta alba including successful

transplantation by priming with a selected chemical

agent, chlorocholine chloride (CCC). This presenta-

tion is the first of its kind in quest of improved

micropropagation of the species.

Healthy Eclipta alba plants were collected from

Bose Institute campus. Shoot tips (0.8–1.0 cm),

containing apical meristem and two successive pairs

of nodal axis were used as explants. They were

surface sterilized in 5% teepol detergent for 5 min,

rinsed with distilled water, treated with a fungicidal/

antibiotic solution (Bavistin (0.2%w/v) and Strepto-

cycline (0.1%w/v)) for 30–40 min in stirrer before

immersion in 0.01% HgCl2 solution for 2 min after

which they were rinsed several time with sterile

distilled water. Thereafter axenic explants were

inoculated in autoclaved MS medium (Murashige

and Skoog 1962), pH-5.6, fortified with 3% sucrose

and gelled with 0.8% agar powder (Type I, Himedia

Laboratories Pvt. Ltd., Mumbai, India). For shoot

induction and multiplication, kinetin (2.3, 4.6 and

9.2 lM), BAP (2.2, 4.4 and 8.8 lM) and TDZ (2.25,

4.5 and 9.0 lM) were used. Cultures were incubated

under 20 ± 2�C and 18 hd-1 photoperiod provided

by cool white fluorescent lamps (30 lmol m-2 s-1).

Routine subculture was made at every 30 days

intervals excepting in shoot regeneration experiment

where cultures were maintained for 60 days without

changing nutrient media.

For rooting and in vitro priming regenerated

shoots (about 1 cm long) were transferred to

autoclaved MS medium containing the growth retar-

dant CCC at different concentrations (0, 0.63, 3.16,

6.33 and 12.66 lM). After 30 days shoot and root

lengths, root numbers, total chlorophyll concentra-

tions and plant biomass were estimated. The

4–5 weeks old plantlets were transferred stepwise to

the soilrite:soil (1:1) and sand:soil (1:1) in plastic

cups keeping in each condition for 2 weeks and

finally the well hardened plants to loamy soil in

earthen pots. Survival percentage was recorded from

two separate sets of plants after 8 weeks. Each set of

experiment of the study was designed with 10 replica

and repeated at least thrice. All data were subjected to

analysis of variance (ANOVA) and comparisons of

means were made with least significant difference

test at the 5% level of probability.

Chlorophyll estimation was made following the

method of Arnon (1949) using 30 day old 0.5 g fresh

leaf tissues crushed in 80% chilled acetone. The

optical density was taken at 645 nm in a spectropho-

tometer (UV-2401PC Spectrophotometer, Shimadzu

Corporation, Japan).

E. alba shoot tip explants responded at high

percentage (88–100%) to all three types of cytokinins

(BAP, kinetin and TDZ) by showing axillary bud

breaking within 5–8 days. The BAP-treated explants

had better potential for shoot regeneration than

others, treated with kinetin or TDZ (Table 1). Within

60 days culture period, highest number of propagules

(23.0 per explant) was induced by 8.8 lM BAP. This

number appears to be the higher compared to all other

previous records on the same plant species (Franca

et al. 1995; Gawde and Paratkar 2004; Dhaka and

Table 1 Effect of various growth regulators at different concentrations on regeneration of E. alba after 60 days of culture period

Growth hormones (lM) Shoot response (%) Number of shoots

emerged (means ± SE)

Rooting response (%)

BAP Kn TDZ

2.2 – – 100a 8.20 ± 1.2a 39 ± 2.3a

4.4 – – 100a 12.00 ± 0.8b 12 ± 1.2b

8.8 – – 100a 23.00 ± 0.45c 0c

– – 2.3 88 ± 2.3b 3.16 ± 0.12def 28.7 ± 0.8d

– – 4.5 94 ± 3.5c 4.67 ± 0.18d 0c

– – 9.0 96 ± 2.3ac 5.30 ± 1.21e 0c

– 2.3 – 100a 2.12 ± 0.20df 34.3 ± 1.8d

– 4.6 – 100a 3.43 ± 0.23ef 49.1 ± 3.5e

– 9.2 – 100a 2.30 ± 0.28df 38.2 ± 2.3a

Means followed by same letter in a column are not significantly different at 5% level

316 Plant Cell Tiss Organ Cult (2008) 92:315–319

123

Kothari 2005). There were no major differences

observed between the frequencies of shoot regener-

ation (per explant), obtained in presence of kinetin

and in TDZ, but it was noticed that kinetin-induced

cultures produced roots along with shoots at all of its

three tested concentrations (2.3, 4.6 and 9.2 lM) at

variable frequencies. Effectiveness of BAP in axillary

shoot regeneration was reported in a number of other

species (Aloufa et al. 2003; Agarwal et al. 2005).

Sprouting of axillary buds from shoot tip explants

(Fig. 1a) was followed by the development of

adventitious buds and formation of shoot clumps

(Fig. 1b). Cent percent shoots showed direct rooting

within 8–10 days without any intervening callus

when transferred to growth regulator-free MS med-

ium (Fig. 1c). Roots were totally absent at higher

BAP and TDZ concentrations (Table 1). Subsequent

to rooting, shoots elongated fast (from 1 cm to

2.18 cm within 30 days). As a result of long inter-

nodal distance, plants were slender and delicate.

In the second phase of the study, different

concentrations of CCC were tested in priming

regenerated shoots of E. alba. The concept of priming

micropropagated plants for better acclimatization is

based on the fact that certain chemicals effectively

pre-sensitize cellular metabolism of plants (Nowak

and Shulaev 2003) increasing adaptive ability of

in vitro plants (Conrath et al. 2002; Nowak and

Pruski 2004). In our experiment, a growth retardant,

CCC was tested to prime in vitro E. alba shoots after

successful multiplication. Out of 4 concentrations

(0.63, 3.16, 6.33 and 12.66 lM), 6.33 lM of CCC

was found most effective for inducing certain bene-

ficial changes in rooting, chlorophyll synthesis, stem

elongation, plant biomass and ex-vitro survival

frequency (Table 2). In 30 days culture regime, roots

per explant increased by number (33.8, in control

13.7) and length (16.2 cm, in control 6.3 cm). Lateral

roots were found to emerge from primary roots at

very early stage of development (Fig. 1d) giving

bushy appearance of the root system. Chlorophyll

content of leaves was increased by about 6 times

(50.3 mg/g leaves) than that in the control (8.89 mg/g

leaves). Checking rapid stem elongation (1.52 cm

after 30 days, 2.18 cm in control) with elevated plant

biomass (488.7 mg, 187.6 mg in control) made

E. alba plants more stouter than the non-treated

plants. The growth regulation manifested by CCC is

probably due to its anti-gibberellin activity (Anon

2003). The in vitro-generated sturdier plants are more

Fig. 1 a–fMicropropagation of

Eclipta alba (a). Emergence

of axillary shoots from

shoot tip explant (b).

Proliferated shoots forming

a clump (c). Root formation

in MS medium without

growth hormone (d). A

CCC-treated shoot showing

increased number of

primary and secondary

roots (e). Young E. albaplants at initial stage of

transplantation in soilrite-

soil mixture (f). An 1-month

old E. alba in soil

Plant Cell Tiss Organ Cult (2008) 92:315–319 317

123

desirable than slender, delicate plants because those

are more resistant to wilting on exposure to environ-

ment (Purohit et al. 1998). Similar to our results,

reduction in shoot and stolon growth and appreciable

increase in chlorophyll content in potato tissue

culture by application of CCC was reported by

Sharma et al. (1999). In the present study, CCC-

treated E. alba plantlets (4–5 weeks old) on trans-

planting to soilrite-soil mixture showed no sign of

environmental shock and started growing normally

(Fig. 1e) without any wilting symptoms. After finally

transplanted to soil enriched with organic manure, the

plants grew with increased vigor (Fig. 1f), acclima-

tized faster and survived by 100% in contrast to the

84% shown by untreated plants.

Acknowledgements Authors gratefully acknowledge the

University Grants Commission, Govt. of India, New Delhi

for financial support in conducting the research. We also thank

Mr. Jadab Ghosh, Dept. of Botany, Bose Institute for providing

technical assistance.

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