Persian Gulf Crop Protection

Available online on: www.cropprotection.ir

ISSN: 2251-9343 (online)
Volume 2 Issue 4, December 2013
Pages 1-6

Studies on In Vitro Propagation of an Important Medicinal Plant- Curcuma

zedoaria Roscoe Using Rhizome Explants

Muhammad Shahinozzaman1*, Muhammad Omar Faruq1, Mustafa Abul Kalam Azad1 and
Muhammad Nurul Amin1

1- Plant Tissue Culture Laboratory, Department of Botany, University of Rajshahi, Rajshahi-6205,

Bangladesh, (*Corresponding author e-mail: mshahin81@gmail.com).

Abstract: A complete protocol for micropropagation of Curcuma zedoaria was developed using
rhizome bud explants. Explants established on Murashige and Skoog (MS) medium were treated with
various concentrations and combinations of BA (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 µM) and NAA (0.0,
0.5, 1.0, 1.5 and 2.0 µM) to determine the best combination for shoot multiplication. MS medium
supplemented with 8.0 µM BA and 1.o µM NAA was determined to be the most suitable for shoot
proliferation. For rooting, the optimal auxin concentration was 4.0 µM NAA. Rooted plantlets were
then transferred to small plastic pots containing a mixture of garden soil and compost (1:1) and
carefully acclimatized. The hardened plants were then successfully established in the soil medium and
can function in the natural environment.

Key Words: Curcuma zedoaria, white turmeric, in vitro propagation, rhizome bud explants.

Persian Gulf Crop Protection, 2(4): 1-6 1

Introduction
Curcuma zedoaria, also known as white
turmeric, is a rhizomatous herbaceous
perennial plant belonging to Zingibraceae
family. It is an important medicinal plant
and its rhizome is commercially exploited
in industry as well as traditional medicinal
practices. It is used traditionally for the
treatment of menstrual disorder, dyspepsia,
vomiting (Prajapati et al., 2003), and for
cancer related disease (Rahman et al.,
2013). Moreover, rural people abundantly
use the rhizome of C. zedoaria due to
having rubifacient, carminative,
expectorant, demulcent, diuretic and
stimulant properties while root is used in
the treatment of flatulence, dyspepsia,
cold, cough, and fever (Wilson et al.,
2005). This plant is multiplied through
vegetative propagation in nature, but takes
a long time to produce rhizome (Stanly and
Keng, 2010). Vegetative propagation
confers low genetic variability to the
species, reducing the chances of selecting a
naturally-occurring clone. Besides, natural
plant resources of this plant are gradually
decreasing due to large scale exploitation
from naturally occurring population for
traditional uses. Taking into account the
aforesaid problems and threats, care should
be taken to protect as well as conserve the
existing germplasm of this plant species
and in vitro propagation may play a
significant role in this regard. Research on
in vitro propagation of C. zedoaria was
carried out by several researchers (Stanly
and Keng, 2010; Stanly and Keng, 2007;
Miachir et al., 2004) who concentrated on
rhizome derived explants as well as in vitro
derived shoot explants. However, tissue
culture techniques have been exploited
widely for in vitro propagation of many
other Curcuma species using rhizome
derived explants (Nadgauda et al., 1978;
Mukhri and Yamaguchi, 1986;
Balachandran et al., 1990; Sit and Tiwari,
1997; Salvi et al., 2002). The overall
objective of the present study was to
develop an optimized protocol for in vitro
propagation of C. zedoaria. The specific
Persian Gulf Crop Protection, 2(4): 1-6
objectives of the current research were to
optimize growth regulator requirements for
consistently high production of shoots
from the primary explants, to select auxin
type and concentration for efficient in vitro
rooting of regenerated shoots and to
acclimatize and transfer of plantlets to
natural conditions.
Materials and Methods
Plant material: The rhizomes of C.
zedoaria were collected during the rainy
season in October and November 2012
from the naturally grown plant population
at the botanical garden of Rajshahi
University, Bangladesh. Rhizome buds
about 1 to 2 cm long were selected as the
initial explants.
Surface sterilization and explants
procurement: About 2.0 to 3.0 cm apical
buds from the C. zedoaria rhizomes were
soaked in a few drops of commercial
detergent and washed thoroughly under
running tap water for a few minutes to
remove all the mud. Then the buds were
immersed in 75% (w/v) ethanol for one
minute before transferring them into
laminar air flow cabinet. Explants were
then surface sterilized with 0.1% HgCl2 for
10 minutes and washed thoroughly 3 to 4
times with sterile distilled water and
soaked with sterile blotting paper under
laminar air flow cabinet. Sterilized
explants were then dissected with a sterile
surgical blade to remove the outer layers of
leaf sheaths under aseptic conditions to
produce 5 to 8 mm sized pieces having at
least one eye (Chirangini and Sharma,
2005).
Culture medium and incubation
conditions: The explants were inoculated
on to Murashige and Skoog (1962)
medium supplemented with 6-Benzyl
adenine (BA) (0.0, 2.0, 4.0, 6.0, 8.0 and
10.0 µM) and α-Naphthalene acetic acid
(NAA) (0.0, 0.5, 1.0, 1.5 and 2.0 µM)
alone or in combination. The pH of the
medium was adjusted to 5.8 prior to
autoclaving. The culture bottles were
sealed with parafilm and were incubated in
the culture room under white fluorescent
2
light with light intensity of 3000 lux at a
photoperiodic16 h at 25 ± 2°C. The growth
of the cultures was observed after 6 weeks.
For adventitious rooting, in vitro derived
shoots were cultured onto MS medium
containing NAA and Indole-3-butyric acid
(IBA) at different concentrations (0.0, 1.0,
2.0, 2.5 and 4.0 µM).
Hardening of in vitro derived plantlets:
Well-rooted plantlets were removed from
the culture tubes and the roots were
washed under running tape water to
remove agar trace. Then the plantlets were
transferred to small plastic pots containing
autoclaved garden soil and compost (1:1)
and maintained inside growth chamber set
at temperature 280C and 70-80% relative
humidity. As to ensure its humidity, the
plants were sprayed periodic ally with
water. After four weeks they were kept in
green house before transferring them to the
Botanical Evaluation Garden of Plant
Tissue Culture Laboratory finally. The
survival rate percentage of the explants
was observed after four weeks.
Experimental design and data analysis:
Ten to fifteen cultures were used per
treatment. All the cultures were examined
periodically and the morphological
changes were recorded carefully. The
effect of different treatments was
determined with respect to number of
shoots, roots and the length of shoots and
roots. The experimental design used was
Randomized complete block design
(RCBD). Data were analyzed using the
Statistical package for the social science
(SPSS) software version 16.0 (Chicago,
Table 1. Effect of MS medium supplemented with BA and NAA on multiple shoot formation of C. zedoaria
after 6 weeks of culture
BA (µM)
0.0 0.5
0.0 1.14 ± 0.69i 1.01 ± 0.69i
2.0 2.17 ± 0.82kl 3.09 ± 0.93ik
ghi fgh
4.0 5.47 ± 1.08 5.89 ± 1.48
efg cde
6.0 6.98 ± 1.04 7.81 ± 1.83
bcde bcd
8.0 8.13 ± 1.61 8.74 ± 1.42
bcd bc
10.0 8.85 ± 1.86 9.05 ± 1.73
Values represent means ± standard error of 10-15 explants per treatment in three repeated experiments. Values
with the same superscripts are not significantly different at 5% probability according to DMRT.
Persian Gulf Crop Protection, 2(4): 1-6
USA). Analysis of variance (ANOVA) was
used to investigate if there is any
significant difference. Mean separation test
was conducted using Duncan’s new
multiple range test (DMRT).
Results and Discussion
The aseptic rhizome explants of C.
zedoaria cultured on MS medium
supplemented with different concentrations
combinations of BA (0.0, 0.5, 1.0, 1.5 and
2.0 µM) and NAA (0.0, 0.5, 1.0, 1.5 and
2.0 µM) produced shoots simultaneously
(Fig 1). Among the various hormone
concentrations and combinations the best
response was produced in culture
containing 8.0 µM BA and 1.0 µM NAA
with an average of 10.17 ± 1.89 shoots
(Tab 1). Shoot formation was found to be
less when explants cultured in MS medium
containing BA or NAA alone. In addition,
when NAA was used in combination with
BA at more concentration than 1.0 µM,
number of shoots was decreased gradually.
The results revealed that NAA at lower
concentration in combination with BA is
optimum for shoot multiplication in C.
zedoaria. Similar results were reported on
C. amada by Ferdous et al. (2012) and
Prakash et al. (2004) who found BA in
combination with NAA as optimal for
multiple shoot induction from rhizome
explants. This study is also supported by
some other reports on zingiberaceous plant
propagation (Jala, 2012; Raihana et al.,
2011; Kambaska et al., 2010; Chougule,
2008; Nasirujjaman et al., 2005; Bharalee
et al., 2005 and Islam et al., 2004).
NAA (µM)
1.0 1.5 2.0
1.08 ± 0.69i 1.04 ± 0.69i 1.00 ± 0.69i
3.96 ± 0.63ij 4.94 ± 1.01hi 4.13 ± 0.91ij
efg efg
6.81± 1.92 6.92 ± 1.72 6.10 ± 1.03fgh
cde def
7.85 ± 1.83 7.24 ± 1.64 6.04 ± 1.19fgh
a ab
10.17 ± 1.89 9.87 ± 1.17 9.59 ± 1.19b
b bc
9.69 ± 1.73 9.01 ± 1.29 8.19 ± 1.65bcde
3
Shoots, more than 1.5 cm in length, were
excised from multiplication culture and
were transferred to MS medium with 0.0,
1.0, 2.0, 2.5 and 4.0 µM NAA and IBA
supplementation for rooting. Out of two
auxins tested, NAA was found to be the
best for root induction (Tab 2). The mean
number of roots as well as mean length of
roots was also highly influenced by the
concentration and type of auxin. The
maximum number of roots per culture was
12.59 ± 2.83 with 5.39 ± 1.92 cm average
length when the shoots were cultured in a
medium containing 4.0 µM NAA.
Although the media containing IBA also
resulted in root formation, the rooting
response was not as good as that in the
media containing NAA. Salvi et al. (2000
& 2001) and Inden et al. (1998) also
reported NAA as best auxin for rooting in
C. longa and in Zingiber officinale
respectively.

Figure 1. Multiple shoots of C. zedoaria produced from rhizome bud explant after
4 weeks of culture.

A variety of morphological abnormalities simple method adopted in this study has

particularly of the stomata and cuticle are
caused by the in vitro conditions, which
results in high mortality rate after transfer
of plants to ex vitro/field conditions (Grout
and Aston, 1977; Rahman et al., 2004). In
order to maximize the survival of in vitro
derived plants, it is routine practice to
acclimatize them under high levels of
relative humidity (Short, 1991). In the
present study in vitro grown plantlets were
acclimatized in humid environment,
following transfer to soil conditions. A
facilitated the successful transfer of 70-
80% of plants from in vitro to ex vitro
conditions (Fig 2). After 20-30 days the
potted plants were nurtured in glass house
under complete sunlight for further
acclimatization of plantlets. After another
two weeks, these plants were transferred
successfully into field. It was further
observed that high humidity and moderate
temperature greatly enhance the initial
survival of potted and field grown
plantlets.

Figure 2. In vitro derived plantlets

after transferring them on to plastic

Persian Gulf Crop Protection, 2(4): 1-6 4

Conclusions
In conclusion, plant tissue culture is now a
well established technology which has
emerged as a potential tool and forms the
backbone of plant biotechnology. Tissue
culture techniques are widely applied for
the improvement of field crops,
horticulture and plantation crops for
increased agricultural production. The
successful production of multiple shoots,
Table 2. Effect of auxins (NAA and IBA) on in vitro root formation of C. zedoaria.
Type of auxin
(µM)
Control
1.0
2.0
NAA
2.5
4.0
1.0
2.0
IBA
2.5
4.0
Values represent means ± standard error of 10–15 explants per treatment in three repeated experiments. Values
with the same superscripts are not significantly different at 5% probability according to DMRT.
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