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: [email protected]).

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 objectives of the current research were to
Curcuma zedoaria, also known as white optimize growth regulator requirements for
turmeric, is a rhizomatous herbaceous consistently high production of shoots
perennial plant belonging to Zingibraceae from the primary explants, to select auxin
family. It is an important medicinal plant type and concentration for efficient in vitro
and its rhizome is commercially exploited rooting of regenerated shoots and to
in industry as well as traditional medicinal acclimatize and transfer of plantlets to
practices. It is used traditionally for the natural conditions.
treatment of menstrual disorder, dyspepsia, Materials and Methods
vomiting (Prajapati et al., 2003), and for Plant material: The rhizomes of C.
cancer related disease (Rahman et al., zedoaria were collected during the rainy
2013). Moreover, rural people abundantly season in October and November 2012
use the rhizome of C. zedoaria due to from the naturally grown plant population
having rubifacient, carminative, at the botanical garden of Rajshahi
expectorant, demulcent, diuretic and University, Bangladesh. Rhizome buds
stimulant properties while root is used in about 1 to 2 cm long were selected as the
the treatment of flatulence, dyspepsia, initial explants.
cold, cough, and fever (Wilson et al., Surface sterilization and explants
2005). This plant is multiplied through procurement: About 2.0 to 3.0 cm apical
vegetative propagation in nature, but takes buds from the C. zedoaria rhizomes were
a long time to produce rhizome (Stanly and soaked in a few drops of commercial
Keng, 2010). Vegetative propagation detergent and washed thoroughly under
confers low genetic variability to the running tap water for a few minutes to
species, reducing the chances of selecting a remove all the mud. Then the buds were
naturally-occurring clone. Besides, natural immersed in 75% (w/v) ethanol for one
plant resources of this plant are gradually minute before transferring them into
decreasing due to large scale exploitation laminar air flow cabinet. Explants were
from naturally occurring population for then surface sterilized with 0.1% HgCl2 for
traditional uses. Taking into account the 10 minutes and washed thoroughly 3 to 4
aforesaid problems and threats, care should times with sterile distilled water and
be taken to protect as well as conserve the soaked with sterile blotting paper under
existing germplasm of this plant species laminar air flow cabinet. Sterilized
and in vitro propagation may play a explants were then dissected with a sterile
significant role in this regard. Research on surgical blade to remove the outer layers of
in vitro propagation of C. zedoaria was leaf sheaths under aseptic conditions to
carried out by several researchers (Stanly produce 5 to 8 mm sized pieces having at
and Keng, 2010; Stanly and Keng, 2007; least one eye (Chirangini and Sharma,
Miachir et al., 2004) who concentrated on 2005).
rhizome derived explants as well as in vitro Culture medium and incubation
derived shoot explants. However, tissue conditions: The explants were inoculated
culture techniques have been exploited on to Murashige and Skoog (1962)
widely for in vitro propagation of many medium supplemented with 6-Benzyl
other Curcuma species using rhizome adenine (BA) (0.0, 2.0, 4.0, 6.0, 8.0 and
derived explants (Nadgauda et al., 1978; 10.0 µM) and α-Naphthalene acetic acid
Mukhri and Yamaguchi, 1986; (NAA) (0.0, 0.5, 1.0, 1.5 and 2.0 µM)
Balachandran et al., 1990; Sit and Tiwari, alone or in combination. The pH of the
1997; Salvi et al., 2002). The overall medium was adjusted to 5.8 prior to
objective of the present study was to autoclaving. The culture bottles were
develop an optimized protocol for in vitro sealed with parafilm and were incubated in
propagation of C. zedoaria. The specific the culture room under white fluorescent

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

light with light intensity of 3000 lux at a USA). Analysis of variance (ANOVA) was
photoperiodic16 h at 25 ± 2°C. The growth used to investigate if there is any
of the cultures was observed after 6 weeks. significant difference. Mean separation test
For adventitious rooting, in vitro derived was conducted using Duncan’s new
shoots were cultured onto MS medium multiple range test (DMRT).
containing NAA and Indole-3-butyric acid Results and Discussion
(IBA) at different concentrations (0.0, 1.0, The aseptic rhizome explants of C.
2.0, 2.5 and 4.0 µM). zedoaria cultured on MS medium
Hardening of in vitro derived plantlets: supplemented with different concentrations
Well-rooted plantlets were removed from combinations of BA (0.0, 0.5, 1.0, 1.5 and
the culture tubes and the roots were 2.0 µM) and NAA (0.0, 0.5, 1.0, 1.5 and
washed under running tape water to 2.0 µM) produced shoots simultaneously
remove agar trace. Then the plantlets were (Fig 1). Among the various hormone
transferred to small plastic pots containing concentrations and combinations the best
autoclaved garden soil and compost (1:1) response was produced in culture
and maintained inside growth chamber set containing 8.0 µM BA and 1.0 µM NAA
at temperature 280C and 70-80% relative with an average of 10.17 ± 1.89 shoots
humidity. As to ensure its humidity, the (Tab 1). Shoot formation was found to be
plants were sprayed periodic ally with less when explants cultured in MS medium
water. After four weeks they were kept in containing BA or NAA alone. In addition,
green house before transferring them to the when NAA was used in combination with
Botanical Evaluation Garden of Plant BA at more concentration than 1.0 µM,
Tissue Culture Laboratory finally. The number of shoots was decreased gradually.
survival rate percentage of the explants The results revealed that NAA at lower
was observed after four weeks. concentration in combination with BA is
Experimental design and data analysis: optimum for shoot multiplication in C.
Ten to fifteen cultures were used per zedoaria. Similar results were reported on
treatment. All the cultures were examined C. amada by Ferdous et al. (2012) and
periodically and the morphological Prakash et al. (2004) who found BA in
changes were recorded carefully. The combination with NAA as optimal for
effect of different treatments was multiple shoot induction from rhizome
determined with respect to number of explants. This study is also supported by
shoots, roots and the length of shoots and some other reports on zingiberaceous plant
roots. The experimental design used was propagation (Jala, 2012; Raihana et al.,
Randomized complete block design 2011; Kambaska et al., 2010; Chougule,
(RCBD). Data were analyzed using the 2008; Nasirujjaman et al., 2005; Bharalee
Statistical package for the social science et al., 2005 and Islam et al., 2004).
(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
NAA (µM)
BA (µM)
0.0 0.5 1.0 1.5 2.0
0.0 1.14 ± 0.69i 1.01 ± 0.69i 1.08 ± 0.69i 1.04 ± 0.69i 1.00 ± 0.69i
2.0 2.17 ± 0.82kl 3.09 ± 0.93ik 3.96 ± 0.63ij 4.94 ± 1.01hi 4.13 ± 0.91ij
ghi fgh efg efg
4.0 5.47 ± 1.08 5.89 ± 1.48 6.81± 1.92 6.92 ± 1.72 6.10 ± 1.03fgh
efg cde cde def
6.0 6.98 ± 1.04 7.81 ± 1.83 7.85 ± 1.83 7.24 ± 1.64 6.04 ± 1.19fgh
bcde bcd a ab
8.0 8.13 ± 1.61 8.74 ± 1.42 10.17 ± 1.89 9.87 ± 1.17 9.59 ± 1.19b
bcd bc b bc
10.0 8.85 ± 1.86 9.05 ± 1.73 9.69 ± 1.73 9.01 ± 1.29 8.19 ± 1.65bcde
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 3

Shoots, more than 1.5 cm in length, were 12.59 ± 2.83 with 5.39 ± 1.92 cm average
excised from multiplication culture and length when the shoots were cultured in a
were transferred to MS medium with 0.0, medium containing 4.0 µM NAA.
1.0, 2.0, 2.5 and 4.0 µM NAA and IBA Although the media containing IBA also
supplementation for rooting. Out of two resulted in root formation, the rooting
auxins tested, NAA was found to be the response was not as good as that in the
best for root induction (Tab 2). The mean media containing NAA. Salvi et al. (2000
number of roots as well as mean length of & 2001) and Inden et al. (1998) also
roots was also highly influenced by the reported NAA as best auxin for rooting in
concentration and type of auxin. The C. longa and in Zingiber officinale
maximum number of roots per culture was 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 facilitated the successful transfer of 70-
caused by the in vitro conditions, which 80% of plants from in vitro to ex vitro
results in high mortality rate after transfer conditions (Fig 2). After 20-30 days the
of plants to ex vitro/field conditions (Grout potted plants were nurtured in glass house
and Aston, 1977; Rahman et al., 2004). In under complete sunlight for further
order to maximize the survival of in vitro acclimatization of plantlets. After another
derived plants, it is routine practice to two weeks, these plants were transferred
acclimatize them under high levels of successfully into field. It was further
relative humidity (Short, 1991). In the observed that high humidity and moderate
present study in vitro grown plantlets were temperature greatly enhance the initial
acclimatized in humid environment, survival of potted and field grown
following transfer to soil conditions. A plantlets.

Figure 2. In vitro derived plantlets

after transferring them on to plastic

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

Conclusions roots depend on the nutrient medium and
In conclusion, plant tissue culture is now a the culture environment. The results
well established technology which has described through this manuscript
emerged as a potential tool and forms the represent a simple and reliable method for
backbone of plant biotechnology. Tissue in vitro propagation of C. zedoaria. This
culture techniques are widely applied for efficient plant propagation system using
the improvement of field crops, rhizome bud explants can be exploited for
horticulture and plantation crops for large scale multiplication of elite plant and
increased agricultural production. The conservation of C. zedoaria.
successful production of multiple shoots,

Table 2. Effect of auxins (NAA and IBA) on in vitro root formation of C. zedoaria.
Type of auxin Mean no. of Mean length of roots
(µM) roots (cm)
Control 2.43 ± 0.13e 1.48 ± 0.26a
1.0 3.72 ± 0.53e 1.94 ± 1.14a
cd
2.0 6.93 ± 1.60 4.88 ± 1.17a
NAA
2.5 9.18 ± 1.61bc 5.03 ± 1.95a
a
4.0 12.59 ± 2.83 5.39 ± 1.92a
1.0 2.96 ± 0.87 1.97 ± 1.10a
de
2.0 4.89 ± 1.17 2.95 ± 1.17bc
IBA bc
2.5 8.48 ± 1.19 3.98 ± 1.32ab
ab
4.0 10.94 ± 1.73 4.84 ± 1.19a
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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