Hort. Environ. Biotechnol. 53(5):410-414. 2012.

DOI 10.1007/s13580-012-0096-1
Research Report

Microrhizome Induction in Acorus calamus Linn. - An Important Medicinal and

Aromatic Plant
*
Ningthoujam Sandhyarani Devi, Rajkumar Kishor, and Gurumayum Jitendra Sharma

Tissue Culture Laboratory, Centre for Advanced Study in Life Sciences, Manipur University, Imphal-795003, India

*Corresponding author: gis1951@yahoo.co.in

Received July 11, 2012 / Revised July 25, 2012 / Accepted July 30, 2012
GKorean Society for Horticultural Science and Springer 2012

Abstract. An effective protocol for in vitro microrhizome induction was developed for Acorus calamus. The explants,
rhizome axillary buds, were cultured on dual phase Murashige and Skoog (MS) medium consisting of agar solidified
phase overlaid by liquid fraction of the same medium. In this study, the effects of indole butyric acid (IBA) and Į–
naphthalene acetic acid (NAA) containing 2-10% (w/v) sucrose were examined on microrhizome induction. Best response
was observed on the medium supplemented with 2.0 mgL-1 IBA and 60% sucrose under 16/8 hours light/dark photo-
period which produced the maximum rhizome fresh weight (0.82 g) and size (length 4.8 cm; diameter 0.55 cm) in 6
weeks. The microrhizomes had 7-8 buds which were developed independent of season and each segment sprouted into
roots and shoots when transplanted to soil. This protocol can be adopted for various applications, viz., large scale
production of propagules of elite cytotype, in vitro conservation of the microrhizome, synthesis of secondary metabolites
and for studying the biosynthetic pathways of the bioactive molecules present in the rhizomes of this important medicinal
and aromatic plant.
Additional key words: dual phase culture, indole butyric acid, microrhizome, Ȼ-naphthalene acetic acid

Introduction calamus (Chopra et al., 1992; Patra and Malik, 1981;

Acorus calamus Linn. (Acoraceae) is a medicinal and
aromatic perennial herb, distributed in Asia, Africa, Europe,
and North America. It is a littoral dweller which grows in
ponds, swamps and on the banks of rivers. The rhizomes
and the leaves are aromatic due to the presence of the
phenylpropanoid asarone. ȕ-asarone is a toxic compound
(Abel, 1987) and its concentration varies between the varieties
of A. calamus (Ahlawat et al., 2010). This plant has been
used as medicine for its anti-spasmodic, anti-diarrhoeic,
carminative, anti-helminthic, anti-depressant and CNS anxiolytic
properties (McGaw et al., 2002). It is also used as tonic,
stimulant and aphrodisiac and for treating rheumatism,
toothache and respiratory ailments (Hutchings et al., 1996).
Dried roots have also been used for flavouring of bitter
liquors and appetizers (Dusek et al., 2007). The use of A.
calamus in various products, viz., shampoo, bath soap,
skincare, perfume, and air refresher has also been reported
(Lee and Han, 2011). Various bioactive compounds, viz.,
acorin, Į- and ȕ-asarone, asaryldehyde, caryophylene, isoasarone,
methyl isoeugenol and safrol have been reported from A.
Venskutoni and Dagilyte, 2003; Wang et al., 1998). It is
also reported that this plant has ecological importance since
it shows to have water purification activity (Kim, 2008;
Vojtiskova et al., 2006) including the removal of nitrogen
and phosphorus (Seo and Park, 2005). Three cytotypes -
diploid, triploid, and tetraploid of this species are found
worldwide. The varieties of A. calamus screened from Manipur
was reported to be triploid (Ahlawat et al., 2010; Sandhyarani
et al., 2011) with relatively low level (7-7.85%) of ȕ
-asarone (Ahlawat et al., 2010).
Research on in vitro induction of storage organ such as
rhizome is encouraged in recent times, because these can be
directly transferred to the field without any acclimatization
or hardening. Microrhizomes of many rhizomatous plants
were found to develop in vitro in liquid medium with inc-
reased sucrose levels, and larger microrhizomes were capable
of survival in the field without any discreet acclimatization
procedure (Shirgurkar et al., 2001). In addition, these organs
can be easily transported as they do not require any culture
medium or any other special measures to prevent contamination.
Microrhizomes of Curcuma longa were also found to pro-
 Hort. Environ. Biotechnol. 53(5):410-414. 2012.
duce secondary metabolites with antioxidant activity equaling
or surpassing commercial dried powdered rhizome preparations
of field-grown plants (Cousins et al., 2007).
A few investigators (Harikrishnan and Hariharan, 1999;
Lee and Han, 2011; Rani et al., 2000) have attempted the
micropagation of A. calamus mainly because it has slow
natural vegetative propagation through rhizome cutting. How-
ever, in vitro microrhizome induction of this plant has not
been reported till now. This communication reports microrhizome
induction in A. calamus under the influence of sucrose and
indole-butyric acid (IBA) using a dual phase culture for the
first time.
Materials and Methods
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Plants of A. calamus (Fig. 1A) were collected from dif-
o o o
ferent natural habitats of Manipur (23 50’-25 42’ NL; 92 58’-
o
94 45’ EL) and grown in the Experimental Garden of the
Department of Life Sciences, Manipur University, India.
The cytotype of the plant was investigated and found to be
A B
C D
Fig. 1. Microrhizome induction in Acorus calamus. (A) Plants
growing in habitat; (B) Harvested microrhizomes; (C) A plantlet
with induced microrhizome in dual phase Murashige and Skoog
-1
medium supplemented with 6% sucrose and IBA (2.0 mgᨿL );
(D) A single plantlet sprouted from transplanted microrhizome.
411
triploid. The determination of the cytotype has been dealt in
our previous report (Sandhyarani et al., 2011).
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The collected young rhizomes of A. calamus were washed
with running tap water to remove the adhering soils. After
removing the leaf sheaths the rhizomes were cut into segments
having single bud each and washed in detergent solution for
10 min. It was followed by a treatment with 5% fungicide
(Dhanustin-50) for 15 min and then with 70% ethanol for 60
s. Subsequently, they were treated with 0.1% HgCl2 for 7
minutes and washed at least three times with autoclaved
distilled water. The rhizomes were finally trimmed and cut
into 6-8 mm sized pieces and used as explants.
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Establishment of aseptic culture and induction of multiple
shoots have been dealt in our previous article (Sandhyarani
et al., 2011). Murashige and Skoog medium (1962) was
used with B5 vitamins (Gamborg et al.,1968), 3% (w/v)
sucrose, 0.8% (w/v) Difco Bacto Agar (Hi-media, Mumbai,
India) and supplemented with various concentrations of
benzylaminopurine (BAP) and in combination with Į
-naphthaleneacetic acid (NAA) for establishment of aseptic
culture and multiple shoot induction. All plant growth regu-
lators (PGRs) were from Sigma (St. Louis, MO, USA). The
pH of the medium was adjusted to 5.8 with 1 N NaOH or
-2
HCl and autoclaved at 121Gand 1.05 kgcm pressure for
20 min. Single rhizome buds were inoculated per culture
tube (32 × 200 mm) (Borosil, India) containing approximately
30 mL of the semi-solid agar medium overlaid with 5 mL of
the sterilized liquid fraction under sterile conditions. Except
for agar, the liquid phase had the same organic, inorganic
and growth regulator regime as that of the semi-solid phase
for each treatment. The cultures were incubated at 25 ± 2G
-2 -1
under 28 ȝmolm s illumination from cool white fluorescent
tubes (Bajaj, India) for 16/8 h light/dark photoperiod.
,QGXFWLRQ RI 5KL]RPHV
In vitro grown microshoots of A. calamus approximately
2-3 cm long which were obtained from the established cul-
-1
ture containing MS medium along with 0.5 mgL NAA
-1
and 2.0 mgL BAP, were used as explants for induction of
rhizomes. In the first experiment, a dual phase MS medium
consisting of different concentrations of sucrose (2, 4, 6, 8,
and 10% w/v) was used for microrhizome induction. Secondly,
based on the result of the first experiment, the dual phase
MS medium containing 6% sucrose was supplemented with
different concentrations of IBA (0.1, 0.5, 1.0, 2.0, and 4.0
-1 -1
mgL ) or NAA (0.1, 0.5, 1.0, 2.0, and 4.0 mgL ) to test
412 Ningthoujam Sandhyarani Devi, Rajkumar Kishor, and Gurumayum Jitendra Sharma
their effect on microrhizome induction. The medium
containing 6% sucrose without any PGR was used as control
for the second experiment.
The culture condition remained the same as described for
the establishment of aseptic cultures. There were fifteen
replicates per treatment and the experiments were repeated
twice.
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The rhizomes were harvested inside the laminar air flow
chamber and the length, diameter and fresh weight of the
rhizomes per plant were measured after six weeks of inoc-
ulation. Rhizomes of each plant with 7-8 buds (Fig. 1B)
were cut into segments having a single bud and washed with
distilled water. Each bud was planted in sterilized sand in
small PVC cups (120 mL volume). The cups were placed in
a plastic tray with a layer of tap water (1 cm) and covered
with another plastic tray to maintain high humidity. Survival
percentage was calculated after four weeks.
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The experiments were set up in a completely randomized
block design and repeated twice. Single explants were inocu-
lated per culture tube and fifteen replicates were taken per
treatment. The recorded data were subjected to analysis of
variance (ANOVA) and the means were separated using
Tukey’s test. All statistical analyses were performed at the
5% level of significance using the software SPSS (Version
14, SPSS Inc., Chicago, USA).
Results and Discussion
Despite being a littoral plant, the explant sources could be
properly sterilized by treating in 5% fungicide for 15 min,
70% alcohol for 60 s followed by 0.1% aqueous HgCl2
solution for 7 min. In the first experiment, different con-
centrations of sucrose (2, 4, 6, 8, and 10% w/v) were tested
to determine the most effective concentration in induction of
microrhizome using dual phase MS medium. The different
treatments showed variable response in terms of size and
fresh weight of the microrhizomes. Although all the tested
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concentrations of sucrose were able to induce microrhizomes
in A. calamus, the medium having 6% sucrose had the
largest size (length 3.9 cm, diameter 0.47 cm) and maximum
fresh weight (0.72 g) (Table 1 and Fig. 1C). Size and fresh
weight gradually decreased with the further increase in
sucrose concentration. An advantage of the dual phase
culture medium is that microrhizomes could be induced
even at a low sucrose concentration of 2% after six weeks of
inoculation while the same could not be observed on
agar-gelled medium (unpublished data). It also has an advan-
tage over liquid culture since it can anchor the explants in
the agar gelled fraction and hence can be kept static without
continuous shaking. Sucrose might act as energy source and
as an osmoticum in inducing rhizome formation (Bhat et al.,
1994). Rhizome serves as sink where assimilates are uploaded,
and in an in vitro culture system assimilates provided as
sucrose may have been transported to the stem for rhizome
formation in gingers (Chirangini et al., 2005). It has also
been reported that sucrose was responsible for storage organ
formation in potato, piper and Curcuma species (Ross and
Davies, 1992; Thorpe, 1982; Tyagi et al., 1998). However,
the research findings of a number of investigators showed
that certain specific concentrations of sucrose were effective
in the induction of microrhizome, viz., 6% in Curcuma
aromatica (Nayak, 2000), 6-8% in Curcuma longa (Shirgurkar
et al., 2001; Sunitibala et al., 2001), 6% in Kaempferia
galanga, 6% and 9% in K. rotunda (Chirangini et al., 2005)
and 6% in C. zedoaria (Anisuzzaman et al., 2008). On the
average, 6% sucrose appears to be the most favourable
concentration for microrhizome induction in a number of
rhizomatous plants. However, the reason why sucrose con-
centrations above these specific levels are inhibitory in
microrhizome induction remains to be determined.
On further supplementation of IBA and NAA in the
medium having 6% sucrose, the former auxin showed better
effect on microrhizome induction. Those formed in the
-1
medium supplemented with 2 mgL IBA and 6% sucrose
produced largest microrhizomes with an average length of
4.8 cm and a diameter of 0.55 cm with the highest average
fresh weight of 0.82 g (Table 2). This treatment produced a
significant increase in fresh weight of the microrhizomes in

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comparison to that of the control (MS + 6% sucrose) as well
as those supplemented with IBA (0.1, 0.5, 1.0, and 4.0 mg
-1
L ). Bigger microrhizomes are desired since plantlets
developed from them grew faster (Shirgurkar et al., 2001).
The microrhizomes had 7-8 axillary buds, as well. Those
treated with different concentrations of NAA also did not
produce any pronounced effect in terms of fresh weight and
size of the microrhizome as compared to those with IBA.
Various investigators reported the positive effect of BAP in
addition to high sucrose concentration in microrhizome in-
duction in C. longa (Raghu, 1997), C. zedoaria (Anisuzzaman
et al., 2008) and other geophytes. However, our previous
study showed that various concentrations of BAP, instead,
induced multiple shoot induction in A. calamus in the dual
phase culture (Sandhyarani et al., 2011). Shirgurkar et al.,
(2001) also reported the inhibitory effect BAP on microrhizome
production in Curcuma longa. Different auxins are known
for their promontory role in rooting, and in the present
study, IBA showed better effect on microrhizome induction
than NAA.
The harvested microrhizomes upon transfer to sterilized
sand could acclimatize with 100% survival (data not shown)
and the buds developed into leaves and simultaneously the
roots also emerged in two weeks (Fig. 1D). Viability of the
microrhizomes was determined by regeneration of the roots
and leaves and subsequent establishment in sterilized sand.
Production of microrhizome is a novel technique for many
rhizomatous plants since they can readily establish in ex
vitro condition, can be transported more conveniently than
plantlets and preserved in vitro over a long period. The
microrhizome production system may also be utilized for
production of secondary metabolites for chemical analysis,
other experimentation, or direct usages.
Acknowledgement : The authors are grateful to the Indian
Council of Medical Research, Government of India, New
Delhi for financial support (Grant No. 4/2-1/2003/CAR/BMS/
TRM) and to the Department of Science and Technology,
Govement of India for award of DST-Woman Scientist-A
fellowship to Ms. N. Sandhyarani Devi.
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