International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 2(5) pp. 129-136, May, 2011
Available online http://www.interesjournals.org/IRJPS
Copyright © 2011 International Research Journals
Full length Research Paper
Micropropagation of the medicinal herb Glycyrrhiza
glabra L., through shoot tip explant culture and
glycyrrhizin detection
1
Wanchat Sawaengsak, 1Tanatorn Saisavoey, 2Petcharat Chuntaratin, and 2Aphichart
Karnchanatat*
1
Program of Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok
10330, Thailand
2
Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan,
Bangkok 10330, Thailand
Accepted 26 May, 2011
Licorice, Glycyrrhiza glabra L., is a perennial plant native to Southern Europe and parts of Asia, used as
a non-nutritional sweetener but with numerous reported pharmacological effects, including antiinflammatory and antiviral properties. This study describes a protocol for the micropropagation of G.
glabra plants from in vitro shoot tips. Three media formulations, Murashige-Skoog (MS), Gamborg (B5)
and Woody Plant Medium (WPM) medium, supplemented with 1-, 1/2- or 1/4- strength B5 salt base were
tested for the ability to support the growth of shoot tips in culture. In this study, 1/2-strength B5 salt
base was found to be the most suitable supplement for G. glabra plant growth and development.
However, MS medium supported a superior explant proliferation rate. MS medium supplemented with
0.5 mgL-1 of the cytokinin benzyladenine (BA) produced the largest average number of shoots (4.75) per
explant. The highest efficiency of root formation occurred in the 1/2-strength B5 medium containing 5.0
-1
mgL of either of the two auxins, indolacetic acid (IAA) or indolebutyric acid (IBA), after six weeks of
culture. The production of glycyrrhizin in culture using 1/2-strength B5 medium supplemented with 5
mgL-1 IAA increased with time up to week eight reaching 27.57 ± 0.66 g g-1 dry wt.
Keywords: Glycyrrhiza glabra L., Licorice, Micropropagation, Shoot tips, Plant growth regulators
INTRODUCTION
Glycyrrhiza glabra Linn., commonly known as “licorice”,
belongs to the family Leguminosae in the genus
*Corresponding author E-mail : i_am_top@hotmail.com ; Tel.:
+662-218-8078; Fax: +662-253-3543
Abbreviations
B5 - Gamborg;
BA - 6-Benzyladenine;
IAA - indole-3-acetic acid;
IBA - indole-3-butyric acid; Kn - kinetin;
MS - Murashige and Skoog; NAA - -naphthalene acetic acid;
TDZ - Thidiazuron; WPM - Woody Plant Medium;
CRD - complete randomized design;
HPLC - high performance liquid chromatography
Glycyrrhiza which contains 30 species native to
subtropical and hot temperate regions. Licorice, as in G.
glabra, is a herbaceous perennial native to Southern
Europe and parts of Asia, the roots and rhizomes of
which have been used commercially as a non-nutritional
sweetening and flavoring agent in some candies and
pharmaceuticals. The principal active component in
licorice extract is glycyrrhizin which is localized
exclusively in the underground woody parts of the
thickening roots and stolons (rhizomes) where it can
comprise from 2-14% of the dry weight (Gibson, 1978;
Steglich et al., 2000; Wang, 2000), whereas soyasaponins, which are also oleanane-type triterpene
saponins, are localized mainly in the seeds and rootlets
of the licorice plant (Hayashi et al. 1996). The roots of the
licorice plant are one of the oldest known botanicals in
Chinese medicine where the beneficial health properties
130 Int. Res. J. Plant Sci.
attributed to licorice include anti-inflammation, laxative,
immunomodulatory, anti-ulcer, anti-allergy and anticarcinogenesis (He et al., 2001; Henry et al., 1991;
Matsui et al., 2004; Takahara et al., 1994; Wang and
Nixon, 2001). In addition, glycyrrhizin has anti-cancer,
anti-bacterial, anti-spasmolytic and anti-viral activities, the
latter against both DNA and RNA viruses (Fiore et al.,
2008; Zalkov et al., 1994). In particular, it has been found
to be highly active in inhibiting replication of the severe
acute respiratory syndrome (SARS)-associated virus as
well as a potential therapeutic agent for chronic hepatitis
and acquired immunodeficiency syndrome (AIDS) (Baba
et al., 1988; Cinatl et al., 2003; Ito et al., 1987).
More than 400 compounds have been isolated from
Glycyrrhiza species. Triterpene saponins and flavonoids
are believed to be responsible for the bioactivities of
licorice. The contents of these saponins and flavonoids
may vary significantly due to different plant species and
geographic sources (Zhang and Ye, 2009). Glycyrhizin is
obtained from the roots and tubers of field grown intact
plants, but the ability to grow the plant is restricted to
certain climates only. In addition, the glycyrrhizin content
may well vary with environmental changes, between
seasons, soils and agricultural conditions, as well as
between cultivar genetics. The qualitative analysis of G.
glabra roots derived from different countries revealed that
some phenolic compounds were indicative of the area of
collection. Sample of licorice roots from Italy (51.91 mg/g)
and China (53.26 mg/g) were the most rich in glycyrrhizin
(Montoro et al., 2011). Although the quantitative of
glycyrrhizin level related species and geographic
sources, it could not keep the highest level of glycyrrhizin
in uniformity.
Even within the problem of these variations lies the more
serious logistics of culture since the conventional method
for propagation of G. glabra, a cross-pollinated plant, is of
course via seed which restricts its multiplication and thus
ease of economically viable agriculture. The alternative
source, and one that offers the opportunity to overcome
the poor seed germination problem, is in vitro culture
which can provide a rapid propagation of new varieties
within an economically viable timescale. To this end,
Syrtanova and Mukhitdinova (1984) attempted the tissue
culture based multiplication of G. glabra and G. uralensis
from seedling cultures. Although multiple shoot formation
with some 90 - 95% plantlet survival upon transplanting
into the field has been reported (CIMAP, 2005), they
gave no experimental details as to how this was attained.
Meanwhile, the development of callus and cell cultures
from G. glabra has increasingly been reported and
included from shoot tips, roots, leaves, stems and
hypocotyl as explant tissue sources (Hayashi et al., 1988;
Hayashi et al., 1992; Henry et al., 1984; Tamaki et al.,
1973; Yoo and Kim, 1986). Moreover, this has included
the production of G. glabra plants from shoot tip and
nodal explant cultures on simple minimal media
(Kohjyouma et al., 1995; Thengane et al., 1998).
Regenerative callus formation, as well as cell
suspensions, have also been reported recently (Mousa et
al., 2007).
Given that plant cell cultures are a
promising alternative source for the production of
valuable secondary metabolites, this potentially paves the
way for potential glycyrrhizin production in tissue culture.
However, despite the initial reports on the successful
tissue culture production of licorice explants, the ability to
produce glycyrrhizin in cell culture is controversial. Initial
reports of its production by Glycyrrhiza sp. callus and
suspension cultures, including within larger scale 80 L
airlift bioreactors, at levels approaching 3-4% of the dry
weight (Scragg & Arias-Castro, 1992; Tamaki et al.,
1973), were not replicated in callus or cell suspension
cultures of a known G. glabra isolate (Hayashi et al.,
1988). Likewise, although Ko et al. (1989) reported the
production of glycyrrhizin in transformed hairy root
cultures of G. ulalensis, they found no production in
transformed roots of G. glabra. Nevertheless, the
formation of glycyrrhizin in G. ulalensis calli cultured in a
Murashige & Skoog (MS) based medium supplemented
with auxin (either naphthalene acetic acid (NAA) or 2,4dichlorophenoxyacetic acid (2,4-D)) could be induced
with
specific
combinations
of
the
cytokinins
benzyladenine (BA), 6-furfuryaminopurine (Kientin) or
thiadiazuron (TDZ) (Shams-Ardakani et al., 2007;
Wongwicha et al., 2008). Thus, in addition to variation in
the genetic source (species and perhaps cultivars) of the
tissue or cell suspension origins, the specific culture
requirements including auxin / cytokinin concentrations or
relative ratio, seem to be potentially important factors in
glycyrrhizin production. To start to clarify this situation,
this paper reports on a study which aimed to evaluate the
optimal culture media and plant growth regulators on
shoot multiplication and root induction for glycyrrhizin
production by tissue culture techniques.
MATERIALS AND METHODS
Plant material and sterilization
Shoots of G. glabra were taken from a single mature mother
plant growing in the nursery of the Institute of Biotechnology
and Genetic Engineering, Chulalongkorn University, Thailand.
Shoot tips of 1.0 - 2.0 cm length were excised and rinsed for 30
min in running tap water, surface-sterilized for 15 min in 10%
(v/v) Clorox containing 0.1% (v/v) Tween 20, and washed three
times with sterile water. The sterilized shoot explants were then
cultured individually on induction medium.
Initiation of cultures and culture condition
Sterilized shoot explants were cultured individually under
aseptic conditions on MS (Murashige and Skoog, 1962) media
containing 3.0% (w/v) sucrose as a carbon source and 0.7%
(w/v) agar. The pH of the medium was adjusted to 5.7 using 1 N
NaOH before autoclaving. The cultures were maintained at 25 ±
Sawaengsak et al. 131
2 °C in the presence of 16/8 h (light/dark) photoperiod, with light
provided by a cool-white fluorescent tube at an intensity of 55
-2 -1
mol m s .
Effect of medium formulations and concentration on shoot
growth and development
To optimize the culture medium for the growth and development
of shoots, the shoot tips of in vitro grown plantlets were
evaluated after 1 month of culture in MS, B5 (Gamborg et al.,
1968) or WPM (Lloyd and McCown, 1981) media. Each media
was varied in the concentration of the salt base supplement
using 1-, 1/2- or 1/4- strength concentrations (still maintaining a
final concentration of 3.0% (w/v) sucrose in 0.7% (w/v) agar).
The pH of each media was adjusted to 5.7, sterilized and plants
cultured in it as described previously above. The number and
length of the shoots, the stem diameter and leaf width were all
recorded after six weeks of culture.
250 × 4.6 mm) by a stepwise gradient elution using a
decreasing acetic acid (1% (v/v)) to acetonitrile (v/v) ratio mobile
phase as follows: 0 min(90:10), 15 min (50:50), 40 min (40:60)
and 50 min (0:100). The flow rate of the mobile phase was 0.9
ml/min with UV absorbance detection at 254 nm. For the time
course study, untreated plants and those supplemented with
plant growth regulators in the root media were harvested at 6, 8,
10 and 12 weeks. To observe the effect of root age in response
to plant growth regulators, roots in different growth stages were
treated with a specific amount of plant growth regulator.
Glycyrrhizin content refers to the total amount of glycyrrhizin
recovered from 0.1 g dry weight of root powder.
Statistical analysis
A complete randomized design (CRD) was used for all
experiments. The data were analyzed using SPSS Version
16.0. ANOVA was used to analyze data. Significant differences
were assessed using Scheffe’s multiple range test and
significance was accepted at p< 0.05.
Effect of cytokinin on shoot multiplication
Shoot explants after four weeks of culture were cut transversely
and transferred to the suitable medium (determined from the
results of the above trails) supplemented with 3.0% (w/v)
sucrose in 0.7% (w/v) agar, and supplemented with different
concentrations (0-2.5 mgL-1) of one of the cytokinins,
benzyladenine (BA), 6-Furfuryaminopurine (Kinetin) or
thiadiazuron (TDZ). The transversely cut shoot explants were
maintained in this culture for six weeks and then the number
and length of shoots per plantlet were measured.
Effect of auxin types on rooting ability
To optimize root induction, in vitro shoot explants after four
weeks of culture for 4 weeks were excised and transferred
individually to medium supplemented with 3.0% (w/v) sucrose in
0.7% (w/v) agar containing different concentrations of auxins (05 mgL-1), being either IAA or IBA. The excised shoot explants
were cultured for six weeks and then the number and length of
the roots measured.
Quantitative analysis
The roots of licorice were dried at 50 °C to constant weight. A
100 mg aliquot of the dried sample was pulverized and then
extracted with 20 ml of 95% methanol: water solution for 12 h
before filtering and evaporating the filtrate to dryness to attain a
crude glycyrrhizin preparation. The sample solution was
prepared by dissolving the crude extract in 1 ml of 95%
methanol: water solution and then a 10 l aliquot of this extract
solution were analyzed by HPLC. The standard solutions were
made up to volume with deionized water to give a series of
standard solutions of 0.000, 0.025, 0.05, 0.1, 0.25, 0.5 and 1.0
mg/ml, used for the calibration curve. Deionized water,
containing varying ratios of 1% (v/v) acetic acid: acetonitrile as
the mobile phase. HPLC analysis was carried out on a
ThermoFinningan HPLC Spectra System equipped with
autosampler model AS3000, Photodiode array UV detector
model UV6000LP and ChomQuest Software. The analysis was
carried out on a Phenomenex column (Luna 5u C18(2) 100A,
RESULTS AND DISCUSSION
Shoots were found to be a suitable explant source for G.
glabra micropropagation with a typical success rate in the
in vitro tissue culture propagation of 90 - 95 % (range) in
this study. After six weeks of culture, morphogenic
changes in the cultured explants were clearly observed.
The favorable effect of a low concentration of MS
macronutrients, also noted in this study (data not shown),
has been discussed by many authors, as has the
increased rate of multiplication when using MS salts at
3/4-, 1/2- and 1/4- strength (Hyndman et al., 1982;
George and Sherrington, 1984). In the present study,
however, media formulation additionally was observed to
display a clear effect upon the morphogenic responses
that were observed. Overall, 1/2-strength B5 salts were
the most suitable media for the in vitro tissue culture
growth and morphogenesis of G. glabra plants (Table 1).
The shoots were healthier and more uniform in length
(Figure 1A) compared to those grown on other media
(data not shown). However, despite the general beneficial
effect of the reduced macronutrient level of media on G.
glabra plant growth and differentiation (Table 1), the
maximum number of shoots per explant (1.5) was found
when explants were grown on full-strength MS salts,
although this was only marginally higher than that on halfstrength MS. Therefore, the effect of cytokinins at
different concentrations upon shoot multiplication rates
was evaluated in explants cultured in full-strength MS
salts.
Certainly, the in vitro multiplication of G. glabra using
different explants sources on modified MS media has
been reported previously for seedling explants (Shah and
Dalai, 1980) and cultures (Syrtanova and Mukhitdinova,
1984), as well as a high frequency regeneration of G.
glabra from nodal explants (CIMAP, 1995). Moreover,
whilst successful shoot tip explant cultivation in simple
132 Int. Res. J. Plant Sci.
Table 1. Effect of different media on the morphogenic responses of shoot explants of G. glabra after six weeks of in vitro tissue
culture. Data are shown as the mean ± 1 S.E. and are derived from 20 replicates. Means with different superscript lower case letters
within a column are significantly different (p<0.05; Scheffe’s multiple means test).
Media
Strength
Number
of
shoot/explant
MS
1
1/2
1/4
1.50 ± 0.20
1.45 ± 0.21A
A
1.10 ± 0.07
B5
1
1/2
1/4
1.40 ± 0.15
AB
1.25 ± 0.12
B
1.00 ± 0.00
WPM
1
1/2
1/4
1.00 ± 000
A
1.05 ± 0.05
1.00 ± 0.00A
A
A
A
Shoot length
(cm)
A
5.45 ± 0.40
5.80 ± 0.42A
A
5.00 ± 0.30
B
5.00 ± 0.27
A
6.58 ± 0.32
AB
5.94 ± 0.38
B
5.12 ± 0.35
AB
6.30 ± 0.30
6.48 ± 0.35A
media has been reported before (Thengane et al., 1998)
study, however, shoot explants were successfully
cultured for six weeks on MS medium, and that
supplemented with 0.5 mgL-1 BA was found to be the
most effective media (Figure 1B), attaining not only the
highest number of shoots per explant but also the highest
shoot length (Figure 2A and 2B). This is in agreement
with previous reports that a low cytokinin concentration
without any exogenously added auxins plays an
important role in shoot induction (Kohjyouma et al., 1995;
Thengane et al., 1998). Therefore, media supplemented
with the cytokinin BA at 0.5 mgL-1 was selected as the
appropriate condition for shoot proliferation assays in the
following experiment.
For the evaluating root induction, shoots of >1.0 cm long
were excised and transferred individually to half-strength
B5 salts supplemented with various concentrations (0 -1
5.0 mgL ) of either IAA or IBA. The highest frequency of
root formation was obtained on the medium
supplemented with 5.0 mgL-1 of either IAA or IBA (Figure
1C and 1D). However, whilst the addition of IBA in the
rooting medium for explants was found to be more
suitable for root induction than IAA containing medium in
terms of the root length attained (Figure 3B), in contrast
the addition of IAA yielded a greater ratio of explants that
actually rooted than that seen with IBA (Figure 3A). The
in vitro raised plantlets were subsequently transferred
individually to plastic cups filled with sterile garden soil
(SC1), vermiculite (SC2), garden soil mixed with
vermiculite and ash husk at 1:1:1 (w/w) (SC3), ash husk
(SC4) and garden soil mixed with ash husk at 1:1 (w/w)
(SC5). These plantlets were kept at room temperature in
natural light for about a week and then transferred to a
greenhouse, where the surviving plantlets resumed
Stem diameter
(cm)
Leaf width
(cm)
Number of
branches/explant
A
0.36 ± 0.02
0.32 ± 0.03B
A
0.54 ± 0.02
A
0.60 ± 0.02
A
0.68 ± 0.03
B
0.54 ± 0.03
0.05 ± 0.03
0.05 ± 0.02A
A
0.06 ± 0.02
0.07 ± 0.00
A
0.06 ± 0.00
B
0.05 ± 0.00
A
0.06 ± 0.00
B
0.05 ± 0.00
0.04 ± 0.00C
B
B
4.95 ± 0.21
5.95 ± 0.35AB
A
6.35 ± 0.22
AB
6.30 ± 0.26
A
8.00 ± 0.33
A
7.35 ± 0.25
AB
3.55 ± 0.48
AB
4.85 ± 0.36
5.90 ± 0.24A
0.43 ± 0.03
A
0.49 ± 0.02
0.38 ± 0.02B
B
B
normal growth and developed healthy leaves after two
weeks. In the case of the SC1 media, some 95% of the
plantlets survived (Figure 1E).
At this stage the contents of glycyrrhizin were then
analyzed by HPLC. The time course of the effect of the
plant growth regulators on glycyrrhizin accumulation in
12-week-old cultures is shown in Figure 4. The maximum
glycyrrhizin content (27.57 ± 0.66 g g-1 dry wt), attained
after eight weeks in culture, was observed in plantlets
cultured using 1/2-strength B5 medium supplemented
with 5 mgL-1 IAA. Indeed, this glycyrrhizin level was very
similar to that reported in G. uralensis calli cultured in MS
media after induction with NAA and BA, but somewhat
less than when induced with TDZ alone (~36.5 + 2.5 g
g-1 dry wt) (Wongwicha et al., 2008).
Until recently, previous work has reported the failure to
detect in vitro production of glycyrrhizin in G. glabra L.
(Wu et al., 1974; Hayashi et al., 1988; Henry et al., 1984),
although they were found to contain several triterpenes.
Indeed, although Ko et al. (1989) reported the production
of glycyrrhizin in transformed hairy root cultures of G.
ulalensis, they found no production in transformed roots
of G. glabra. Wongwicha et al. (2008) recently reported
that G. ulalensis calli induced using MS + NAA and BA or
TDZ alone produced glycyrrhizin, as discussed above.
However, Shams-Ardakani et al. (2007) similarly reported
the formation of glycyrrhizin from G. glabra var.
glandulifera calli tissue induced using MS + 2,4-D or NAA
+ 2,4-D + kinetin. This reported glycyrrhizin production in
G. glabra, as opposed to G. ulalensis, is supported by
this study reported here, but furthermore the production
of glycyrrhizin from G. glabra shoot explants attained
both a high level and also was observed changed as the
concentration of plant growth regulator hormone, used to
Sawaengsak et al. 133
Figure 1. G. glabra explants after culture in different media for six weeks. (A) Shoot explants of licorice on 1/2-strength B5
media; (B) Shoot multiplication of explants cultured on MS + 0.5 mgL-1BA; (C & D) rooted plantlets on 1/2-strength B5 salt
media supplemented with 5.0 mgL-1of either (C) IAA or (D) IBA, and (E) three week old in vitro raised plantlets of G. glabra after
transfer to a plastic bag containing SC1, SC2, SC3, SC4 and SC5. Bar = 1 cm.
Figure 2. Effect of different concentrations of the three cytokinins, BA, Kinetin and TDZ, as supplements in MS media) on the average (A) number of shoots produced per explant
and (B) shoot length, of G. glabra shoot explants, after in vitro culture for six weeks. Data are shown as the mean + 1 SE, and are derived from 20 replicates. Means with different
lower case letters differ significantly (p< 0.05; Scheffe’s multiple range test).
134 Int. Res. J. Plant Sci.
Figure 3. Effect of different concentrations of the two auxins, IAA and IBA, as supplements in half-strength B5 salt media on the average (A) ratio (%) of explants that rooted and
(B) root length, of G. glabra shoot explants after in vitro culture for six weeks. Data are shown as the mean + 1 SE, and are derived from 20 replicates. Means with different lower
case letters differ significantly (p< 0.05; Scheffe’s multiple range test).
Figure 4. Time course of glycyrrhizin accumulation in G. glabra shoot explant cultures grown in vitro in 1/2-strength B5 salt media
supplemented or not with either IAA or IBA at 5 mgL-1. Data are shown as the mean + 1 SE, and are derived from 20 replicates. Means
with different lower case letters differ significantl y (p< 0.05; Scheffe’s multiple range test).
Sawaengsak et al. 135
give a chemical stress to the explants at different growth
stages, also changed. Indeed, the variation in the
glycyrrhizin content was clearly attributed to the type and
concentration of the plant growth regulator (auxin or
cytokinin or the auxin/cytokinin ratio) used, which
dramatically altered both the growth of explants and their
glycyrrhizin formation in plant tissue culture. This is
potential dependence upon the auxin/cytokinin ratio
reported here in accord with the broad trend also
observed for tissue culture of G. ulalensis explants
(Wongwicha et al. 2008). In addition, the time course for
glycyrrhizin accumulation in the cultured explants varied
significantly, presumably reflecting large differences in
glycyrrhizin formation during tissue development and
differentiation.
CONCLUSIONS
A simple minimal medium of MS or B5 suitable for the in
vitro tissue culture based growth and development of G.
glabra shoot explants was developed. A high frequency
of multiple shoots was accomplished on MS media
supplemented with 0.5 mgL-1 BA. In addition, when
supplemented with 5 mgL-1 IAA, the media was effective
not only for root induction but also for glycyrrhizin
formation, attaining the highest glycyrrhizin content after
eight weeks. Using this procedure has the potential then
to not regenerate plants on a large scale in a short time,
but also to produce relatively high glycyrrhizin contents at
the same time. This protocol will thus serve as a basis to
enable future research into developing G. glabra
transformed root cultures for the mass production of
glycyrrhizin.
ACKNOWLEDGMENTS
The authors thank the Chulalongkorn University
Graduate School thesis grant, the National Research
University Project of CHE, the Ratchadaphiseksomphot
Endowment Fund (AG001B, AM1019A, and AS613A),
and the Thai Government Stimulus Package 2
(TKK2555), for financial support of this research, as well
as the Institute of Biotechnology and Genetic Engineering
for support and facilities. We also, thank Dr. Robert
Butcher (Publication Counseling Unit, Chulalongkorn
University) for his constructive comments in preparing
this manuscript.
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