Plant Growth Regul (2011) 64:141–145
DOI 10.1007/s10725-010-9548-8
ORIGINAL PAPER
In vitro plant regeneration from organogenic callus
of Curcuma kwangsiensis Lindl. (Zingiberaceae)
Shijun Zhang • Nian Liu • Aiwu Sheng •
Guohua Ma • Guojiang Wu
Received: 18 April 2010 / Accepted: 13 November 2010 / Published online: 24 November 2010
Ó Springer Science+Business Media B.V. 2010
Abstract A novel protocol for callus-mediated shoot
regeneration was established for an important medicinal and
ornamental plant native to South China, Curcuma kwangsi-
ensis, using shoot base sections excised from seedlings in vitro
as explant sources. The frequency of callus formation reached
91% for explants cultured on MS medium containing 1.4 lM
TDZ, 4.4 lM BA and 2.3 lM 2,4-D. 8.2 shoots per callus was
achieved on MS medium supplemented with 1.4 lM TDZ,
17.8 lM BA and 2.7 lM NAA. Single shoots transferred into
MS medium free of plant growth regulator rooted well.
Regenerated plants acclimatized ex vitro at 100%, and grew
vigorously under shaded greenhouse conditions.
Keywords Curcuma kwangsiensis
Rhizome

Plant regeneration
Callus
Abbreviations
BA 6 Benzylaminopurine
NAA Naphtaleneacetic acid
2,4-D 2,4 Dichlorophenoxyacetic acid
TDZ Thidiazuron
PGR Plant growth regulator
S. Zhang
G. Ma
G. Wu (&)
South China Botanical Garden, Chinese Academy of Sciences,
510650 Guangzhou, China
e-mail: wugj@scbg.ac.cn
S. Zhang
N. Liu
A. Sheng
College of Horticulture and Landscape Architecture,
Zhongkai University of Agriculture and Engineering,
510225 Guangzhou, China
S. Zhang
Graduate University of the Chinese Academy of Sciences,
100049 Beijing, China
Introduction
Curcuma kwangsiensis belongs to the monocotyledonous
family Zingiberaceae, genus Curcuma that contains over
60 species (Tyagi et al. 2007). C. kwangsiensis cultivated
exclusively in South China since the ancient times, is used
not only in traditional medicine but also as a subtropical-
tropical ornamental plant. It exhibits anti-inflammatory,
anti-tumor, anti-allergy and other properties through its
main active components, curcuminoids and volatile oil
(Deng et al. 2006; Salvioli et al. 2007). According to the
ornamental value by its green slender foliage and showy
colorful cylindrical inflorescences, C. kwangsiensis is also
commonly used as bedding plant, pot plant and cut flower
with over 3–4 weeks of post-harvest life in China.
C. kwangisiensis is normally propagated vegetatively using
portions of underground rhizomes known as seed rhizomes.
However, slow propagation rate, soil-born disease infec-
tion, deterioration of rhizomes caused by bacteria and
fungi, and insect attacks are the most common problems in
horticultural production. These same problems face other
Curcuma species, such as Curcuma aromatica (Nayak
2000) and Curcuma amada (Prakash et al. 2004). In
addition, rhizome dormancy and infrequent flowering
considerably inhibit annual production of C. kwangsiensis.
For better utilization of this important economical plant in
China, efficient propagation and regeneration systems
through tissue culture could not only increase propagation
rate dramatically for horticultural production but also
provide an in vitro regeneration protocol for transgenic
manipulation to introduce genes conferring bacterial and
fungal resistance into C. kwangsiensis.
Previous studies have reported in vitro culture in some
Curcuma species, such as C. longa (Shirgurkar et al. 2001;
Salvi et al. 2000, 2001, 2002; Prathanturarug et al. 2003,
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2005; Tyagi et al. 2004, 2007), C. aromatica (Nayak 2000),
C. alismatifolia (Mahadtanapuk et al. 2006), and C. zedoaria
(Loc et al. 2005). So far success with callus induction of
Curcuma genus is restricted to C. longa (Salvi et al. 2001),
C. amada (Prakash et al. 2004) and C. aromatica (Mohanty
et al. 2008). However, we cannot search any reports available
for callus induction in C. kwangsiensis. Therefore, the aim of
this investigation is to apply a callus-mediated plantlet
regeneration protocol, which would provide a prerequisite to
the horticultural production and genetic improvement such
as transgenic engineering for C. kwangsiensis.
Materials and methods
Plant materials
The rhizome buds of C. kwangsiensis were collected from
plants growing in South China Botanical Garden for in
vitro shoot cultures.
Shoot culture
The buds (1–2 cm) were excised and sterilized in 70%
alcohol for 10 s and 0.1% mercuric chloride for 10 min,
subsequently washed seven to eight times with sterilized
distilled water, then inoculated on Murashige and Skoog
(1962) medium (MS) supplemented with 4.4 lM BA and
0.5 lM NAA for shoot initiation for 3 weeks. Then leaves
of elongated shoots were excised and remnant shoot bases
in 5 mm long were aseptically transferred into MS media
supplemented with 22.2 lM BA and 0.5 lM NAA for
further multiple shoot proliferation at 30-day interval.
Callus culture
In vitro single shoot base sections were cut into five mm long
explants, then inoculated on MS media supplemented with
different concentrations of 2,4-D (0, 2.3, 4.5 lM), NAA
(0, 21.6, 32.4 lM), TDZ (0, 1.4 lM) and BA (0, 4.4 lM) for
callus induction and further proliferation in darkness. They
were subcultured within an interval of 30 days. The number
of explants forming calli was scored to calculate callus
formation frequency at 60 days of culture.
Shoot regeneration
Calli produced on callus induction medium containing
1.4 lM TDZ, 4.4 lM BA and 2.3 lM 2,4-D (the medium
yielding optimal callus induction) were used to evaluate
medium for shoot formation from callus. Calli were sepa-
rated from shoot base explants after 8 weeks of callus
production and divided into small pieces with a fresh
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Plant Growth Regul (2011) 64:141–145
weight of approximately 150 mg, then transferred to light
culture on different medium combinations containing BA
(17.8 lM), NAA (2.7 lM) or TDZ (1.4 lM). Green shoot
primordia developed on the surface of calli within 2 weeks.
The leaves and shoots elongated subsequently, so the
number of shoots formed per callus was investigated after
4 weeks of culturing. Regenerated shoots were then moved
to MS medium with TDZ (1.4 lM) for shoot multiplication
and finally to MS medium free of plant growth regulators
(PGR) for leaf elongation and induction of roots.
Culture conditions
The MS basal media with different PGR in culture jars
containing 30 g l-1 sucrose and adjusted to pH 5.8 before
they were solidified with 0.55% agar, were autoclaved at
121°C and 104 kPa for 20 min. The light culture jars were
placed in a growth chamber at 27 ± 1°C with 16 h pho-
toperiod providing 80 lmol m-2 s-1 fluorescent light. For
callus induction, the culture jars were kept in the dark. The
well-developed, regenerated plants longer than 3 cm with
2–3 leaves were transplanted into a mixture of Chinese
peat: coconut chaff: perlite in the ratio 1: 1: 1 in a green-
house at 27 ± 2°C with 14 h photoperiod providing
400 lmol m-2 s-1 fluorescent light.
Data collection and statistics
Each treatment was repeated three times and at least 15
explants per replication were used (i.e., a total of 45
explants per treatment). The data were statistically ana-
lyzed by one-way ANOVA and Duncan’s test (P = 0.05)
to separate treatment means. For the statistical analyses the
program SPSS version 11.0 (SPSS, Inc., 2001) was used.
Results and discussion
Callus induction
In order to explore the possibility of callus induction, in
vitro shoot base sections as explants (Fig. 1a) measuring
about 5 mm in length were cultured horizontally on
different media with a wide range of PGR combinations
and concentrations (Table 1). On the media containing
NAA either alone or in combination with BA, only around
30% explant response was obtained with brown friable
callus developing from the cut surface in 2 weeks. It
indicated that NAA alone could trigger callus production,
BA did not show synergy with NAA during callus forma-
tion. However, with increased culture time the calli could
not proliferate continuously and turned gradually into white
roots and no shoot primordia regenerated. Furthermore,
Plant Growth Regul (2011) 64:141–145
Fig. 1 Different stages of callus induction and plant regeneration in
C. kwangsiensis (bar = 5 mm). a The shoot base sections used as
explants. b Callus formation at the cut surface of the explants on the
medium containing 1.4 lM TDZ, 4.4 lM BA and 2.3 lM 2,4-D for
2 weeks. c A mass of callus developed from bud base after 3-week
substitution of 2,4-D for NAA could not trigger callus
formation at all. Even if 2,4-D and BA combinations gave
rise to a few soft calli in 62.3% of the explants, the
induced callus could not proliferate and wilted gradually,
indicating that callus development was further blocked.
Accordingly, neither NAA and 2,4-D (both auxins), nor
BA (cytokinin) would be sufficient when they are used
alone or together for C. kwangsienesis embryogenic callus
survival.
Similarly, TDZ alone or in combination with BA, NAA,
2,4-D respectively, failed to produce callus. Interestingly,
if TDZ was supplemented with BA and 2,4-D explants
responded remarkably. Expansion and swelling of explants
at the cut surface were noted and soft mucilaginous yel-
lowish callus emerged from the cut surface within 2 weeks
(Fig. 1b). 1.4 lM TDZ ? 4.4 lM BA ?2.3 lM 2,4-D led
to the highest response (91%). A mass of callus formed and
multiplied constantly (Fig. 1c). This indicated that TDZ in
combination with BA and 2,4-D could induce a continu-
ously growing, embryogenic callus. An increase in 2,4-D
concentration to 4.5 lM resulting in reduced callus for-
mation to 61%, 2.3 lM 2,4-D concentration would be
suitable.
Shoot regeneration from the callus explant
Calli obtained on MS media with TDZ, BA and 2,4-D
combinations when transferred to light culture on MS
143
inoculation. d Green shoot primordial formed on the callus on MS
containing1.4 lM TDZ, 17.8 lM BA and 2.7 lM NAA 3 weeks
later. e Single shoot after 2-week culture. f Well-developed plantlet
with roots. g Acclimatized plant growing in pot for 2 months
media with BA (17.8 lM) or TDZ (1.4 lM) alone or BA
and TDZ combinations, failed to induce green shoot
primordia (Table 2). Whereas, 68.3% of callus explants
gave rise to green shoot primordia on medium supple-
mented with 17.8 lM BA and 2.7 lM NAA after 2 weeks
light culture, and about 3.3 shoots per explant developed.
However, addition of 1.4 lM TDZ into the former medium
significantly increased the regeneration frequency to about
95.3% and more green shoot primordia formed (Fig. 1d).
The leaves and shoots elongated subsequently. About 8.2
shoots per explant developed 4 weeks later. A prerequisite
for horticultural production of C. kwangsiensis is repro-
duction of plantlets in large quantity. So we transferred the
shoots regenerated from callus (Fig. 1e) to MS medium
supplemented with 1.4 lM TDZ to produce multiple
shoots. TDZ could effectively trigger shoot multiplication,
without the influence of BA and NAA. 13.84 shoots per
explant were obtained on average after 30 days of culture.
Subsequently the shoots were subcultured in MS basal
medium and rooting was spontaneous (Fig. 1f). Regener-
ated plantlets are proved to be easily adaptable for ex vitro
conditions with 100% survival rate in a soilless substrate
(Fig. 1g). And these disease-free plants have well growth.
It indicated that huge amounts of tissue culture raising
plants were good supplements to disease-free planting
material and it could help farmers to solve the problem of
germplasm deterioration by low propagation rate and dis-
ease infection. The high survival rate is same as the results
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Table 1 Effects of different combinations of BA, NAA, 2,4-D and TDZ on callus induction of C. kwangsiensis on MS medium
Plant growth regulators (lM) Callus induction Nature of callus Regeneration results
frequency ± SE (%)
BA NAA 2,4-D TDZ

0 21.6 0 0 30.7 ± 2.3 b Brown, compact, friable Root organogenesis

0 32.4 0 0 28.7 ± 4.3 b Brown, compact, friable Root organogenesis
4.4 32.4 0 0 28.9 ± 3.8 b Brown, compact, friable Root organogenesis
0 0 2.3 0 0a n.t. n.t.
4.4 0 2.3 0 62.3 ± 2.3 c Few, yellowish, meri-friable Calli turned brown and finally died
0 0 0 1.4 0a n.t. n.t.
4.4 0 0 1.4 0a n.t. n.t.
0 32.4 0 1.4 0a n.t. n.t.
0 0 2.3 1.4 0a n.t. n.t.
4.4 0 2.3 1.4 91.0 ± 2.0 d Soft, yellowish, mucilaginous Shoot organogenesis
4.4 0 4.5 1.4 61.0 ± 3.2 c Soft, yellowish, mucilaginous Shoot organogenesis
Data represented average ± SE of three replicates, each with 15 explants. Means having the same letter in a column were not significantly
different by Duncan’s multiple-range test (P = 0.05)
n.t. not tested

from in vitro protocols of other Curcuma species (Salvi
et al. 2002; Tyagi et al. 2004).
In this study, we focused on establishing a callus-
mediated plant regeneration system for C. kwangsiensis.
Till now, there are a number of protocols of direct in vitro
propagation of C. species. For example, Mahatanapuk et al.
(2006) reported shoot regeneration of C. alismatifolia using
retarded shoots method. The researchers found that
0.5 mg L-1 TDZ mixed 4 mg L-1 IMA could generate
30–40 new shoots from one explant by using retarded shoot
method. In their studies, they chose TDZ as the inducer of
direct shoot regeneration and multiplication. TDZ in
combination with IAA could induce direct shoot regener-
ation from cultured immature inflorescence of C. longa
(Salvi et al. 2000). There are other selectable plant-growth-
regulators besides TDZ, such as BA and NAA (Tyagi et al.
2004; Das et al. 2010). However, we had found the infor-
mation of embryogenic callus formation and the informa-
tion of subsequent plantlet regeneration in C. species was
much less. And we were unable to search any reports about
callus induction of C. kwangsiensis.
From our observation, it was obvious that the
supplement of TDZ into the culture media is critical for
embryogenic callus formation and more efficient plantlet
regeneration in C. kwangsiensis. TDZ, a cotton defoliant,
has both auxin- and cytokinin-like activity and can be
substituted for auxins or combinations of auxins and
cytokinins (Shen et al. 2007; Singh et al. 2003).
The promoting effect of TDZ on in vitro development
has been lately reported over a wide range species
(Yucesan et al. 2007; Jones et al. 2007; Roy et al. 2007;
Wang et al. 2007). With respect to in vitro culture of
Curcuma species, it is known that TDZ is mainly used to
induce direct shoot regeneration (Prathanturarug et al.
2003, 2005; Mahadtanapuk et al. 2006). Our results
also exhibited that the application of TDZ alone could
effectively trigger direct shoot multiplication in
C. kwangsiensis.

Table 2 Effects of different combinations of BA, NAA and TDZ on regenerative response of callus of C. kwangsiensis on MS medium
Plant growth regulators (lM) Frequency of shoot Shoots/explant ± SE Shoot length ± SE (cm)
regeneration ± SE (%)
BA NAA TDZ

17.8 0 0 0a 0a 0a
0 0 1.4 0a 0a 0a
17.8 0 1.4 0a 0a 0a
17.8 2.7 0 68.3 ± 2.3 b 3.3 ± 0.2 b 6.3 ± 0.2 b
17.8 2.7 1.4 95.3 ± 2.3 c 8.2 ± 0.2 c 8.8 ± 0.2 c
Data represented average ± SE of three replicates, each with 15 explants. Means with the same letter in a column were not significantly different
by Duncan’s multiple-range test (P = 0.05)

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However, the utilization of TDZ for callus induction in
the Zingiberaceae family had never been employed before.
Several reports mentioned plant regeneration and callus
formation. They used BA mixed with NAA for callus
formation from leaf bases of turmeric (Salvi et al. (2001)),
2,4-D only to induce semi-friable callus from leaf sheath
explants of C. amada (Prakash et al. (2004)), and 2,4-D
only or with Kn for plant regeneration from callus culture
of C. aromatic (Mohanty et al. (2008). 2,4-D also improved
callus formation from shoot tips of ginger, another member
of Zingiberaceae (Guo et al. 2007). So far, this is the first
report of using TDZ combined with auxin (BA) and
cytokinin (2,4-D, NAA) for embryogenic callus formation
and plantlet regeneration of C. kwangsiensis.
In summary, the present work demonstrates the estab-
lishment of a promising in vitro culture system of
C. kwangsiensis, an important plant native to South China.
The protocol can not only help to enhance the conservation
of the Chinese native germplasm but also to create a
chance to use this crop in genetic manipulation for intro-
duction of genes conferring bacterial and fungal resistance
using transgenic engineering. The evaluation of this
regeneration protocol for genetic transformation will be
described in a subsequent study.
Acknowledgements
This work was supported by the Key Technology Program
of Guangdong Province (2008B020400001) and the CAS/
SAFEA International Partnership Program for Creative
Research Teams.
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