AsPac J. Mol.

2010
Mol. Biol.
Biol.Biotechnol.
Biotechnol.
Vol. 18 (1), 2010
Vol. 18 (1) : 127-130

Micropropagation of red ginger

127

Micropropagation of red ginger (Zingiber montanum Koenig), a medicinal plant

M.N. Hamirah1*, H.B. Sani2, P.C. Boyce3 and S.L. Sim2
Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak.
2
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota, Samarahan, Sarawak.
3
Malesian Tropicals, Suite 9-04, Tun Jugah Tower, No.18 Jalan Tunku Abdul Rahman, 93100 Kuching, Sarawak.

Proceedings Asia Pacific Conference on Plant Tissue and Agribiotechnology (APaCPA) 17-21 June 2007

Abstract. A protocol has been developed for the in vitro regeneration of red ginger (Zingiber montanum Koenig), a valuable
medicinal plant. Rhizome buds of 1.5 to 2 cm long were surface sterilized with commercial bleach prior to culture on Gamborg
B5 medium incorporated with Tetracycline at 15 ml/L and Plant Preservative Mixture at 2 ml/L. For induction of multiple
shoots, three studies were conducted, (1) effects of different plant growth regulators, (2) effect sof whole and sectioned buds and
(3) effects of culture phase. Thidiazuron (TDZ) at 0.5 mg/L was found to induce the highest shoot multiplication with a mean of
8.1 shoots per explant. Sectioned buds produced a mean of 4.6 shoots from each explant. As far the culture phase, liquid medium
was found to be superior to solid medium. Rooting of propagules was conducted on B5 medium devoid of growth regulators.
Acclimatization was conducted on medium containing a mixture of 1:1:1 soil, sand and peat with about 85% survivability.
Keywords: In vitro regeneration; Whole buds; Sectioned buds; Culture phase.

INTRODUCTION

MATERIALS AND METHODS

Zingiber montanum Koenig syn. Zingiber cassumunar

Roxb. belongs to the family Zingiberaceae. The species is native to India. It is known as bonglai in peninsular Malaysia,
bangle in Java and plai in Thai. There are two forms of Z.
montanum, one with yellow rhizome skin but creamy white
flesh and another with red skin but yellow flesh. In this
study, the red skin type was chosen. It has a pungent odour
and a foul-smelling flower (Boyce, 2006). This plant is highly valued for its medicinal properties. In Malaysia, the rhizome is used for post-natal treatment, swelling, rheumatism.
In Thailand they are applied for joint pain, intestinal disorders and numb feet (Sirirugsa, 1999). They were reported
to have anti-fungal, anti-inflammatory, analgesic and antioxidant activity. This probably is due to the presence of certain secondary metabolites such as zerumbone, curcuminoid
and (E)-1-(3,4-dimethoxyphenyl)but-1-ene (Kishore and
Dwivedi, 1992, Ozaki et al., 1991, Habsah et al., 2000).
There is a need to exploit its medicinal properties, therefore
more planting material is needed. Slow propagation rate and
the risk of disease transmittance through division by sectioning of the rhizomes have hampered propagation by conventional means. Thus in vitro technique is considered the best
alternative that can supply a large number of planting materials for commercial planting and further study to discover
their chemical properties.

Explant sources and sterilization. The stock plants for

this study were collected from Kampung Serambu, Bau
District in the Kuching Division, Sarawak. Rhizome buds
about 1 to 2 cm long were selected as the initial explants.
The fresh buds collected were cleaned of soil dirt and left
under running tap water for one to one and a half hour.
Then the buds were immersed in 75% (w/v) ethanol for one
minute. Without rinsing, they were agitate din 20, 30 or 40
% (w/v) Clorox (5.25 % w/v sodium hypochlorite) added
with 0.1ml/L Tween 20 and four drops of 25% HCl for 20
minutes with constant agitation. After that they rinsed with
sterile distilled water four times. Under aseptic conditions
the bud scales were peeled off and then trimmed to about
0.5 cm long.
Culture medium. The medium used was the Gamborg B5
medium, gelled with 2.8g/L Gelrite and 30% sucrose as carbon source. The pH was adjusted to 5.7 -5.8 with 1N KOH
or 0.1N HCl prior to autoclaving. Tetracycline at 15 mg/L
and 2 ml/L Plant Preservative Mixture (PPM) were added
to the medium to check the contamination. Trimmed buds
were inoculated onto the medium and the cultures were
* Author for correspondence:
M.N. Hamirah, Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak.

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AsPac J. Mol. Biol. Biotechnol. Vol. 18 (1), 2010

illuminated with white fluorescent light (3000 lux), at 16

h photoperiod with temperature of 252C. After 15 days
the aseptic explants were transferred to B5 medium supplemented with BAP at 1 mg/L for 4 weeks to induce more
shoots. The shoots were sub-cultured on B5 medium for two
weeks before they were used in the subsequent experiments.
Induction of multiple shoots. Three experiments were conducted for the induction of multiple shoots. In experiment
1, four types of plant growth regulators were tested , i.e.
6-Benzykaminopurine (BAP), kinetin (Kn), 2-isopentyl adenine riboside (2ip-R) each at 1,2, 3 and 4 mg/L and thidiazuron (TDZ) at 0.1, 0.3, 0.5 and 0.7 mg/L. In experiment
2, the study was conducted to examine if sectioned buds will
have a different response to multiple shoot induction. Two
types of explants were tested, one is the whole bud and the
other is a bud section longitudinally into two halves. These
explants were cultured onto B5 medium supplemented with
BAP at 3 mg/L in solid and liquid phases.

RESULTS AND DISCUSSION

Surface sterilization. The best regime for surface sterilization was rhizome buds was using 20% Clorox for 20
minutes. This resulted 75% of axenic explant. Even though
there were no differences statistically increase in Clorox concentration brought about decrease in the number of axenic
explants. This is due to the high number of damaged explant resulted form the high Clorox concentration, i.e. 40%
which probably is too harsh for the cells. The damaged explant can be detected by the discolouration of the explants
and healthy explants turned reddish after about a week.
Contamination was still a problem but only by bacteria. Incorporation of an antibiotic Tetracycline and biocide PPM
into culture medium helped to check contamination if compared to medium without the supplement of antibiotic and
biocide. This finding was supported by previous research by
Jiminez et al. (2006) in which PPM was able to reduce contamination in bamboo micropropagation. Incorporation of
antibiotics also managed to counter bacterial contamination
in Curcuma longa (Salvi et al., 2002).

A
C
D

Concentration of disinfectant (%)

20
30
40
3 (75)
2.80
2.40
1.00
0.8
0.60
0.20b
0.60ab
1.20a

Table 1. Mean number of axenic (A), contaminated (C) and

damaged explant (D) on B5 media incorporated with Tetracycline at 15 mg/l and PPM at 2 ml/l.
Values are mean from 5 replicates, 4 explants each replicate. Within a row means having a letter in common are

Micropropagation of red ginger

not significantly different at 5% level by Duncans multiple

range test.
Induction of multiple shoot. Effecst of different plant
growth regulators. In the media supplemented with BAP,
the highest shoot multiplication was obtained from medium
added with BAP at 3 mg/L with a mean of 3.5 shoots per
explant. Propagules were able to root on the same medium
after one month in culture. The plantlets produced were
morphologically normal with strong roots and healty leaves.
BAP have successfully induced shoot multiplication in other
Zingiberacea species such as Curcuma zedoaria and Curcuma
longa (Nyguyen et al., 2005, Rahman et al., 2004).
In TDZ treatment, the optimum shoot multiplication,
i.e. a mean of 8.1 shoots per explant was found in medium
incorporated with 0.5mg/L TDZ. However, the morphology is slightly abnormal; the shoots are in paler in shade,
shorter and with a broader base. Single shoot developed into
clumps and when they are separated, they slowly formed
miniature shoots after two months in culture. Similar observation was reported by Tefera and Wannakrairoj (2006)
in which inclusion of TDZ reduced shoot length and resulted miniature shoots. These can be detected especially on
plants cultured on higher TDZ concentrations, i.e. 0.5 and
0.7 mg/L. For the plants cultured on lower TDZ concentration i.e. 0.1 mg/L, less or no clump was formed. Among the
cytokinins, TDZ is able to induce higher number of shoots
even at a much lower concentration. In micropropagation
of carnation, it was found that TDZ induced six times more
the number of shoots compared to BAP at an equal concentration (Genkov and Ivanova, 1995).

TDZ

BAP

2ip-R

Concentration
of PGR
(mg/l)
0.1
0.3
0.5
0.7
1
2
3
4
1
2
3
4

Number of
shoots

Shoot
length

4.43b
4.86b
8.14a
3.71b
1.86b
2.43ab
3.57a
2.86ab
3.14a
1.71b
1.85b
2.14b

1.72
1.47
1.28
1.56
2.63
3.2
2.48
2.41
2.69
2.78
2.91
2.5

Table 2. Effects of TDZ, BAP and 2ip-R on shoot multiplication.

Within a column means having a letter in common are

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Figure 4 (a) Shoot formation on medium with BAP 3 mg/l

after 1 month, (b) After 2 months.
Figure 5. Shoot regeneration after 2 months on Kinetin
supplemented medium (a) Kinetin at 1 mg/l, (b) Kinetin at 3 mg/l.
Figure 6. Shoot regeneration after 2 months on 2ip-R supplemented media (a) 2ip-R at 2 mg/l, (b) 2ip-R at 4
mg/l.
Figure 7. Shoot regeneration on TDZ supplemented media
(a) Formation of new shoot (note the fat bottom). (b)
Formation of shoot clusters after 3 months, (c) Miniture shoot formation after 4 months.
Figure 8. Rooting of plantlets from BAP treatment after 3
months.
Figure 9. Sectioned bud cultured on BAP 3 mg/l supplemented media after 1 month.
Figure 10. (a) and (b) Shoot formation on liquid medium
after 6 weeks, (c) On solid medium after 6 weeks.

Micropropagation of red ginger

129

not significantly different at 5% level by Duncans multiple

range test.
2-ip-R at 1 mg/L produced 3.1 shoots per explant was
the highest among all the concentrations tested. A slightly
broader stem was found on plants treated with 2ip-R. However, kinetin probably has the weakest effect on shoot multiplication where each explant produced only 1-2 shoots.
Effects of whole and sectioned buds. Whole and sectioned-bud explants produced a mean of 4.6 and 5 shoots
respectively. However, by sectioning the bud explant into
half, it will be able to double the production when compared
to using the whole bud. This approach has been reported
in pineapple micropropagation in which the shoot multiplication rate has improved significantly (De Almeida et al.,
2002).
Effects of culture phase. Liquid medium was found to be
superior to solid medium. About 4.8 shoots were produced
from a single explant. In fact, shoots produced from liquid
medium were greater in length and they rooted faster compared to shoots culture on the solid medium.

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Rooting and acclimatization. Rooting was induced on

B5 medium without plant growth regulators for one month.
Plantlets 3 to 4 cm high were taken out from the culture vessels and washed thoroughly with tap water and treated with
fungicide, Benomyl at 2 mg/L before they were transferred
to potting medium containing 1:1:1 mixture of soil, peat
and vermiculite. The plants were covered with plastic bags to
retain moisture. About 85% plantlets survived.

CONCLUSIONS
In vitro technique is a useful approach for propagating plants on large scale. For ginger species, propagation
through conventional technique is time consuming and the
risk of disease transmittance is there.
Shoot multiplication of Zingiber montanum Koenig can
be obtained using TDZ, BAP or 2ip-R. However, in this
study, BAP produced healthier plantlets as compared other
types of cytokinins tested. Sectioning of explants longitudinally and culturing then in liquid medium has helped to
improve shoot multiplication rate of this species.

ACKNOWLEGEMENTS
The authors acknowledge the financial support for this
research by the IGS fund (Grant No.L18403 101 00 Zingiberaceae) from the Ministry of Science, Technology and
Innovation (MOSTI), Malaysia.

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Micropropagation of red ginger

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