BIOTROPIA VOL. 15 NO. 1, 2008BIOTROPIA VOL. 15 NO. 1, 2008 : 12 - 24

SOMATIC EMBRYOGENESIS FROM MERISTEM
EXPLANTS OF GINGER

OTIH ROSTIANA* AND SITTI FATIMAH SYAHID

*Division of Plant Breeding, Indonesian Medicinal and Aromatic Crops Research Institute
Jalan Tentara Pelajar No. 3 Bogor 16111, Indonesia

ABSTRACT

The use of planting materials from in vitro culture, especially derived from somatic em-
bryos has some advantages such as genetically stable and pathogen-free. Meristem culture of
ginger through somatic embryogenesis could be a potential method for producing pathogen-free
planting materials. Somatic embryogenesis on ginger was performed to obtain vigorous plantlets
having the same rhizome size as the mother plant. Callus was induced from meristem tissue of
inner bud of Indonesian ginger rhizome Var. Cimanggu-1 and consecutively subcultured into
certain media at each steps of experiments. The vigorous embryogenic calli were observed on MS
medium containing 100 mgl-1 glutamine and 2% sucrose with addition of 1.0 mgl-1 2,4-D + 3.0
mgl-1 BA. The highest number of somatic embryos (about 82.0.g-1 friable calli) was achieved on
that medium, 4 weeks after culturing. Furthermore, the optimum growth of embryogenic calli
containing somatic embryo was obtained on MS medium enriched with 6% sucrose. The high-
est number of mature somatic embryos (57.2 embryos) was achieved on MS medium, 18 days
after incubation. The regeneration potency of somatic embryos obtained from ginger meristem
was 51.20%.g-1 friable callus. The valuable result of this study was the achievement of normal
rhizome size of regenerated plantlets, instead of micro rhizome.

Key words: Zingiber officinale Rosc., meristem culture, somatic embryogenesis.

INTRODUCTION

Ginger (Zingiber officinale Rosc.) is one of the most important export commodities
of Indonesia. This crop is also one of the important components of Indonesian herbal
medicine, locally called jamu, phytopharmaca and is contributing to the Indonesian
foreign exchange and work opportunity for labors. The lack of market stock and serious
damages due to pest and disease, have caused high fluctuations to Indonesian ginger
exports and prices within the last decades. To overcome the limiting factors, the use of
healthy planting material is necessary to be developed.
Plant tissue culture has been adopted for the purpose of in vitro mass propagation
on various crop species. In case of the Big-White Ginger Cultivar of Indonesian Variety
Cimanggu-1, the use of shoot bud-derived plantlets, either by direct organogenesis or
through callusing, has been developed to obtain disease-free planting materials (Mariska

* Corresponding author : otihrostiana@yahoo.com

Somatic embryo development of ginger meristem – O. Rostiana & S.F. Syahid

and Syahid 1994). In further field experiments, the healthy plantlets and vigorous plants
were not able to produce normal rhizomes (the same-big rhizome as their mother plants),
eventhough they were repeatedly planted in three consecutive generations (Syahid and
Hobir 1996). It is suggested that genetic alteration or epigenetic change during the in
vitro culture and regeneration have been performed. In order to eliminate the genetic
change during the in vitro culture, other regeneration pathway should be considered.
Somatic embryogenesis has been accomplished in some species for producing
millions of seeds. The form of somatic seeds through somatic embryogenesis, is more
efficient than organogenesis. Besides, somatic embryogenesis is more preferable in plant
genetic improvement through in vitro culture and genetic transformation as well, because
single cell derived-plant is eassier to be controlled as somatic embryo derived-plant.
In general, somatic embryogenesis and meristem derived plants are true-to-type and
genetically identical to the mother plants (Evans and Sharp 1986; Jimenez 2001),
however, certain differences might appear according to the plant species characteristics.
The true-to-type of somatic embryo derived plantlet has been found in in vitro culture of
Picea abies (Heinze and Schmidt 1995). On the other hand, among recalcitrant species
such as serealia crops and conifers, good results have been performed by the induction
of somatic embryogenesis.
Achievements in inducing somatic embryogenesis in monocots are mostly
derived from generative explants. Meanwhile, in dicotyl vegetative explant, such as
leaves, are commonly applied for inducing somatic embryo derived-plantlet. Leaf
explant of orchard grass completely induces somatic embryo through the formation of
embryogenic calli with addition of a strong auxin like dicamba (Bhojwani and Razdan
1996). On the other hand, Kackar et al. (1993) performed the somatic embryo culture
of Indian ginger Var. Eruttupetta by using an aseptic leaf-explant with the addition of
2,4-D and dicamba. The same explant source was also performed in inducing somatic
embryogenesis of fingerroot (Boesenbergia rotunda L.) and galangal (Kaempferia galanga
L.) (Tan et al. 2005; Rahman et al. 2004).
This study was aimed at obtaining the normal-size of rhizome (the same size
as their mother plant) through induction of somatic embryos from meristem explant
of Indonesian Var. Cimanggu-1. Most meristematic cells of meristem tissue derived-
somatic embryo are genetically stable and not easy to be mutated (Bach and Pawlowska
2003). Therefore, to eliminate somaclonal variation and other genetic alteration during
the in vitro culture, meristem tissue will be applied for the induction of somatic
embryogenesis on ginger.

BIOTROPIA VOL. 15 NO. 1, 2008

MATERIALS AND METHODS

Explant preparation
The inner shoot bud (meristem) of Indonesian Var. Cimanggu-1, was excised
and simultaneously sterilised by using sterilizing agents such as 70% EtOH, 5% sodium
hypochlorite and 0.2% mercury chloride, for about 5-10 minutes, followed by rinsing
with sterile-aquadest.

Callus induction
Sterilized meristems were placed on MS basal medium (Murashige and Skoog
1962) consisting of 8% agar, 2% sucrose, 100 mg l-1 L-glutamine, 1.0 to 3.0 mg l-1
2,4-dichlorophenoxy acetic acid (2,4-D) and 0 to 5.0 mg l-1 N6-benzyl adenin (BA).
A single factor experiments were arranged in completely randomized design, replicated
three times.

Subculturing.
Embryogenic calli were formed 8 weeks after culturing on callus induction
medium, and then transferred on to hormone-free MS or N6 basal media with the
addition of 3% mannitol for calli proliferation. A single factor experiments were arranged
in completely randomized design, replicated four times for each treatments.
Pro-embryos were then subcultured into the best basal medium, either MS or N6
basal media, according to the best result of the previous stage of experiment, with the
addition of 6% sucrose (6S) for obtaining mature embryo. Single-factor experiments
were arranged in completely randomized design, replicated four times.

Regeneration
Mature embryos (torpedo-like structure-embryos) were subcultured into
regeneration medium for further development. MS basal medium with addition of 3%
sucrose and 200 mgl-1 L-proline were applied in combination with PGRs such as BA at
the concentration of 0, 0.1, 0.5 and 1.0 mgl-1, GA3 at the concentration of 0, 0.1, 0.3,
0.5 and 1.0 mgl-1. Single-factor experiments were arranged in completely randomized
design, replicate three times.

Histology
The callus at each stage of development was subjected to histological analysis
according to Sass (1951) by using paraffin-embedded callus dissection following
Formaldehyde-glacial Acetic acid-Alcohol (FAA) fixation series.

Statistical analysis
All collected data were analyzed by using ANOVA (P0.05) followed by Duncan’s
Multiple Range Test (P. 0.05) (Windows Computers, 2000). Observation results on
embryo proliferation were analyzed by using t-test according to Furlong et al. (2000).

Somatic embryo development of ginger meristem – O. Rostiana & S.F. Syahid

RESULTS AND DISCUSSION

Callus induction
Callus initiation was on tract when the basal-edge of the meristems enlarged,
followed by the change of shoot-dome colour from white to yellowish, at two weeks after
culturing. Eight weeks after culturing, friable callus (embryogenic callus) formation of
about 93.33%/explant was formed on MS basal medium enriched with 1 mg l-1 2,4-D
in combination with 3 mg l-1 BA. Though, that combination was statistically the same
as the treatment without BA (Table 1).

Table 1. Percentage of embryogenic calli from ginger meristem cultured on MS medium enriched with
various concentration of 2,4-D and BA, 8 weeks after culturing

Treatment
Percentage of embryogenic calli (%)

2,4-D (mg.L-1) BA (mg.L-1)
1 0 86.67 a*)

1.0 8.33 e
3.0 93.33 a
5.0 70.00 b

2 0 46.67 c
1.0 25.00 d
3.0 63.33 b
5.0 61.67 b

3 0 28.33 d
1.0 36.67 cd
3.0 63.33 b
5.0 61.67 b

Note:
Numbers followed by the same letters are not significantly different according to DMRT (5%).

The application of 2,4-D on ginger meristem culture showed that the higher
the concentration applied, the more compact calli were observed. Furthermore, when
2,4-D was applied at 2-3 mg.l-1 browning and necrotic calli were observed. An addition
of 1.0 mg.l-1 BA into medium containing 2,4-D neither vigorous embryogenic calli
were obtained, low quantity and compact calli were observed (Table 1).

Embryogenesis from the subcultured callus
For obtaining globular somatic embryos structure the formed embryogenic
calli were cultured on either MS or N6

basal media with the addition of 3% mannitol.
Transparent globular embryos were developed on both media in one week after
subculturing. The shape of ginger embryos at globular phase was generally round or
oval (Fig. 1a).

BIOTROPIA VOL. 15 NO. 1, 2008

f g

cm cm

Figure 1. The development of ginger meristem into newly regenerated plant with normal rhizome size
through somatic embryogenesis.

a. The structure of globular somatic embryo of ginger, 4 weeks after subculturing into prolif-
eration medium (30 times enlargement).

b. Globular embryo, 2 weeks after proliferation (40 times enlargement). Protoderm layer
started to differentiate (arrowed).

c. The structure of torpedo somatic embryo of ginger, 18 days after subculturing into matura-
tion medium (10 times enlargement).

d. Torpedo embryo, 18 days after subculturing into maturation medium (40 times enlarge-
ment). Arrowed: differentiated procambium.

e. Embryo somatic-derived seedlings on MS medium supplemented with 1 mgl-1 BA (Left)
and embryo somatic-forming adventitious roots on hormone free-MS medium (Right),
30 days after subcultured (1 : 1.4 scaled).

f. Embryo somatic-derived normal plantlet, 8 weeks after subcultured on to hormone-free
MS medium (1: 1.3 scaled).

g. Normal rhizome size of ginger-regenerated plants obtained from meristem culture through
somatic embryogenesis.

Somatic embryo development of ginger meristem – O. Rostiana & S.F. Syahid

Numbers of globular phase of somatic embryos increased due to the age
of cultures. The highest number of somatic embryo was obtained at 4 weeks after
subculturing, 82.00 ± 12.25 embryo.g-1 embryogenic callus on MS medium and 70.00
± 19.26 embryo.g-1 embryogenic callus on N6 medium. Statistical analysis showed that
MS basal medium was more capable in inducing higher numbers of somatic embryo
than that of N6 basal medium (P0.05). The number of somatic embryo decreased by the
increase of age of cultures (Table 2). Histological analysis conformed that the somatic
embryo was induced from cortex tissue and that the globular phase developed at 2
weeks after culturing on proliferation medium (Fig. 1b).

Table 2. Number of somatic embryos derived from ginger meristem culture on MS and N6 basal media
enriched with 3% mannitol

Period (weeks)
Average number of somatic embryos/g of embryogenic callus

MS N6
1 32.75 ± 11.97 39.75 ± 5.31
2 54.50 ± 20.16 51.50 ± 9.66

3 70.50 ± 31.97 48.75 ± 15.16

4 82.00 ± 12.251) 70.00 ± 19.261)

5 53.75 ± 16.32 36.25 ± 9.28

6 51.50 ± 10.92 22.75 ± 15.28

7 43.50 ± 19.55 11.00 ± 7.65

Note: Based on t-test at 5% level.

Mature embryo formation was performed on MS basal medium, enriched with
6% sucrose. Cylindrical form of mature embryo started to develop into 2 different
domes, i.e. cotyledons and apical shoot-forming dome as well as root-forming dome.
Two days after subculturing into MS medium enriched with 6% sucrose, about 10.60
± 4.76 torpedo-shape somatic embryos were initiated from 1 g of embryogenic calli
(Table 3).

BIOTROPIA VOL. 15 NO. 1, 2008

Table 3. Number of mature somatic embryos on MS basal medium supplemented with 6% sucrose

Age(day) Average number of mature embryo/g of embriogenic calli1)

2 10.60 ± 4.76
6 33.40 ± 6.74
10 55.80 ± 12.73
14 38.60 ± 6.53
18 57.20 ± 15.99
22 45.80 ± 10.50
26 28.00 ± 8.65
30 16.20 ± 8.91

The highest number of torpedo-shape somatic embryo (57.20 ± 15.99 embryos)
was obtained at 18 days after subculturing. Mature somatic embryo was capable to
form two different domes, i.e. shoot and root (Fig. 1c-d). However, in some cases apical
meristem structure was more dominant than shoot meristem.
Histological section showed that during the formation of torpedo-shape somatic
embryo, root-apical, procambium and scutellum differentiations were performed (Fig.
1c-d).

Regeneration
Germination of somatic embryo was characterized by the formation of the
triangle-shape at meristem apical dome with yellow greenish in color, at 6 days after
subcultured. At the second week of culturing, germinating seeds started to appear and
developed to become normal seedlings/plantlets, especially when BA was added into
MS basal medium.
Three weeks after culturing, various shapes of somatic embryos either a single
normal or fused embryos, had been differentiated into plantlets. Meanwhile, the
abnormal embryos underwent necrosis and then aborted. Embryo germination was
indicated by the presence of shoot and root buds. Four weeks after culturing, the
morphogenesis of somatic embryos were clearly visible, nevertheless the low ability
of somatic embryos to germinate was still a limiting factor in meristem culture of
ginger.
MS basal medium supplemented with 1 mgl-1 BA was found to be the best
culture medium for obtaining normal plantlets at the age of 30 days, to which the
highest number of seedlings was obtained (22.1 seedlings). On the other hand, instead
of growing plantlet, vigorous adventitious roots were obtained on hormone-free MS
medium (Fig. 1e). Furthermore, it was found that both BA and GA3 significantly affected
the somatic embryo regeneration (P 0.05). However, the higher the concentration of GA3,
the lower the number of seedlings was obtained (Table 4).

Somatic embryo development of ginger meristem – O. Rostiana & S.F. Syahid

Table 4. Number of seedlings derived from somatic embryo of ginger meristem cultured on MS medium
supplemented with BA and GA3

Treatment
Average number of seedlings

BA (mgl-1) GA3 (mgl
-1)

0 0 7.3 cd*

0.1 16.4 ab
0.3 16.4 ab
0.5 9.3 bcd
1.0 9.3 bcd

0.1 0 7.3 cd
0.1 10.0 bcd
0.3 3.7 de
0.5 16.1 ab
1.0 11.5 bc

0.5 0 0.5 f
0.1 0.5 f
0.3 0.5 f
0.5 12.8 abc
1.0 17.4 ab

1 0 22.1 a
0.1 12.6 abc
0.3 12.5 abc
0.5 0.5 f
1.0 1.2 ef

Notes : Numbers followed by the same letter are not significantly different according to DMRT (5%).
For statistical analysis data were transformed into √(y + 0.5).

Based on this study, it was found that regeneration potency of meristem culture
of ginger through somatic embryogenesis was 51.20%.g-1 of embryogenic calli when
cultured on MS medium supplemented with 1 mg l-1 BA. The addition of GA3 into MS
basal medium produced only 34.13% of regeneration potency, and 1.13 to 31.84% of
regeneration potency when BA was combined with GA3. Therefore, to gain an optimal
germination of ginger somatic embryo, higher concentration of BA (> 1 mg.l-1) without
addition of GA3, was necessary.
Germinating somatic embryos from MS medium supplemented with either 1
mg l-1 BA, 0.5 mg l-1 BA, 0.1 mg l-1 BA + 0.3 mg l-1 GA3 or 0.1 mg l

-1 GA3 were
then transferred onto hormone-free MS medium with the addition of 3% sucrose.
Among them, the most vigorous plantlets were obtained from MS + 0.1 mg l-1 BA + 0.3
mg l-1 GA3 culture medium-derived seedlings (Fig. 1f ), at 8 weeks after subculturing.
However, the number of embryo somatic-generated plantlets remained low, of which
only about 2-6 plantlets were observed.

BIOTROPIA VOL. 15 NO. 1, 2008

Abundant embryogenic calli (93.33%/explant) from meristem culture of ginger
were achieved on MS basal medium supplemented with 1 mg l-12,4-D in combination
with 3 mg l-1 BA, and 100 mg l-1 L-glutamine, at 8 weeks after incubation. This result
showed that 2,4-D was necessary for initiation of embryogenic callus of ginger, with the
addition of BA at proper concentration. The same result was also found in embryogenic
callus initiation of Kaempferia galanga L. (Vincent et al. 1992).
Callus proliferations were obtained on hormone-free MS medium consisting of
3% mannitol. A globular-shape pro-embryo started to develop at the first week after
subculturing into proliferation medium. Quantities of globular embryo increased due
to the age of culturing up to seven weeks, then gradually decreased.
Ammonium-rich basal media such as MS, which consists of high concentration
of NH4NO3 and myo-inositol, supported well the differentiation of ginger somatic
embryo, though an adverse effect was observed on other Grammineae species (Talwar
and Rashid 1989; Adkins et al. 2002). Salt nutrients in MS medium with addition of
3 to 12% sucrose are usually sufficient to support embryo differentiation (Raghavan
2003; Anbazhagan and Ganapathi 1999). An addition of 3% mannitol into MS basal
medium, assumed that it acted as an osmotic regulator which in turn affected the rate
of cell differentiation and stimulated embryogenic cells morphogenesis in meristem
culture of ginger. Torres et al. (2001) reported that the development of synchronized
embryo and the increase of its quantity were observed when mannitol was added into
MS medium. The same results were also found on embryogenic culture of papaya, celery,
alfalfa and maize (Ziv 1999; Bronsema et al. 1997).
Somatic embryo maturation in meristem culture of ginger was indicated by the
change of embryo color and formation of the brown spots. These brown spots suggest the
presence of an active substance i.e. amylum, protein and lipid, secreted by somatic embryo
from the osmotic cell pressure. The increase of secretion of active substances suggests its
correlation with the desiccation process when somatic embryo enters the germination
phase. Bach and Pawlowska (2003) suggested that the presence of amylum, protein
and lipid around the vascular cells was an indicator in somatic embryo development.
Similarly, Oropeza et al. (2001) reported that the development of embryogenic calli was
associated with the number and type of intracellular protein and callus cells. Hence,
somatic embryo maturation from proliferated calli of ginger meristem is interrelated
with the type and number of active substance among the embryogenic cells.
The growth and development of ginger mature somatic embryo into complete
plantlets (seedlings) sometimes go along with the abnormal germinated seeds, such
unexpected growth of hairy roots, hereafter we named as an early germinating seeds.
According to Bronsema et al. (1997), somatic embryo maturation is associated with
the presence of scutellum, a coleoptile and root-bud like structures. Somatic embryo
maturation was also affected by the composition of applied culture medium. The presence
of ammonium ion and nitrate at a high concentration is a prerequisite in ginger somatic
embryo development. High concentration of ammonium ion supported the process of

Somatic embryo development of ginger meristem – O. Rostiana & S.F. Syahid

somatic embryo differentiation into normal plantlets (George 1993). These results were
in agreement with the findings of Krikorian (1995), Adkins et al. (2002) and Ramage
and Williams (2002). However, the needs of ammonium ion for the growth of somatic
embryo and its morphogenesis depend on the explant sources and the initial plant
growth regulators applied.
According to Percy et al. (2000), high osmotic pressure in culture medium would
increase the somatic embryo formation. Sucrose, usually applied as a carbon source, is
also a potent osmotic agent (van Creij et al. 1999). Another potential osmotic agent
is Abscisic Acid (ABA), that at a proper concentration it would enable to control the
medium osmolarity (Raghavan 2003; Mohan and Krishnamurthy 2002). To overcome
the early seeds germination (the formation of unexpected hairy roots) in somatic
embryogenesis of ginger, an experiment on addition of ABA, aside from sucrose, into
culture medium of somatic embryo maturation is now in progress.
Somatic embryo germination was indicated by the simultaneous formation of
shoots and roots. Goh et al. (1999) stated that embryo germinated when the plumullae
started to emerge. In this research, adventitious roots formation was observed at first
stage of regeneration and increased by the increase of age of culture up to the fourth
week.
Addition of plant growth regulators into regeneration medium would accelerate
the germination process and increase the quantity and quality of seedlings. In this
research, a high percentage of somatic embryo germination was observed on the
medium containing 1 mg.l-1 BA, at 30 days after culturing. Regeneration potency of
ginger meristem somatic embryo reached 51.20%.g-1 of embryogenic calli cultured
on MS basal medium enriched with 1 mgl-1 BA, of which the number of seedlings
was 8.98 times higher than that of the control medium. Meanwhile, GA3 alone or in
combination with BA, remained ineffective in supporting somatic embryo germination
of ginger. However, to obtain the optimum growth of plantlet, the presence of BA is a
prerequisite in meristem culture derived somatic embryo of ginger. Similar result was
also found in the development of ginger leaf derived embryogenic callus (Kackar et
al. 1993), though simultaneous development of somatic embryo derived either root or
shoot meristems remained unclear.
The development of somatic embryo into normal plantlet commonly occurs
through four stages i.e. callus induction, callus proliferation, embryo maturation and
germination. In case of somatic embryo derived from ginger meristem culture, its
development into normal plantlet needs a transfer process of germinated seeds from the
regeneration medium into new medium. Such new medium is called growth medium
for plantlet development. In this experiment the medium used for the growth of plantlet
was hormone-free MS medium with the addition of 3% sucrose.
Although the development of protocol in ginger somatic embryogenesis required
more researches, the results of this experiment, somehow, have a definite advantage
in in vitro studies of ginger, especially in generating healthy planting material with
normal rhizome size, a bigger size than the usual micro rhizomes resulted from shoot

BIOTROPIA VOL. 15 NO. 1, 2008

tip-derived in vitro plantlets (Fig. 1 g). Therefore, the encouraging results of this study
gave great possibility for development of ginger resistant variety either through in vitro
selection, somatic hybridization or genetic transformation.

CONCLUSIONS

The addition of 1 mg.l-1 2,4-D and 3 mg.1-1 BA into MS basal medium enriched
with 100 mg.l-1 of glutamine and 2% sucrose, was effective in inducing embryogenic
calli from meristem explants of Indonesian ginger. In further embryo development,
MS medium was found to be more capable to increase mature somatic embryo than
N6 medium.
The regeneration potency of somatic embryos obtained from Indonesian ginger
meristem was 51.20%/g friable callus. Though, further researches are needed to be
conducted, especially to eliminate early germinating seeds. The protocol for generating
somatic embryo derived plantlet from meristem culture of ginger was developed. The
most valuable result of this study was the achievement of normal rhizome size of
regenerated plantlet, instead of micro rhizome.

ACKNOWLEDGEMENT

The authors would like to thank Dr. Ika Mariska for her assistance and
encouragement during the research work and the manuscript preparation and also to
Dr. D. Sitepu for his critical reading of the manuscript, and Ms. R.R. Sitinjak for her
contribution on the research work.

REFERENCES

Adkins, S.W., A.L. Adkins, C.M. Ramage and R.R. Williams. 2002. In vitro ecology: Modification
of headspace and medium conditions can optimize tissue and plant development. In:
Taji A, R. Williams (eds) The importance of plant tissue culture and biotechnology
in plant sciences. University of New England Unit, Australia, pp. 55-77.

Anbazhagan V.R. and A. Ganapathi. 1999. Somatic embryogenesis in cell suspension cultures of
pigeon pea (Cajanus cajan). Plant Cell, Tissue Organ Cult. 56: 179-184.

Bach, A and B. Pawlowska. 2003. Somatic embryogenesis in Gentiana pneumonanthe L. Acta
Bio. Crac. Series Bot. 45 (2): 79-86.

Bhojwani, S.S. and M. Razdan. 1996. Plant tissue culture: Theory and practice. Elsevier Amster-
dam, Oxford, New York, Tokyo.

Somatic embryo development of ginger meristem – O. Rostiana & S.F. Syahid

Bronsema, F.B.F., W.J.E. van Oostveen and A.A.M. van Lammeren. 1997. Comparative analysis
of callus formation and regeneration on cultured immature maize embryos of the
inbred lines A188 and A632. Plant Cell, Tissue Organ Cult. 50: 57-65.

Evans, D.E and W.R. Sharp. 1986. Somaclonal and gametoclonal variation. In: Evans et al. (eds)
Hand book of plant cell culture, Vol. 4, Technique and application. MacMillan Pub.
Co., New York, pp. 97-132.

Furlong, N.E., E.A, Lovelace and K.L. Lovelace. 2000. Research methods and statistics an
integrated approach. Harcourt College, Tokyo.

George, E.F. 1993. Plant propagation by tissue culture. 2nd ed. Exegetics Ltd, England.
Goh, D.K.S., N. Michaux-ferriere, O. Monteuuis and M.C. Bon. 1999. Evidence of somatic

embryogenesis from root tip explants of the rattan Calamus manan. In Vitro Cell.
Dev. Biol. Plant. 35: 424-427.

Heinze, B and J. Schmidt. 1995. Monitoring genetic fidelity vs somaclonal variation in Norway
spruce (Picea abies) somatic embryogenesis by RAPD analysis. Euphytica 85: 341-
345.

Jimenez, V.M. 2001. Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous
hormones. R. Bras. Fisiol. Veg. 13 (2): 196-223.

Kackar, A., S.R. Baht, K.P.S. Chandel and S.K. Malik. 1993. Plant regeneration via somatic embryogenesis
in ginger. Plant Cell. Tissue Organ Cult. 32: 289-292.

Krikorian, A.D. 1995. Hormones in tissue culture and micropropagation. In: P.J. Davies (ed.) Plant hor-
mones: Physiology, biochemistry and molecular biology. Kluwer Academic, Dordrecht, Boston,
London, pp. 774-793.

Mariska, I and S.F. Syahid. 1994. Propagation of ginger through meristem culture. J. Indust. Crops. Res. 7
(1): 1-6.

Murashige, T and F. Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue
cultures. Physiol. Plant. 15: 473-497.

Mohan, M.L and K.V. Krishnamurthy. 2002. Somatic embryogenesis and plant regeneration in pigeon pea.
Biol. Plant. 45 (1): 19-25.

Oropeza, M., A.K. Marcano and E. De Garcia. 2001. Proteins related with embryogenic potential in callus
and cell suspensions of sugarcane (Saccharum sp.). In Vitro Cell Dev. Biol. Plant. 37: 211-216.

Percy, R.E., K. Klimaszewska and D.R. Cyr. 2000. Evaluation of somatic embryogenesis for clonal propaga-
tion of western white pine. Can. J. For. Res. 30: 1867-1876.

Raghavan, V. 2003. One hundred years of zygotic embryo culture investigations. In Vitro Cell. Dev. Biol.
Plant. 89: 437-442.

Rahman, N.N., M.N. Amin, T. Ahamed, M.R. Ali and A. Habib. 2004. Efficient plant regeneration through
somatic embryogenesis from leaf base-derived callus of Kaempferia galanga L. Asian J. Plant
Sci. 3 (6): 675-678.

Ramage, C.M and R.R. Williams. 2002. Mineral nutrition and plant morphogenesis. In Vitro Cell. Dev.
Biol. Plant. 38: 116-124.

Sass, J.E. 1951. Botanical microtechnique. Iowa State University Press, Ames.

BIOTROPIA VOL. 15 NO. 1, 2008

Syahid, S.F and Hobir. 1996. The growth and rhizome yield of ginger derived from in vitro culture. J. Indust.
Crops. Res. 2 (2): 95-100.

Talwar, M and A. Rashid. 1989. Somatic embryo formation from unmerged inflorescences and immature
embryos of a graminaceous crop echinochloa. Ann. Bot. 64: 195-199.

Tan, S.K., R. Pippen, R. Yusof, H. Ibrahim, N. Rahman and N. Khalid. 2005. Simple one-medium formulation
regeneration of fingerroot [Boesenbergia rotunda (L.) Mansf. Kulturfl.] via somatic embryogenesis.
In Vitro Cell Dev. Biol. Plant 41: 757-761.

Torres, A.C., N. Mfëe-Ze and D.J. Cantliffe. 2001. Abscisic acid and osmotic induction of synchronous
somatic embryo development of sweet potato. In Vitro Cell. Dev. Biol. Plant. 37: 262-267.

Van Creij, M.G.M., D.M.F. Kerckhoffs, S.M. De Bruijn, D. Vreugdenhil and J.M. Van Tuyl. 1999. The effect
of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana
L. crosses. Available at <http://www.liliumbreeding.ne/crey-med.htm>[12/06/04].

Vincent, K.A., M. Hariharan and K.M. Mathew. 1992. Embryogenesis and plantlet formation in tissue culture
of Kaempferia galanga L. a medicinal plant. Phytomorphology. 42 (3 & 4): 253-256.

Ziv, M. 1999, Developmental and structural patterns of in vitro plants. In: Soh W, Bhojwani SS (ed.) Morpho-
genesis in plant tissue culture. Kluwer Academic, Dordrecht, Boston, London, pp. 235-253.