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European Journal of Applied Sciences – Vol. 11, No. 5
Publication Date: October 25, 2023
DOI:10.14738/aivp.115.15522
Ab Rahman, Z., Ahmad, N. A. S., Basirun, N. N. A., & Othman, A. N. (2023). Regeneration of kaempferia parviflora from Meristem
Through Somatic Embryogenesis. European Journal of Applied Sciences, Vol - 11(5). 84-93.
Services for Science and Education – United Kingdom
Regeneration of kaempferia parviflora from Meristem Through
Somatic Embryogenesis
Zuraida Ab Rahman
Biotechnology & Nanotechnology Research Centre,
MARDI Headquarters, Persiaran MARDI-UPM, 43400 Serdang, Selangor, Malaysia
Nur Auni Syazalien Ahmad
Universiti Malaysia Pahang, Gambang Campus,
Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia
Nur Najwa Arifah Basirun
Biotechnology & Nanotechnology Research Centre,
MARDI Headquarters, Persiaran MARDI-UPM, 43400 Serdang, Selangor, Malaysia
Ayu Nazreena Othman
Biotechnology & Nanotechnology Research Centre,
MARDI Headquarters, Persiaran MARDI-UPM, 43400 Serdang, Selangor, Malaysia
ABSTRACT
The most effective combination of growth regulators for inducing callus formation
in the meristem tissue of Kaempferia parviflora was found to be 0.5 mg/L of 6-
Benzylaminopurine (BAP) combined with 2.0-5.0 mg/L of Naphthaleneacetic acid
(NAA). This combination resulted in the formation of calluses weighing between 5.7
g and 5.8 g. These calluses were predominantly friable and had a yellowish colour.
To promote the proliferation of calluses, two different carbon sources, namely
sucrose and maltose, were tested. The best results were obtained when using
sucrose at a concentration of 30 g/L, supplemented with 0.5 mg/L of Thidiazuron
(TDZ) and a range of 1.0-3.0 mg/L of BAP. Under these conditions, the callus weight
reached the highest values recorded, ranging from 10.37 g to 11.50 g. Additionally,
this medium exhibited the highest efficiency in developing green somatic embryos
(2.1-5.5%), with no browning observed, unlike with maltose. For regeneration, the
optimal medium consisted of a combination of 5.0 mg/L of NAA, 0.5 mg/L of TDZ,
and 1.0 mg/L of BAP. This medium resulted in the highest production of somatic
embryos (21.5 g) and the highest number of plantlets (7.8). Overall, the study
demonstrated the successful manipulation of media composition to induce callus
formation, promote callus proliferation, and achieve efficient somatic embryo
production and plantlet regeneration in Kaempferia parviflora.
Keywords: Regeneration, Kaempferia parviflora, callus, somatic embryogenesis
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Ab Rahman, Z., Ahmad, N. A. S., Basirun, N. N. A., & Othman, A. N. (2023). Regeneration of kaempferia parviflora from Meristem Through Somatic
Embryogenesis. European Journal of Applied Sciences, Vol - 11(5). 84-93.
URL: http://dx.doi.org/10.14738/aivp.115.15522
INTRODUCTION
Kaempferia parviflora is a medicinal plant of the Zingiberaceae family. This medicinal plant is
also known as "Krachaidum" by Thai locals, while in Malaysia it is called "Kunyit hitam". It can
be found in tropical regions like Thailand, Malaysia, Sumatra, and Borneo Island (Zuraida et al.,
2014). For many generations, the fresh rhizomes have had a distinctive scent and a mildly
pungent flavour, and they are frequently used as a food supplement and a folk remedy in
Southeast Asia to treat a variety of illnesses, including leukorrhea, dysentery, aphthous ulcers,
dry mouth, and stomach discomfort(Tewtrakul et al., 2009). A meristem is a form of planttissue
that is made up of meristematic cells, which are undifferentiated cells capable of cell division.
Cells in the meristem might develop into any of the several tissues and organs that are present
in plants. These cells continue to divide until they are differentiated, at which point they cease
to divide altogether ("Meristem," 2022). Thus, this study is focused on the regeneration of
Kaempferia parviflora from the meristem through somatic embryogenesis.
In certain species, somatic embryogenesis has proven successful in creating millions of seeds.
Somatic embryogenesis is less effective in producing the shape of somatic seeds. Furthermore,
because in vitro single cell produced plants are simpler to handle than somatic embryo derived
plants, somatic embryogenesis is preferable for plant genetic improvement through culture and
genetic transformation as well. Plants created through somatic embryogenesis and meristems
are true to type and genetically identical to their mother plants (Rostiana & Syahid, 2008).
Furthermore, exogenous hormones are frequently regarded as the most crucial component in
the establishment of in vitro regeneration protocols (Azad et al., 2004; Suboti et al., 2009).
Auxin and cytokinin are essential for controlling organ regeneration, and their hormone
concentration ratios are significant in identifying particular organogenesis processes in various
somatic tissues, including leaves and roots (Christianson & Warnick, 1985; Su et al., 2011).
Therefore, the objective of this study is to generate a Kaempferia parviflora regeneration
system from meristem tissue through somatic embryogenesis.
MATERIALS AND METHODS
Plant Material and Callus Induction
Kaempferia parviflora was cultivated inside a glasshouse at Malaysian Agriculture Research
and Development (MARDI), Serdang, for initial culture establishment. Germinated
meristematic buds of Kaempferia parviflora were collected and cleaned under running tap
water for a duration of one hour. Further cleaning of the explants was done with commercial
laboratory detergent, which is Decon 5% (v/v), and the explants were rinsed meticulously. The
steps were followed by the immersion of the explants in 1% (v/v) fungicide for one hour, and
afterward, the immersed explants were washed out under running tap water for five minutes.
Subsequently, the explants underwent surface sterilization, where 10–20% of Clorox® with a
few drops of Tween 20 was inserted inside the flask containing the explants under sterile
conditions. The explants were then rinsed with sterile distilled water several times to ensure
thorough cleaning. Meristem parts were obtained by the removal of the outer layer of leaf
sheaths from the explant with a sterile surgical blade under aseptic conditions. The sterile
meristem was then inoculated onto Murashige and Skoog's (1962) basal medium containing
3% sucrose and 3.0 mg/L of BAP. The obtained plantlets (Fig-1a) were used for explants, which
were in vitro meristem isolated (Fig-1b) and cultured on MS basal medium containing various
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European Journal of Applied Sciences (EJAS) Vol. 11, Issue 5, October-2023
concentrations of BAP (0.0, 0.5, 2.0, and 5.0 mg/L) and NAA (0.0, 0.5, 2.0, and 5.0 mg/L) for
callus initiation. The pH of the medium was adjusted to 5.8 before autoclaving (15 minutes,
121 ̊C). The cultured flasks were wrapped with parafilm to prevent contamination and
incubated in a culture room under white fluorescent light with a light intensity of 3000 lux and
a photoperiodic period of 16 hours at 25 ± 2 ̊C. The growth of the callus was observed and
subcultured at monthly intervals, and the weight of the callus was recorded after 3 months of
culture. The obtained calluses were kept subcultured on the same medium, and their fresh
weight and morphology were recorded.
b d
h i j
k l m n
Figure 1: Regeneration of In vitro plantlets of Kaempferia palviflora via somatic
embryogenesis. In vitro plantlets (a), isolated meristem (b), Friable and yellowish
callus cultured on medium contaning 0.5mg/L BAP+ 2.0-5.0 mg/L NAA (c,d), somatic
embryos (e,f,g), histological observation of somatic embryos, meristematic cell and in
early stage of globular pro-embryo (h), early phase regeneration of somatic with
shoot apical meristema (i), regeneration of plantlets in regeneration medium (j,k),
rooted plantlets ready to transfer into glasshouse (l,m), 6 weeks old plantlets kept at
the glasshouse under 75% shading (n).
e f g
a c