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Procedia Chemistry 14 (2015) 122 129

2nd Humboldt Kolleg in conjunction with International Conference on Natural Sciences,

HK-ICONS 2014

Micropropagation of Embryogenic Callus of Oil Palm

(Elaeis guineensis Jacq.) Using Temporary Immersion System
Cynthia Lendria Magdalena Marbuna, Nurita Toruan-Mathiusa, Reflinia,
Condro Utomoa*, Tony Liwanga
a
Plant Production and Biotechnology Division, PT SMART Tbk., Sinar Mas Land Plaza, 2nd Tower, 10th Fl., Jl. M.H. Thamrin No 51,
Jakarta 10350, Indonesia.

Abstract

Tissue culture technique has been used to produce elite oil palm clonal materials in commercial scale. Proliferation of
embryogenic callus in oil palm commonly utilizes solid and liquid medium, but the result are still not optimum. Recently,
researchers have developed an improved technique called Temporary Immersion System (TIS) and this technique has shown
great potential to increase proliferation rate of the embryogenic callus. This research can be divided into two groups i.e. selection
of liquid medium using shaker to increase the proliferation rate and proliferation of embryogenic callus using TIS. The objectives
of this research are to obtain the best medium for increasing embryogenic proliferation rate using shaker and to obtain the best
medium and time interval of immersion to increase the proliferation rate using TIS. Approximately 0.3 g of embryogenic callus
from embryoid-line A1 were weighted and cultured in twenty two types of liquid medium for the first research and two types of
medium with the composition of MSD and MSA for the second research. The proliferation rate based on fresh weight obtained
from week 8 observations. The data was analysed with Anova of F ( = 5 %) and then with DMRT in = 5 %. TIS observation
was done every 1 h, 3 h, 6 h of immersion time with time interval of 3 min. The results showed that the best medium
composition to increase proliferation of embryogenic callus using shaker was MSA9, the best medium composition to increase
proliferation of embryogenic callus using TIS was MSD, and the best time interval of immersion was every 3 h for 3 min.
2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
2015 C.L.M. Marbun, N.T. Mathius, Reflini, C. Utomo,T. Liwang. Published by Elsevier B.V.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014.
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
Keywords: Temporary Immersion System; somatic embryogenesis; in vitro culture; Elaeis guieensis Jaqc.; liquid medium.

* Corresponding author. Tel.: +62 21 392 5720 ; cell phone : +62 881 123 8675; +62 815 1670 140
E-mail address: condro.utomo@sinarmas-agri.com

1876-6196 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
doi:10.1016/j.proche.2015.03.018
 Cynthia Lendria Magdalena Marbun et al. / Procedia Chemistry 14 (2015) 122 129 123

Nomenclature

MSA MS Medium with Amino Acid

MSM Medium modified of Murashige and Skoog
MSK MS medium without Amino Acid
TIS Temporary Immersion System

1. Introduction

Microprogation of oil palm through tissue culture tends to increase the oil production. This can be proven by the
fact that production from tissue culture is 30 % higher than commercial plant Dura Pisifera (DP) in the field1.
Tissue culture is still not efficient because it requires laborious procedures and more time to produce the palm
clones. From all explants, only 15 % will produce callus and only 3 % of callus produce somatic embryo2. The
induction, maturation, and germination stages of somatic embryo were influenced by medium composition, vitamin,
amino acid, and plant growth regulator, such as auxin and cytokinin3.
In general, callus induction and callus embryogenic formation, which followed by germination to produce
planlet, are done in solid medium. Alternative approaches involving the use of liquid culture for oil palm
propagation have been attempted recently with the aim of improving growth and uniformity of callus and somatic
embryos4,,5. Liquid medium is suitable for micropropagation to reduce plantlet production costs and automation6.
Liquid culture system provides more uniform culture conditions along with easier medium replacement, and can
utilize bigger vessel than solid culture. While the results have been promising, in vitro culture in liquid medium still
have some disadvantages, such as vitrification, cell damages by propeller rotary, and lack of oxygen. To prevent
these problems, a new technique called temporary immersion system (TIS), has been introduced. This system uses
periodic contact between plant materials and liquid medium rather than continuous contact. Time interval and
duration of immersion are controlled automatically by a timer. This system provides adequate oxygen transfer and
sufficient mixing. The result showed that nodular callus production of somatic embryo in TIS was higher than those
in solid medium and the strutures of somatic embryos were more uniform5. TIS has been used in commercial
micropropagation of many kinds of plant species. Micropropagation through in vitro culture has been reported in
Hevea brasiliensis Mull. Arg7, Coffea arabica L.8,9,10, Musa parasidiaca L.11, Theobroma cacao L.12, Ananas
comosus (L.) Merr.13, Camptotheca acuminata Decne.14, Phoenix dactylifera L.15, and Elaeis guineensis Jacq.16.
Productivity of somatic embryo from liquid medium can be increased by addition of amino acid. Amino acid is
one of organic nitrogenous sources, which is important for callus induction, adventive shoot regeneration,
embryogenesis, and androgenesis17. Several amino acids have been used intensively for medium enrichment, such as
alanine, arginine, asparagine, glutamine, glycine, and tryptophan18,19.
Vitamins are nitrogenous substances required in trace amounts to serve as catalyst in enzyme systems. Specific
medium components, such as amino acids and vitamins were found to exert a profound effect on tissue culture
systems of certain species and optimization of such compounds can stimulate regeneration in recalcitrant cultivars20.
Plant cells grown in vitro are capable of synthesizing essential vitamins in suboptimal quantities; thus culture
medium is often supplemented with vitamins to enhance growth21.
The objectives of the research were to obtain the best medium to increase the proliferation of embryogenic callus
from oil palm using shaker and to obtain the best medium and time interval of immersion to increase the
proliferation of embryogenic callus using TIS.

2. Material and methods

2.1. Materials of embryogenic callus and culture conditions

Embryogenic callus initiated from young leaves of oil palm was obtained from the Sinar Mas Plantation,
Indonesia. Embryogenic callus from embryoid-line A1 with size range from 0.5 mm to 1.0 mm in globular stage
were cultured in erlenmeyer flask containing liquid MS medium supplemented with 2,4-D and NAA22. The pH of
the culture medium was adjusted to 5.7. The medium was autoclaved at 121 C and 1 atm for 20 min (atm =
124 Cynthia Lendria Magdalena Marbun et al. / Procedia Chemistry 14 (2015) 122 129

standard atmosphere equal 101 325 Pa). All cultures were incubated in dark room at 28 C. Callus was subcultured
three times in the same medium to achieve embryogenic callus before being used as material source for liquid
cultures in TIS. For somatic embryo initiation and maturation, all cultures were incubated in the culture room at
28 C under cool-white fluorescent lamps with light intensity of 1 000 lux (1 lux = 1 lm m2) over a 16 h
photoperiod. Flask of TIS, which consisted of an upper containment for the plant propagules and a lower
containment for the medium, was used. In this research, the volume for liquid culture was 150 mL per flask. When
the lower compartment was pressured with sterile air, liquid medium was pumped into the upper compartment to
immerse the embryogenic callus for specific period and time interval adjusted automatically by a timer (Fig 1).

b. Timer
a. Pump c. RITA Vessel

Fig. 1. The tools of TIS consists of (a). pump, (b). timer, and (c). RITA vessel.

2.2. Selection of liquid medium using shaker for embryogenic callus proliferation

Approximately 0.3 g of embryogenic callus from embryoid-line A1 with size range from 0.5 mm to1.0 mm in
globular stage weighted and cultured in sterile medium. Twenty two medium treatments had been used in this
research (Table 1). Each medium consisted of modified MS (MSM) and 5.0 mg L1 of each amino acid. MSM was
composed of 0.52 % biotin, 3.11 % D-Ca-Panthotenat, 4.68 % ascorbid acid, 3.64 % choline chloride, 49.35 %
L-cystein- HCL, 2.34 % 2.4-D, 10.39 % NAA.

Table 1. The composition of proliferation medium

Treatment Composition Treatment Composition Treatment Composition
MSM without
MSK0 MSA7 MSM + aspartic acid MSA14 MSM + cystein
amino acid

MSA1 MSM + glycine MSA8 MSM + glutamate MSA15 MSM + methionine

MSA2 MSM + valine MSA9 MSM + asparagine MSA16 MSM + proline

MSM +
MSA3 MSM + leucine MSA10 MSM + glutamine MSA17
phenilalanine
MSM +
MSA4 MSA11 MSM + lycine MSA18 MSM + tyrosine
ornithine
MSA5 MSM + serine MSA12 MSM + arginine MSA19 MSM + triptophan
MSM +
MSA6 MSA13 MSM + histidine MSA20 MSM + alanine
threonine

Each treatment had ten replications using 100 mL Erlenmeyer and 21 10 = 210 observation units. The
proliferation rate based on fresh weight was obtained from eight weeks observation. Embryogenic callus from
proliferatin stage were plated on plating medium and the percentage rate of maturation observed from eight weeks
observation. Data were analyzed statistically using Anova of F( = 5 %) and any significant difference using DMRT23.
 Cynthia Lendria Magdalena Marbun et al. / Procedia Chemistry 14 (2015) 122 129 125

2.3. Proliferation of embryogenic callus using Temporary Immersion System

Approximately 0.3 g of embryogenic callus from embrioid-line A1 with size range from 1.0 mm to 2.0 mm in
globular stage weighted and cultured in sterile medium. Two types of medium, MSD and MSA were used. MSD
(modified MS consisted of 0.52 % biotin, 3.11 % D-Ca-Panthotenat, 4.68 % ascorbid acid, 3.64 % choline chloride,
49.35 % L-cystein- HCL, 2.34 % 2.4-D, 10.39 % NAA, and 25.97 % alanine) and MSA (modified MS medium
combined with 25.97 % asparagine). This research used 1 h, 3 h, and 6 h immersion time with time interval of
3 min. Each treatment had 10 replications and had 2 3 10 = 60 observation units. The proliferation rate based on
fresh weight was obtained from 8 wk observation. Embryogenic callus were plated on plating medium and the
percentage rate of maturation observed from 8 wk observation. Data were analysed statistically using Anova of
F( = 5%) and any significant difference using DMRT23.

3. Results and discussion

3.1. Selection of liquid medium using shaker for embryogenic callus proliferation

a
a ab ab ab ab ab ab ab ab ab ab
a a ab ab
a ab ab
bc
c

Figure 2. The effect of medium for the fresh weight of embryogenic callus

Fig. 2. The effect of medium for the fresh weight of embryogenic callus. The same letters are not significantly different
according to DMRT at = 0.05.

There was no significant difference among those treatments. The highest rate of fresh weight obtained from
MSA9 (Fig. 2). The proliferation rate of embryogenic callus reached 3.38 g. This medium produced the highest
mean of fresh weight because medium was added by vitamins (biotin, D-Ca-Panthotenat, ascorbid acid, choline
chloride, L-cystein-HCL) and amino acid of asparagine 5.0 mg L 1.
This result was similar with the result from De Touchet et al.24, which showed the embryogenic callus reached
1.2 g, which meant four times increased; and the result from Soh et al.2 showed the embryogenic callus reached 1.8
g, which meant six times increased.
Amino acid is one of organic nitrogen source and has important role in callus induction, embryogenesis, and
explant androgenesis17. Amino acids such as alanine, arginine, proline, serine, threonine, tyrosin, and vitamins can
increase culture productivity19. According to Stasolla and Yeung25 , exogen amino acid will enter cell and stimulate
protein synthesis accumulated in cell then embryogenic cell division occured. Therefore this process is believed to
increase the cell proliferation.
Asparagine 150 mg L1 promotes the process of somatic embryogenesis and regeneration in wheat (Triticum
aestivum). During embryogenesis (from globular embryo onwards), a few amino acids like Phe, Trp, Lys, Arg, Leu,
Tyr, Asp and Ser, present amino ion higher levels compared to regenerated plantlets26.
126 Cynthia Lendria Magdalena Marbun et al. / Procedia Chemistry 14 (2015) 122 129

Vitamins are nitrogenous substances required in trace amounts to serve catalytic functions in enzyme systems.
Specific medium components, such as amino acids and vitamins have shown to exert a profound effect on tissue
culture systems of certain species and optimization of such compounds can stimulate regeneration in recalcitrant
cultivars20. Plant cells grown in vitro are capable of synthesizing essential vitamins in suboptimal quantities; thus
culture media are often supplemented with vitamins to enhance growth21.
Thiamine (vitamin B) at concentration ranging from 0.1 to 5.0 mg L1 is an important cofactor in carbohydrate
metabolism, and generally considered as an important component in culture medium. Biotin is important in
carboxylation reaction and less common in culture medium, usually biotin was added at (0.01 to 1.0) mg L1 27
concentration to the culture medium. Both biotin and thiamine biosynthesis pathways utilize transfer of sulphur from
cysteine to cofactor precursors28. Culture medium of existing date palm regeneration systems are supplemented with
arbitrarily selected vitamins at variable concentrations, including inositol, calcium pantothenate, pyridoxine,
nicotinic acid, thiamine, biotin, etc.29. A study conducted by Al-Kharyi21 on Pheonix dactylifera L. has shown that
callus growth and embryo formation were best achieved on a medium containing 0.5 mg L1 thiamine combined
with 2.0 mg L1 biotin.

Fig. 3. The Morphology of embryogenic callus after cultured on plating medium

( = 2 mm). The arrows showed the mature somatic embryos.

Embryogenic calluses have small nodular structure and are transparent. Their somatic embryos have bigger
globular structure and also have whitish or yellowish colour. Figure 3 showed that embryogenic calusses from
medium of MSA 3, MSA 4, MSA 5, MSA 6, MSA 7, MSA 8, MSA 9, MSA 10, MSA 15, MSA 16, MSA 17, and
MSA 18 were converted into somatic embryos because they had bigger globular structure and whitish or yellowish
colour.
Comparing the effect of amino acid supplementations on the callus growth of Pheonix dactyifera L., tryptophan
and methionine had inhibitory effect on callus growth while arginine, alanine, asparagine and glutamic acid gave the
highest values of both fresh weight and growth rate 30. However, their control experiment, without any amino acid,
provided higher rate of callus growth. Mohamed31 reported that glutamic acid, methionine, tryptophan,
phenylalanine and arginine inhibited the growth of callus tissue cultures of fenugreek. In contrast, Abou El-Nil32
 Cynthia Lendria Magdalena Marbun et al. / Procedia Chemistry 14 (2015) 122 129 127

reported that, growth of date palm callus tissue was stimulated by the addition of amino acids, specifically
glutamine. Embryogenic callus could be converted into somatic embryo in some treatments (Fig. 3).

3.2. Proliferation of embryogenic callus using Temporary Immersion System

There was significant difference between medium treatment and time interval of immersion. The best medium
composition for increasing the rate of embryogenic proliferation was MSD (Fig. 4a), which contained MS medium
modification consisted of 0.52 % biotin, 3.11 % D-Ca-Panthotenat, 4.68 % ascorbid acid, 3.64 % choline chloride,
49.35 % L-cystein-HCL, 2.34 % 2.4-D, 10.39 % NAA, and 25.97 % alanine. The result showed that the best
immersion time was every 3 h with time interval of 3 min (Fig. 4b). All of the embryogenic calluses did not get
browning (Fig. 5) and all embryogenic calluses in every treatment matured on plating medium observation (Fig. 6).
The fresh weight of embryogenic callus was increased seven times using TIS. This result is different from the
result from reference16, which stated that the immersion time of every 6 h with time interval of 3 min gave the
highest proliferation rate of E. guineensis somatic embryos and increased 2.2 times using TIS. It showed that the
embryogenic callus differentiation to somatic embryos had occured. Somatic embryo had bigger size than
embryogenic callus with globular structure. Most of somatic embryos were yellowish in color, with smooth and
compact structures (Fig 4).

a b
A a B aa
a
bb b bb

Fig. 4. (A) The effect of medium; (B) the effect of time interval of immersion for increasing the fresh weight.
The same letters are not significantly different according to DMRT at = 0.05.

Fig. 5. The morphology of embryogenic callus after cultured in TIS.

128 Cynthia Lendria Magdalena Marbun et al. / Procedia Chemistry 14 (2015) 122 129

Fig. 6. The morphology of embryogenic callus after cultured on plating medium.

The arrow shows embryogenic callus converted to somatic embryo.

Periodic immersion in the medium made the embryogenic callus capable in absorbing nutrients more efficiently.
When the liquid medium is immersing the explants, all the surface of the explants is exposed to the medium, thus
absorption occurred at all parts of the explants. Therefore, the level of growth and development in all parts of
explants were more uniform because the nutrients were absorbed simultaneously in an equal proportion7. TIS could
create different forms of stress, such as temporary hypoxia, which is sensed by the cells as sort of stress triggering
short-term metabolic adaptations. Stress is an important factor related to the acquisition of embryogenic
competence33.
The higher percentage of E. guineensis somatic embryo in this research was achieved from time interval of 6 h
and immersed for 1 min. Somatic embryo cultured in TIS would increase 13 times higher than cultured in solid
medium. In contrast, the higher percentage of somatic embryo from coffee was obtained from time interval of 4 h
and immersed for 1 min and produced 2 250 somatic embryos8.
Time interval of 2 h and immersed for 15 min gave the best micropropagation in pineapple (Ananas comosus
(L.) Merr.). It could increase 2.3 times by using TIS and 1.3 times by using liquid culture34. Eucalyptus globulus
Labill. gave the best result in fresh weight of shoot from the time interval of 12 h and immersed for 2 min 35.

Conclusion

This work resulted several information regarding the best medium composition to increase proliferation of
embryogenic callus in different technique. The best medium composition to increase proliferation of embryogenic
callus using shaker was MSA9, which consisted of MS modification medium added with 0.52 % biotin, 3.11 %
D-Ca-Panthotenat, 4.68 % ascorbid acid, 3.64 % choline chloride, 49.35 % L-cystein- HCL, 2.34 % 2.4-D, 10.39 %
NAA, and 5.0 mg L1 asparagine. The best medium composition to increase proliferation of embryogenic callus
using TIS was MSD, which consisted of MS modification medium added with 0.52 % biotin, 3.11 % D-Ca-
Panthotenat, 4.68 % ascorbid acid, 3.64 % choline chloride, 49.35 % L-cystein- HCL, 2.34 % 2.4-D, 10.39 % NAA,
and 25.97 % alanine. The best time interval of immersion to increase proliferation of embryogenic callus using TIS
was every 3 h for 3 min.

Acknowledgements

Utmost gratitude for the management of PT SMART Tbk., Sinar Mas Agribusiness, for publishing this research
paper. The authors also would like to thank to tissue culture technicians for their assistance.
 Cynthia Lendria Magdalena Marbun et al. / Procedia Chemistry 14 (2015) 122 129 129

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