close

Вход

Забыли?

вход по аккаунту

?

1.4991220

код для вставкиСкачать
Morphology and protein molecular weight analysis from three variant of oil palm
pollen; Dura, Pisifera, and Tenera
R. Priambodo, A. B. Witarto, A. Salamah, Setiorini, D. Triyono, and A. Bowolaksono
Citation: AIP Conference Proceedings 1862, 030116 (2017);
View online: https://doi.org/10.1063/1.4991220
View Table of Contents: http://aip.scitation.org/toc/apc/1862/1
Published by the American Institute of Physics
Articles you may be interested in
Studies of adaptive traits of Bali cattle in Buleleng district, Bali and Barru district, South Sulawesi
AIP Conference Proceedings 1844, 060003 (2017); 10.1063/1.4983443
Parameterization of form factors of a K p interaction model based on one hadron exchange
AIP Conference Proceedings 1862, 030006 (2017); 10.1063/1.4991110
Application of k-means clustering algorithm in grouping the DNA sequences of hepatitis B virus (HBV)
AIP Conference Proceedings 1862, 030134 (2017); 10.1063/1.4991238
Hypergraph partitioning implementation for parallelizing matrix-vector multiplication using CUDA GPU-based
parallel computing
AIP Conference Proceedings 1862, 030153 (2017); 10.1063/1.4991257
A time series model: First-order integer-valued autoregressive (INAR(1))
AIP Conference Proceedings 1862, 030157 (2017); 10.1063/1.4991261
Electrical properties study of La2FeTiO6 double perovskite material at high temperature
AIP Conference Proceedings 1862, 030028 (2017); 10.1063/1.4991132
Morphology and Protein Molecular Weight Analysis from
Three Variant of Oil Palm Pollen;
Dura, Pisifera, and Tenera
R. Priambodo1, A. B. Witarto1, A. Salamah1, Setiorini1, D. Triyono2,
and A. Bowolaksono1, a)
1
Department of Biology, Faculty of Mathematics and Natural Sciences (FMIPA),
Universitas Indonesia, Depok 16424, Indonesia
2
Department of Physics, Faculty of Mathematics and Natural Sciences (FMIPA),
Universitas Indonesia, Depok 16424, Indonesia
a)
Corresponding author: alaksono@sci.ui.ac.id
Abstract. Oil palm is a plant that widely cultivated in Indonesia, with an area of about 11 million hectares in 2014. There
are three main variants that most cultivated; Dura, Pisifera, and Tenera. Oil palm pollen was spread through the wind. The
very wide area of oil palm plantation and those characteristics of oil palm pollen dispersion makes oil palm pollen may
give negative effect to the people around plantation, such as an allergy. The research on the morphology and protein
characters of the oil palm pollen from three variants has not done yet. This research aims to observe the morphology and
protein character from three variants of oil palm pollen. The study begins with the pollen collection from three variants of
oil palm. Oil palm pollen was observed using the light and scanning electron microscope. Oil palm pollen protein was
extracted and the molecular weight of these proteins was analyzed. The result of this research was the morphology character
from three variants of oil palm pollen have successfully been observed. Those three variant of oil palm have no differences
structures; triangular shaped with round edge, tricolpate with connected colpus aperture, psilate exine ornamentation at the
front side and peripheral side, while at the back side has microreticulate exine ornamentation. Three variants of oil palm
pollen protein show the same characteristics. The molecular weight of the protein was ranged from 10-00 KDa. The
information can be useful for the next research to figure out component of proteins inside the oil palm pollen.
INTRODUCTION
Palm oil is the largest plantation crops in Indonesia. Tree plantations have reached approximately 11 million
hectares in 2014. In a big cities, oil palm plant itself is used as a shade plant road, which is generally placed on some
roads and housing in big cities like Jakarta [1]. Oil palm has pollen that can be spread by the wind and insects, because
the structure of the pollen that is relatively small, light, and have a quite attractive smell. Pollen is the male
gametophyte structure in higher plants, such as the group Angiospermae plants. Pollen can be spread by various
factors, one of them winds. Characters of pollen that spread by the wind, among others, is lightweight [2].
Oil palm pollen has a soft structure, so it easy to fly because of the wind, but there are also lumpy in the other
hand. Grains of pollen often also quite sticky so easily attached to the animal's body, such as insects that come to visit
the flower and pollinate the flowers. Pollen is composed of the outer wall of an external layer (exine) and the inner
layer (intine) adjacent to the cytoplasm. Exine consists of a highly resistant material called sporopollenin, evolved in
part from some of the tapetum and microspores. Intine consists of cellulose material and pectin. The pollen has a
diameter ranging between 20-250 µm. Characteristics of pollen such as size, the number of holes, and sculpture
(carving), can be used for identification of flowering plant taxonomy [3].
International Symposium on Current Progress in Mathematics and Sciences 2016 (ISCPMS 2016)
AIP Conf. Proc. 1862, 030116-1–030116-6; doi: 10.1063/1.4991220
Published by AIP Publishing. 978-0-7354-1536-2/$30.00
030116-1
Research on oil palm pollen has been done by some previous researchers. Kimura et al. [4] and Chew et al. [5] has
done research about oil palm protein pollen as an allergen. Their research focused on the small molecular weight of
protein from oil palm pollen. Rengganis [6] also has done research about allergenic pollen from several trees in Jakarta,
one of them is oil palm. The research results show the pollen morphology and protein molecular weight from oil palm
pollen. Morphology and protein analysis from three variants of oil palm pollen are still not known yet. The information
of that character can help the further researches that related with the protein component in oil palm pollen.
The purposes of the research are to analyze the morphology and protein character from three variants of oil palm pollen.
MATERIALS AND METHODS
Pollen Materials. The object of the research was oil palm (Elaeis guineensis, Jacq.) from three variants; Dura,
Pisifera, and Tenera. Oil palm that used in this research was aged 6-10 years and was taken from Jakarta, Depok (West
Java), and Dharmasraya (West Sumatera). The pollen was taken from the male flower of oil palm. Each oil palm tree
may produce more than one male flower that already flowering.
Pollen Collection. Pollen was taken directly from the male flower of oil palm plants from three variants; Dura,
Pisifera, and Tenera, then treated based on Ching & Ching [7] methods. Crude pollen, that was already taken from the
male flower, was kept in a room with a temperature around 19 °C for 1-2 days. Crude pollen was purified using a
special filter which has diameter 45 µm. Oil palm pollen that has been filtered was collected and was stored in a freezer
with temperature around -20 °C.
Pollen Visualization. Pollen morphology was observed at UI-Olympus Bioimaging Center, using a light
microscope (Olympus-IX71) and was observed at PT. Fajar Mas Murni, using a scanning electron microscope
(Hitachi-SU3500). The pollen observation with a light microscope was used two methods, which is unstained and
stained with safranin. Oil palm pollen was put on object glass and was heated at 95-100 °C until all the water
evaporated. Acetolysis solution was given 3-5 drops to the pollen and was mixed until the contaminant was separated.
Absolute ethanol was given around 4 drops to the pollen. Contaminants were removed and 1 drop of safranin was
added to the pollen. The cover glass was put to cover the region of observation and pollen was observed under the
microscope. The pollen observation with scanning electron microscope was used fresh pollen. Oil palm pollen was
put into double sided carbon adhesive tape, then it was observed by optimizing the brightness, contrast, magnification,
and also focus.
Protein Extraction. Oil palm pollen protein was extracted using the fresh pollen storage from previous steps.
Pollen was weighed 5-10 gram and then was located on a grinder. Pollen was added with 100 ml liquid nitrogen, and
then pollen was grind using a mortar. The process was repeated two times to make sure that the pollen was totally
ground. Absolute ethanol (10 % w/v) was added to pollen directly after the grinding process finished. The mixture of
pollen and absolute ethanol was stirred using a magnetic stirrer for 30 minutes at room temperature (25 °C). The
mixture was put into 1.5 ml collection tube, and was centrifuged at 14,000 rpm for 20 minutes at 4 °C. Pellet from
that process was dried at a room temperature for 2-3 hours to dry the pellet completely. The pellet was weighed and
0.5 M NaCl (5 % w/v) was added to the pellet. The mixture of the pellet and 0.5 M NaCl was vortexed and was put
into the homogenizer machine for 2 minutes. The process was repeated two times to make sure that the mixture
completely homogeny. The homogeny mixture was centrifuged at 14,000 rpm for 20 minutes at 4 °C. Supernatant
was transferred to a sterile 15 ml collection tube. The supernatant was dialyzed using a dialyzed membrane with PBS
solution as a buffer. Dialyzed process was done using a magnetic stirrer at 4 °C for 12-18 hours. The solution inside
the dialyzed membrane was transferred to a sterile 15 ml collection tube and can be stored in a freezer with temperature
around -20 °C.
Protein Concentration Measurement. The concentration of protein extract of oil palm pollen from the previous
step was measured using Bi-Cinchoninic Acid assay (BCA assay). Albumin was used as the standard in the
measurement. Samples, standard, and working reagents were mixed in a 96-microplate well and were incubated in an
incubator at 37 °C for 30 minutes. The microplate was cooled at room temperature and the absorbance was measured
using microplate reader.
Protein Visualization using SDS-PAGE (Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis).
Oil palm pollen protein extract was visualized using the SDS-PAGE method. Novex protein standard from Invitrogen
was used as a standard in the SDS-PAGE process. Oil palm pollen protein was mixed with LDS and Reducing Agents,
then was incubated at 70 °C for 10 minutes. Protein samples and protein standard were put into
4-12 % bis tris gel. MES SDS Running Buffer was mixed with an Anti-oxidant agent to perform running buffer in
030116-2
SDS-PAGE process. The process was performed on 200 volts with constant impedance around 100-200 mA for
approximately 35 minutes. Gel from SDS-PAGE process was stained using Coomassie Brilliant Blue (CBB) and silver
nitrate. In CBB staining, the gel was mixed with CBB solution and was shaken for 5-10 minutes. CBB solution was
removed, and the gel was destained with pure water and was shaken for 2-5 hours until the protein band looks clear
enough to analyze. Silver staining kit from Invitrogen was used to perform silver nitrate stained. The gel was mixed
with Fixing Solution and was shaken at room temperature (25 °C) for 10 minutes. Fixing Solution was removed and
was replaced with Sensitizing Solution. The gel was mixed with Sensitizing Solution and was shaken at room
temperature for 30 minutes. Sensitizing Solution was removed and was replaced with pure water. The gel was mixed
with pure water and was shaken at room temperature for 10 minutes. Pure water was removed and was replaced with
Staining Solution. The gel was mixed with Staining Solution and was shaken at room temperature for 15 minutes.
Staining Solution was removed and was replaced with pure water. The gel was mixed with pure water and was shaken
at room temperature for 5 minutes. Pure water was removed and was replaced with Developing Solution. The gel was
mixed with Developing Solution and was shaken at room temperature for 3-15 minutes until protein band looks clear
enough to analyze. Stopping Solution was mixed with gel to stop the reaction. The remaining solutions on the gel
were removed and were replaced with pure water. The gel was mixed with pure water and was shaken at room
temperature for 5-30 minutes to recover the gel from the reagents that still remaining.
RESULTS AND DISCUSSION
Oil palm pollen was collected from a male flower that already flowering, which has yellowish colored as described
in Fig. 1a. The male flower was cut and pollen was collected based on Ching and Ching [7] method. The result of that
process was called crude pollen. One male flower can produce 25-200 gram crude pollen based on its aged and size.
Older oil palm tree and the bigger male flower will produce more pollen. Crude pollen was filtered using specially
filtered with pore diameter around 45 μm. One male flower can produce 8-10 gram pollen. This result was related to
the flowering age of oil palm male flower. Oil palm male flower only flowering for 3-6 days. In maximum flowering
stages, one oil palm male flower can produce 20-30 gram of pollen [8].
Identification of oil palm variants can simply be seen on its fruit [9]. Oil palm fruit cross section from three variants
is shown in Fig. 2. The region of mesocarp, endocarp, and endosperm can be analyzed to identify the variant of oil
palm. Dura variant has the thickest endocarp than others, which is around 2-8 mm. Thick endocarp makes Dura variant
quite a resistance to environmental change and disease, but this variant has a low level of oil production. Pisifera
variant has the thickest mesocarp than others, which is around 7-13 mm. Think mesocarp makes Pisifera variant may
produce a lot of oil, but unfortunately Pisifera variant can easily die and not resistance enough to environmental change
and disease. Tenera variant has the combination characteristics of Dura and Pisifera variant. Tenera variant has thick
endocarp and mesocarp, so it still has a good protection and good oil production.
Morphology from three variants of oil palm pollen was observed using a light microscope and scanning electron
microscope. The results of the microscope observation are shown in Fig. 3. Three variants of oil palm pollen show
(a)
(b)
(c)
FIGURE 1. Pollen collection from oil palm plant
(a) male flower in an oil palm plant; (b) oil palm male flower; (c) filterized oil palm pollen (crude pollen).
030116-3
(a)
(b)
(c)
FIGURE 2. Oil palm fruit cross section (a) Dura variant; (b) Pisifera variant; (c) Tenera variant,
Inside picture : a. mesocarp, b. endocarp, c. endosperm
1
2
3
(a)
(b)
(c)
FIGURE 3. Morphology from three variants of oil palm pollen,
1. using light microscope with unstained method, 2. using light microscope
with safranin stained, 3. using scanning electron microscope, a. front side
(Dura variant), b. peripheral side (Pisifera variant), c. back side (Tenera
variant), d. aperture, e. exine.
very similar pollen morphology. Oil palm pollen has a triangular shape with a rounded edge. The aperture of the
pollen was observed as tricolpate with connected colpus. The ornamentation of exine shows that those three variants
have psilate ornamentation (smooth) at the front side and peripheral side, while at the back side has microreticulate
ornamentation (small pores). Safranin stained shows that oil palm pollens are more stained in intine than the exine. It
shows that exine not easily broken by acetolysis solution, so this region is not stained as much as intine. While intine
is more fragile to acetolysis solution than the other part of the pollen, so it stained most than the other part of the
pollen.
Protein was extracted from three variants oil palm pollen. The protein concentration was measured using BCA
assay with albumin as a protein standard. The result of the protein measurement is shown in Table 1. The absorbance
o f samples were compared with standard curved from albumin standard. The result shows that the concentration
average for Dura variant is about 4,298 μg/ml, Pisifera variant is 3,772 μg/ml, and Tenera variant is 4,485 μg/ml.
Those results show that the protein concentration is more than 1 mg/ml and possible to perform protein visualization
using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Some of the protein was diluted
to perform SDS-PAGE, while the rest was stored.
Pollen proteins from three variants of oil palm were analyzed using SDS-PAGE method to observe molecular
weight character of the protein. The result of SDS-PAGE process is shown in Fig. 4. Molecular weight from three
variants of oil palm pollen shows high similarity. The protein molecular weight is ranged from 10 KDa to 100 KDa.
Two staining methods shows small differences. The protein molecular weight in silver nitrate stained shows more
030116-4
TABLE 1. Protein concentration from three variants of oil
palm pollen using BCA assay
Sample
Dura
Pisifera
Tenera
Abs. 570 nm
0.079
0.081
0.075
Abs. 570 nm
0.086
0.081
0.086
Abs. 570 nm
Average
0.073
0.079
0.070
0.073
0.079
0.080
Delta Blank
Concentration (µg/ml)
0
4,298
0.059
3,772
0.062
4,485
(a)
(b)
FIGURE 4. Protein molecular weight visualization using SDS-PAGE method,
silver nitrate stained; (b) Coomassie Brilliant Blue stained of 1. oil palm pollen
protein Dura variant, 2. oil palm pollen protein Pisifera variant, 3. oil palm
pollen protein Tenera variant, 4. non-template control, M: Novex protein
molecular marker
protein band than in Coomassie Brilliant Blue (CBB) stained. These results show that oil palm pollen protein has a
relatively small amount of molecular weight. One of the developments from this result was to observe the allergic
protein from oil palm pollen. Kimura et al. [4] and Chew et al. [5] observed that protein that can cause allergic to
human is ranged from 14 KDa to 31 KDa. Based on this protein molecular weight characteristic result, oil palm pollen
has several potential protein candidates as allergens for humans. It still needs further research to observed that
potential.
CONCLUSIONS
The result of this research was the morphology character from three variants of oil palm pollen have successfully
been observed. Those three variants of oil palm have no differences and show the triangular shaped with round edge.
The aperture of the pollen was observed as tricolpate with connected colpus. The ornamentation of exine shows that
those three variants have psilate ornamentation (smooth) at the front side and peripheral side, while at the back side
has microreticulate ornamentation (small pores). Three variants of oil palm pollen protein show the same
characteristics. The molecular weight of the protein was ranged from 10 KDa to 100 KDa.
REFERENCES
1.
2.
I. R. Baratawidjaja et al., Asian Pac. J. Allergy Immunol. 17, 9-12 (1999).
W. G. Hopkins and N. P. A. Huner, Introduction to Plant Physiology (John Wiley & Sons, Hoboken, New Jersey,
2009), pp. xviii + 503.
030116-5
3.
4.
5.
6.
7.
8.
9.
P. H. Raven, R. F. Evert, and S. E. Eichhorn, Biology of Plants (Worth Publishers Inc., New York, 1992), pp.
xvii + 791.
Y. Kimura et al., Biosci. Biotechnol. Biochem. 66, 820-827 (2002).
F. T. Chew et al., Allergy 55, 340-347 (2000).
I. Rengganis, Kealergenikan Serbuk Sari Indonesia pada Manusia, Ph.D. thesis, Institut Pertanian Bogor, Bogor,
2009.
T. M. Ching and K. K. Ching, Plant. Physiol. 39, 705-709 (1964).
Y. Fauzi, Y. E. Widyastuti, I. Satyawibawa, and R. H. Paeru, Kelapa sawit (Penebar Swadaya, Jakarta, 2012),
pp. iv + 236.
I. Pahan, Panduan Lengkap Kelapa Sawit; Manajemen Agribisnis dari Hulu Hingga Hilir (Penebar Swadaya,
Jakarta, 2013), pp. xx + 412.
030116-6
Документ
Категория
Без категории
Просмотров
0
Размер файла
385 Кб
Теги
4991220
1/--страниц
Пожаловаться на содержимое документа