CHAPTER 1

INTRODUCTION TO STRUCTURED INTERNSHIP PROGRAM (SIP)

1.1 INTRODUCTION
Structured Internship Program (SIP) is an attachment program between students of Faculty of
Engineering Technology with Industry. This program aimed to implement the students
technical and soft skills.

1.2 OBJECTIVE OF SIP

The main objective of SIP is to give the students early exposure the reality of working and
industrial world. The students also gain the better understanding the application of the
technology and engineering principles in the field.
i.

To produce competent students in the term of technical and soft skills in the working
world.

ii.

To apply the theory that had been learnt in class in the industry.

iii.

To increase the employability of the students after graduation.

iv.

To gain knowledge and experience in the professional organization.

1.3 OBJECTIVE OF THE REPORT.

i.

To document all the activities and training along the internship period.

ii.

To be as a proof of the internship program for industry and faculty evaluation of

student performance.

1.4 PROGRAM OUTCOMES

At the end of program, the students are enables to apply the knowledge and skills:
i.

To increase the competitiveness in the engineering technology field.

ii.

To relate the experiences in working world with the theory in the learning world.

iii.

To apply the theory and knowledge.

iv.

To improve the interaction and communication skills.

v.

To gain as much experience that beneficial for jobs scope after graduation.

CHAPTER 2:
ORGANIZATION BACKGROUND.

2.1 MALAYSIAN PALM OIL BOARD (MPOB)

2.1.1 Premier Government Agency
MPOB is the premier government agency entrusted to serve the countrys oil palm
industry. Its main role is to promote and develop national objectives, policies and
priorities for the wellbeing of the Malaysian oil palm industry.
It was incorporated by an Act of Parliament (Act 582) and established on 1 May 2000,
taking over, through a merger, the functions of the Palm Oil Research Institute of
Malaysia (PORIM) and the Palm Oil Registration and Licensing Authority (PORLA).
Each of these respective organisations has been involved in the oil palm industry for
more than 20 years and it is to render more effective services as well as to give greater
national and international focus to the industry that MPOB was instituted.
2.1.2 Funding
MPOB derives its funding mainly from cess imposed on the industry for every tonne of
palm oil and palm kernel oil produced. In addition, MPOB receives budget allocations
from the government to fund development projects and for approved research projects
under the Intensification of Research in Priority Areas (IRPA) programme.
2.1.3 Vision
To become the premier Nobel Laureate - producing research and development
institution, providing leadership and impetus for the development of a highly
diversified, value-added, globally competitive and sustainable oil palm industry.
2.1.4 Mission
To enhance the well-being of the Malaysian oil palm industry through research,
development and excellent services.
3

2.1.5 Strategies
i.

Expand and improve the current uses of oil palm products.

ii.

Find new uses for the products.

iii.

Improve production efficiency and quality of products.

iv.

Optimise land utilisation in oil palm areas.

v.

Promote the use, consumption and marketability of oil palm.

2.1.6 Functions
i.

Implement policies and development programmes to ensure the viability of the

oil palm industry of Malaysia.

ii.

Conduct and promote research and development activities relating to the oil
palm industry.

iii.

Regulate, register, co-ordinate and promote all activities relating to the oil palm
industry.

iv.

Develop, promote and commercialise research findings as well as provide

technical, advisory and consultancy services to the oil palm industry.

v.

Develop and maintain markets for oil palm products as well as promote efficient
marketing.

vi.

Liaise and co-ordinate with other organisations inside or outside Malaysia to

further enhances the oil palm industry of Malaysia.

vii.

Plan and implement training programmes and human resource development in

line with the needs of the oil palm industry.

viii.

Be the resource and information centre of the oil palm industry including the
publication and dissemination of information on oil palm as well as other oils
and fats.

2.2 MALAYSIAN PALM OIL BOARD (MPOB) KLUANG.

2.2.1 History
The MPOB Research Station Kluang, Johor was established in September 1979 and it
was formerly known as the PORIM Research Station. It is located 13 km and 115 km
from Kluang and Johor Bahru respectively. The station is makeup of two adjacent
areas, main station (486 ha) and Bukit Lawiang (404 ha). The main function of the
station is for research and development of oil palm. The oil palm breeding research is
the main R&D activities of the station and this station has been recognized to have the
largest oil palm germplasm collection in the world.

2.3 AGRONOMY & GEOSPATIAL TECHNOLOGY UNIT

2.3.1 Background
AGT was established in December 2012, through a restructuring of the Agronomy &
Mechanisation Unit to focus on specific research and development for agronomy and
geospatial technology applications. AGT focuses research in the agronomic and
fertiliser aspects for increasing yield, biomass utilization, sustainable FFB production,
geospatial information technologies and remote sensing for precision agriculture.

2.3.2 Vision
To make the oil palm industry more competitive by applying knowledge and
technological know-how towards an efficient oil palm production system, in harmony
with the environment.

2.3.3

Mission

i.

To maximize land utilization, labour productivity and sustain high profitability

ii.

To ensure sustainable oil palm production through efficient oil palm production
system

iii.

To promote integrated resources management, leading to sustainable and higher

productivity of soil through optimum nutrient utilization and recycling of plant
biomass and effluents

iv.

To develop and promote geospatial technologies for precision agriculture

application to enhance profitability and sustainability of oil palm management

2.4 ADVANCE BIOTECHNOLOGY AND BREEDING CENTRE

2.4.1 Background
The Biology Research Division undertakes research activities, which are
implemented by three units: Advanced Biotechnology and Breeding Centre (ABBC),
Crop Production and Management (CPM) Unit, and Smallholders Development and
Technology Transfer (SDTT) Unit. The ABBC was officially established in
November 2000 and it was formerly known as the Plant Science and Biotechnology
Unit. The mission of the ABBC is to provide the Malaysian oil palm industry with
improved and novel planting materials and services through R&D in breeding and
biotechnology. The Unit operates through six groups, namely Metabolics, Gene
Expresion, Transformation, Genomics, Tissue Culture and Breeding and Genetics.
The Unit drives forward new research areas that profit from multi-disciplinary
approaches. Recent advances in biotechnology, especially in the areas of genomics
and genetic engineering, have opened opportunities and expedited progress in crop
improvement. Genomic tools are being developed to enhance the speed and
precision of breeding for improved oil palm varieties. The tools and techniques of
genetic engineering are being developed to channel the inherent high productivity of
the oil palm towards value-added products. These include high oleate and high
stearate oil, bioplastics, nutraceuticals and industrial oils. Tissue culture research is
6

conducted with the view to using this technique both as a means of producing
transgenic oil palm and as a tool for the propagation of elite plants. An important
element of a comprehensive strategy for biotechnology is collaboration. The unit has
formed strategic alliances through contract and collaborative research with
international and national centres of excellence.

2.4.2 Mission
The mission of the ABBC is to provide the Malaysian oil palm industry with
improved and novel planting materials and services through R&D in breeding and
biotechnology.
Continuous improvement and generation of elite oil palm planting materials
2.4.3 Objective
Generation of improved and elite planting material through breeding and clonal
propagation, enhanced through the use of molecular tools developed.
2.4.3 Strategic Research Areas
1. Diversity studies for oil palm
2. Establishment of a DNA database for oil palm germplasm material
3. Establishment of a cryopreservation method for storage of germplasm and
clonal materials
4. DNA fingerprinting for our planting material
5. Marker assisted selection towards improving oil palm breeding material
6. Genetic resources
7. Improve the selection methodology
8. Establishment of advanced breeding populations
9. Further exploitation of Elaeis oleifera, interspecific hybrids and backcrosses
10. Introgression of germplasm into breeding research
11. Genotyping analysis

12. Use of omics technologies to discover biomarkers for improvement of clonal

propagation
13. Epigenetics studies towards understanding the oil palm clonal abnormality
14. Mitochondria research with relations to yield production and clonal
abnormality
15. Production of elite clonal palms and specialty palms such as Bactris and Elaeis
oleifera.
16. Development of novel methods for improvement of the tissue culture process.
17. Development of liquid cultures for improving the efficiency of the tissue
culture protocol.
18. Clonal propagation and breeding to produce planting materials with the
following priority traits:
a) High oil yield
b) Ganoderma tolerance
c) High bunch index
d) Low height
e) Long stalk

CHAPTER 3:
TRAINING ACTIVITIES

3.1 AGRONOMY & GEOSPATIAL LABORATORY.

3.1.1 Soil Analysis
Soil Analysis is the analysis of a soil sample to determine nutrient and contaminated
content, composition, and other characteristics such as the acidity or pH level. A soil
test can determine fertility, or the expected growth potential of the soil which indicates
nutrient deficiencies, potential toxicities from excessive fertility and inhibitions from
the presence of non-essential trace minerals. The test is used to mimic the function of
roots to assimilate minerals. The expected rate of growth is modelled by the Law of the
Maximum.
i.

Nitrogen Determination.

Procedure:

1. Weight 0.25g soil samples and put into Digestion tube.

2. Add a tablet of Kjeldahl catalyst into each Digestion tube.
3. Add 2ml concentrated H2SO4
4. Add 0.3g Na2S2O3
5. Place Digestion tube into Digestion block and set for 2 hours at 420oC
6. After 2 hours, allow the Digestion tube to cool.
7. Run the FOSS Distillation unit for each Digestion tube.
8. Collect the distillate.
9. Titrate the distillate with 0.01M HCl.
10. Record the reading.
11. Repeat the steps for each Digestion tubes.

Results:
Samples

Depth of soil

Volume of HCL

(cm)

(ml)

0-15

2.71

0.152

15-30

2.47

0.138

30-45

2.88

0.161

0-15

2.78

0.156

15-30

2.28

0.128

30-45

2.83

0.158

ii.

%N

Phosphorus Determination.
Procedure:
1. Weight 2g of soils sample in the bottles.
2. Add 20 ml extracting solution.
3. Put the bottle in orbital shaker and shake for 1 minute.
4. Pour the content into the filter paper and left for an hour.
5. Pipette 0.2ml of the filtrate into glass tube.
6. Add 0.8 Reagent B to all tube and add 4ml distilled water.
7. Prepare standard and blank.
8. Analyze the samples with UV-Vis
9. Record all data.

* Extraction solution: Add 30ml 2M NH4F, 400ml 0.5M HCl in 2L flask and
make up to volume with water.
* Reagent A: 12g Ammonium Molybdate dissolves in distilled water and add
148ml concentrated H2SO4. Pour in 1L volumetric flask. Dissolves 0.2908g
Potassium Antimony in distilled water and add to the flask and make up to
volume with distilled water.
10

* Reagent B: 25ml of Reagent A add to 0.132g Ascorbic Acid.

Preparation of standard solution for Calibration.
Extracting

Reagent B

solution

Stock

Water

Total (ml)

solution

Std 1

0.2

0.8

0.1

3.9

Std 2

0.2

0.8

0.3

3.7

Std 3

0.2

0.8

0.5

3.5

Results: Refer Appendix 1

iii.

Magnesium, Calcium and Potassium Determination.

Procedure:
1. Weight 1g soil samples in beaker.
2. Add 50ml of 1M Ammonium acetate.
3. Place in orbital shaker and shake for 2 hours.
4. After 2 hours, filter the mixture and left overnight.
5. After left the samples overnight, the filtrate is taken for analysis.
6. For K analysis, take 5ml filtrate and put it in th test tube.
7. For Ca analysis, 5ml of filtrate is added in the beaker with 1ml SrCl2.
5ml of the mixture is added into the test tube.
8. For Mg analysis, 4ml SrCl2 and 5ml filtrate is added to 25ml conical
flask and add up d.H2O and make up to volume. 5ml of the mixture is
added into test tube.
9. All the test tube prepares is ready for AAS analysis.
10. All the samples, blank and standard is placed in the autoanalyzer.
11. Set the AAS instrument and run the analysis.
12. Record all the reading.
Results: Refer Appendix 2

11

iv.

pH Determination.

Procedure:
1. Weight 10g soil samples in beaker and add 25ml d.H2O.
2. Stir the mixture and left overnight
3. Before start to analyse pH value, warm up the pH meter for about 1
hour.
4. Calibrate the pH meter with stock solution of pH4 and pH7.
5. After the samples are left overnight, the sample was stirred before being
analyse.
6. Read the pH value, wait until the reading stable.
7. Record all the data.
Results:

v.

Sample

pH

6.25

6.15

4.93

6.45

5.73

5.01

Carbon Determination.

Procedure:
1. Weight 1g of soil samples in beaker.
2. Add 5ml Sodium Dichromate.
3. Add 10ml conc. H2SO4
4. Dilute with 50ml distilled water and left overnight.
5. After left overnight, the sample is taken and put into test tubes.
6. Prepare the sucrose standard solution for calibration. Dissolves 2.3750g
of glucose in 1L volumetric flask and make up with distilled water to
volume.
12

7. Pipette 0, 2.5, 5.0, 7.5ml of sucrose solution in beaker and label as

blank, std1, std2 and std3 respectively.
8. Add 2.5ml of Sodium Dichromate solution into each beaker.
9. Add 10ml concentrated H2SO4 and left for 10minutes.
10. Dilute with 25,22.5, 20 and 17.5ml of distilled water into respective
beaker.
11. Put the standard solution in test tube.
12. Set up the UV-Vis with the absorbance wavelength of 600m and read
the samples starting with blank, standard and samples.
13. Record the results

Results: Refer Appendix 3

vi.

Task: Comparative Study of Carbon Determination in Soils using Loss On

Ignition, Total Organic Carbon Unit and Wet Chemistry (Walkley-Blake)
-

Refer Appendix 4

13

3.1.2 Leaf Analysis

Leaf analysis (also called stem leaf analysis, tissue analysis or foliar analysis) is the
most precise method of monitoring plant nutrient levels. While soil analysis reveals the
levels of essential soil nutrients, leaf analysis shows exactly what the plant has
successfully absorbed. Leaf analysis can be helpful in detecting nutrient deficiencies
before they affect plant health and yield.
i.

Phosphorus, Potassium, Calcium, Magnesium and Boron Determination.

Procedure:
1. Prepare crucible and labeled.
2. Weight 1g of leaf in the crucible.
3. Place the crucible + leaf samples in the furnace and burnt it at 500oC for
6 hours.
4. After 6 hours, off the furnace and left the samples overnight.
5. Take out the crucible from furnace.
6. Add 10ml of 20% HCl into each crucible.
7. Pour the mixture into plastic tube and tighten the cap.
8. Place in centrifuge at 500rpm for 5minutes.
9. Carefully pipette 5ml of supernatant and place into 25ml volumetric
flask.
10. Add distilled water and add up to volume.
11. Shake the flask to allow the solution mix.
12. The solution is considered as Original Solution.
13. For Potassium, Calcium, Magnesium.
a. Pipette 0.5ml of the Original Solution and put in 25ml
volumetric flask.
b. Add 2.5ml SrCL2 and add distilled water up to volume.
c. Shake the flask to mix the solution well.
d. Pour 14ml of the solution into tube.
e. Place the test tube in the auto analyzer and set all the parameters.
Run the AAS test.
f. For K analysis, the standard solution for K is analyzed first and
used for calibration.
14

g. Read all the samples and record the data

h. Repeat step 5 to 7 using Ca solution standard for Ca analysis and
Mg standard solution for Mg analysis.
14. For Phosphorus
a. Take 1ml of Original Solution (prepared before) and put in the
test tube.
b. Add Vanado solution and distilled water.
c. Volume of solution need to prepare sample for P analysis as
shown in Table 1.
d. Run the samples analysis using UV-Vis.
Table 1: Samples and standard preparation

All
samples
Standard
0.06%
Standard
0.075%
Standard
0.15%
Standard
0.30%

Original
Solution
(ml)

Vanado
solution
(ml)

Distilled
water (ml)

Total
Volume
(ml)

1.5

2.5

1.5

2.5

1.5

2.5

1.5

2.5

15. For Boron

a. Pipette 1m supernatant from Original solution prepared before.
b. Put the solution in the test tube.
c. Add 2ml buffer solution with 1ml azomethine reagent.
d. Shake the test tube to mix the solution.
e. Left for 2 hours.
f. After 2hours, analyze the samples using the UV-Vis.

Results: Refer Appendix 5 (K, Ca, Mg), Appendix 6 (B)

15

ii.

Nitrogen Determination
Procedure:
1.

Weight 0.25g leaf samples and put into Digestion tube.

2.

Add a tablet of Kjeldahl catalyst into each Digestion tube.

3.

Add 2ml concentrated H2SO4

4.

Place Digestion tube into Digestion block and set for 2 hours at 420oC

5.

After 2 hours, allow the Digestion tube to cool.

6.

Run the FOSS Distillation unit for each Digestion tube.

7.

Collect the distillate.

8.

Titrate the distillate with 0.01M HCl.

9.

Record the reading.

10. Repeat the steps for each Digestion tubes.

Results:
Samples
1
2
3
4
5
6
7
8

Volume of HCL (ml)

18.6
8.35
17.35
16.64
18.56
15.70
16.82
17.00

%N
2.604
1.169
2.429
2.33
2.60
2.19
2.35
2.33

16

3.1.3 Discussion

Laboratory tests often check for plant nutrients in three categories:

a) Major nutrients: nitrogen (N), phosphorus (P), and potassium (K)
b) Secondary nutrients: sulfur, calcium, magnesium
c) Minor nutrients (traced elements) : iron, manganese, copper, zinc, boron,
molybdenum, chlorine.
Soil testing is used to facilitate fertilizer composition and dosage selection for land
employed in both agricultural especially in the oil palm planting. The soil quality also
can be determined, as the soil was taken from different depth. If the nutrient content
in the deepest depth is less, but at the surface the nutrient is more, it can be concluded
that the soil absorption is weak. If the nutrient level is low, the fertilizer should be
added.

The concentration of each element in the analysed tissue is compared with established
desired ranges for healthy, productive plants or crops of the same species. A report is
given with the analysis which clearly defines both the nutrient deficiencies and/or
excesses that may be limiting plant health and yield.

Leaf Analysis is valuable for:

Trouble shooting for diagnosing and correcting existing nutrient problems.

Monitoring crop nutrient status for optimal crop production

Ensuring balanced plant nutrient levels for top produce quality.

Predicting plant nutrient problems during the growing season before they cause
production loss.

Understanding interactions between the soil and plant nutrient uptake.

Formulating foliar spray programs to address nutrient deficiencies and

imbalances.

17

3.2 FRUIT BUNCH ANALYSIS LABORATORY.

3.2.1 Bunch Analysis
Bunch analysis in oil palm is essential for evaluation of a palm for its oil yield. Oil
palm being a cross pollinated crop, performance of individual palm is utmost
important. Evaluation of palms is needed for different purposes, like for selection of
superior and elite mother palm hybrid.
3.2.1.1Fruit Bunch Analysis Procedure:
1. The type of matured oil palm bunches was determined either Dura, Tenera or
Pisifera.
2. The oil palm bunches samples was weight and the data was recorded.
3. The spikelet and stalk of matured oil palm bunches was separated manually
using axe.
4. The stalk was weighted and the data was recorded.
5. The stalk was disposed and the spikelet was mix on the Compartment Box to
obtain 2 samples partition randomly.
6. The samples partition:
7. FC Fruit Component
8. FB Fruit Bunch
9. The formula for samples partition
Weight of Bunch (kg)
1 11.9
12 15.9
16 23.9
24 39.9
More than 40

FB / FC
3/1
4/1
3/1
2/1
1/1

10. FB samples was weighted and then transferred into trolley box to remove
moisture.
11. FC samples were placed in smaller tray to separate fruit and spikelet for
mesocarp preparation.

18

i. FB Samples
1. FB samples will be stored in trolley compartment at room temperature for
about 48-72 hours to remove the moisture.
2. The data was obtain to determine the moisture content by sorting the FB
samples into 3 parts and then weight again the parts.
a. Normal Fruits
b. Parthenocarpic Fruits
c. Empty Spikelet

ii. FC Samples
1. Sorting the fruit from spikelets
a. Fruit and spikelet was separated with knife.
b. Fruit that already separated with spikelet will be cast into Fruit
Compartment to separate the fruit randomly into 2 parts.
c. 1 part of the fruit will be again sorted to obtain about 30 to 60 fruits
according to its size, weight and genus (E.guineensis or E.oleifera).
2. Depericarping
a. 30 to 60 fruits were weighted and the data was recorded.
b. The fruit was depericarped and sorted into 2 parts:
- Wet Mesocarp.
- Wet Nut
c. Wet Mesocarps and Wet Nuts were weight and the data were
recorded.
d. Wet Mesocarps and Wet Nuts were then placed overnight in the
oven at 105oC for 16 hours.
e. The mesocarps were taken out from the oven and left to cool for
about 30 minutes.
f. The nuts were taken out from oven and were broken to remove the
shell and obtain the kernels.
g. Kernels without the shells was then reweighted and the data were
recorded.

19

3. Mesocarp Grinder and Sampling

a. Dried mesocarps were weighted and the data were recorded.
b. The dried mesocaps were fine-grinded using blender machine.
c. Grinded mesocarps were then filtered to obtain fine mesocarps
powder and placed into smaller tray.
d. Prepare Thimble paper (chromatography) and fold to make
envelops 6 x 3 inch and weight.
e. Record the data.
f. Weights 5g of mesocarp powder and put in the thimble envelops.
g. The weight of mesocarp powder and thimble paper was recorded.

4. Extraction Process
a. 5L Hexane was filled in the Soxhlet Flask
b.

Assembled the Extractor and greased the entire connective joint so

it easier to open and avoid evaporation.

c. Soxhlet Isopad Heater was heated.

d. Put the thimble envelop into the extractor (80 to 120 samples
envelops).
e. Extraction for about 20hours.
f. Once the oil is extracted from the sample, the remaining sample is
weighed as the dry weight and hence the amount of oil yield per
sample can be calculated.

3.2.2Fatty Acids Analysis

Procedure:
1. The fruit samples are taken from the apical, middle and basal of the bunch
and weighted.
2. The samples were steamed at 15-20psi for 45 minutes to soften the
mesocarp..
3. The mesocarps were grinded and put into 250ml beaker until 2/3 full.

20

4. In fume hood, 300mL hexane was added to dilute and extract the oil. The
mixture was then filtered using filter paper with anhydrous Sodium
Sulphate.
5. The oil and hexane mixture were separated through distillation using
rotary evaporator.
6. The oil obtained after separation process.
7. The oil were taken and melted at 60oC and poured into vial.
8. 1.9mL hexane was added into the vial as solvent.
9. 0.1mL of sodium methoxide was pippetted into the vial.
10. Capped the vial and spin vortex the vial.
11. Distilled water was added until the vial was 3/4 full.
12. Double layer solution of oil and glyceroxide was formed.
13. The top layer was transferred into gas chromatography vial and sealed
with Teflon cap.
14. Screening of fatty acids with Gas Chromatography.

3.2.3 Carotene Content Analysis

1. 0.10 g of the oils were taken and put in the volumetric flask.
2. The samples are dissolves and diluted with isooctane.
3. The solutions were pour into the cuvette.
4. UV-ViS was used to determine the beta-carotene content at wavelength
set at 446nm.

21

3.2.4 Discussion.
Oil palm bunch analysis is vital for the determination of the quantity and quality of oil
palm breed in term of oil yield. The data collected at the end of the analysis will lead
to the find out of the best breed. Besides that, bunch analysis is also important to
describe in detail the oil composition of each bunches for the identification of the best
mother palm.
The data obtain from the FB samples moisture contents is to determine the quantity of
production of fertile fruit, parthenocarpic fruit and empty spikelet in term of weight in
kg per bunch. The percentage of fertile fruit in a bunch should be greater than the
other components that indicate an optimum production of oil yield.
While fatty acid composition test is used to determine the quality of the selected
mother palm that producing high unsaturated oil. The method adopted by MPOB in
the analysis is proposed by Timms (1978) for routine palm oil analysis that using Gas
chromatography (GC). Same goes with the carotene content.

3.3

CRYOPRESERVATION

AND

MOLECULAR

BREEDING

LABORATORY
3.3.1Moisture Content
Procedure:
1. Hammer was used to break 20 palm seeds/nuts from each sample number to
obtain the palm oil kernels.
2. Surgical blade was used to excise the embryos from the kernels (15 healthy
and fresh embryos)
3. Weight the container, embryo plus container
4. Put the samples in the oven at 105oC for 16 hours (4.45pm 8.45am)
5. Take out the embryo from the oven.
6. Weight the dried embryo and record the data.
Results: Refer Appendix 7

22

3.3.2 Cryopreservation
# Based on the Moisture Content results, the sample number with moisture content
that is less than 20% is suitable for cryopreservation. The sample number with higher
moisture content will be placed in the desiccator with silica gel for further moisture
removal.
Procedure:
1. Hammer was used to break all palm seeds/nuts (apporx. 400 seeds) from each
sample to obtain the palm oil kernels.
2. After all the seed were broken, soak all seeds samples in 0.05% Tween20 and
Mercuric chloride mixture for about 10min.
3. Rinse 3 times with sterilized distilled water.
4. Soak the seeds in sterilized distilled water for 10min.
5. Rinse again with sterilized distilled water.
6. Dried the seeds in petri dish with sterilized Whattman paper. (left overnight)
7. Applied aseptic techniques, process in the laminar flow cabinets in clean
room, sterilized equipment.
8. After the seeds dried, surgical blade was used to excise the embryo from the
kernels.
9. For each samples, 150 healthy embryos were needed to be place in the 15
cryovial (10 embryos each)
10. After 15 cryovial filled, the cryovial was placed in Cryosystem tank (in liquid
N2 at -196oC). Repeat the step for all samples.
11. Take another 5 healthy and fresh embryos for fresh culture on the MS Basal
Medium. Kept in the culture room. Monitor the growth every week.

23

3.3.3 DNA Extraction

Modified CTAB Method
i.

Day 1
1. Pre-warmed 2X CTAB at 60oC water bath.
2. Grind 4g of leaf samples in mortar with acid wash sand and liquid N2
until become powder.
3. 0.2ml of 0.5M Ascorbic acid, 0.2ml of 0.4M DIECA and 0.2ml
mercaptoethanol was added (in fume hood).
4. 20ml CTAB buffer was transferred (use falcon tube) into each
mortaronce basin with added of warmed H2O (in fume hood).
5.

Transfer the solution into 50ml corex tube (uncap)

6.

Incubate at 60oC water bath for about 30min.

7.

Take out from water bath and left to cool at room temperature.
(30min)

8.

Add15ml of Chloroform and Isoamyl Alcohol mixture C:I (24:1) and

shake the mixture well. Centrifuge at 25oC/12000rpm for 20min.

9.

Transfer [16ml] of the aqueous phase into another corex tube (using
pipette with cut tips). Add [9.6ml] of isopropanol. Mix gently. Allow to
precipitate at -20oC for 1hour. [16ml x 0.6 = 9.6ml]

10. Centrifuge at 4oC/12000rpm for 15min.

11. Decant the supernatant and tap at tissue paper. Wash the pallet in 5ml
wash buffer, incubate at 4oC.
ii.

Day 2
1. The washed pellet was observed, if the pellet still greenish in colour, replace
and change new wash buffer. Incubate at room temperature until the pallet is
white.
2. The pellet was white in colour so the wash buffer was removed with pipette.
3. Cover the corex tube with parafilm and make a few holes, put the tube in the
speed vacuum machine for 1 hour to dry the pallet.
4. After the pellet dried, TE buffer was used to re-dissolve the pellet.
5. Incubate the tube in 50oC water bath. Check and flip every 2 hours (incubate
until all the pellet dissolves takes more than 1day).
# before going back, incubate the tube at 4oC. Continue the next day.
24

iii.

Day 3
1. After the entire pellet re-dissolved, add 6.25L RNAse (10mg/ml) for 5ml
TE buffer.
2. Incubate at room temperature for 30min.
3. Add 2.5mL of chilled 7.5M NH4 Acetate (pH7.7). Capped, inverted the tube
and incubate in ice for 30min.
4. Centrifuge at 4oC/12000rpm for 15min.
5. Transfer the supernatant into another tube by pipette using cut tips and
discard the pellet (the RNA)
6. Add 18.8mL of -20oC 100%EtOH, invert tube slowly.
7. Incubate at -20oC for 1hour.
8. Centrifuge at 4oC/12000rpm for 15min.
9. Wash pellet with 6.3mL of chilled -20oC 70% EtOH. Incubate at room
temperature for 15min.
10. Pipette out EtOH.
11. Dry pellet in speed vacuum for 1hour
12. Re-dissolves pellet in 1.3mL TE buffer.
13. Incubate at 50oC water bath. Before going back, incubate tube at 4oC.
Continue next day.

iv.

Day 4
1. Took out the corex tube and check the pellet.
2. Continue to incubate the tubes in 50oC water bath until the pellet completely
dissolves.
3. Transfer the solution into 1.5mL tubes using pipette with cut off tips. Short
spin down.
4. The DNA was ready to be determined its quality and quantity using
Spetrophotometer. (Optical Density)
5. If it not used, stored in the freezer at 4oC.

Results of Optical Density: Refer Appendix 8

25

3.3.4 Discussion
Cryopreservation or cryoconservation is a process where cells, whole tissues, or any
other substances susceptible to damage caused by chemical reactivity or time are
preserved by cooling to sub-zero temperatures. At low enough temperatures, any
enzymatic or chemical activity which might cause damage to the material in question
is effectively stopped. Cryopreservation methods seek to reach low temperatures
without causing additional damage caused by the formation of ice during freezing. In
palm oil cryopreservation, the method is used to study the effect or consequences on
the embryo growth, quality and palm oil defect genetically or physically when the oil
palm embryo was stored in the liquid Nitrogen at -196oC for years. Before the embryo
can be preserve in the liquid Nitrogen, the embryo must contain moisture at most 20%
to avoid ice frost that can damage the embryo and the growth
Preparation of large quantity and high quality genomic DNA from a large number of
plant samples is a major bottleneck for most genetic and genomic analyses, such as,
genetic mapping, and next-generation sequencing directly from sheared genomic
DNA. A variety of DNA preparation methods and commercial kits are available. The
development of a high throughput DNA isolation method by modified CTAB
extraction method is aclean up procedure which this method yielded large quantity
and high quality DNA from the oil palm. The DNA obtain need to be analysed the
quality and quantity using Spectrophotometer so the DNA is good enough for further
genetically processes.

26

CHAPTER 4
CONCLUSION
At the end of this industrial training, much knowledge, techniques, procedures can be learnt
in the palm oil research field. The experiences that can be gained is crucial to increase the
competitiveness in the engineering technology field especially in biotechnology and
molecular genetics. This training also give the chances to apply the theory learn in the class
on the real working world by running the sophisticated and high technology instruments and
procedures in the research field. This training program also boast my soft skills, including
confident level and communication skills which gained by socialize and interaction with the
Research Officers and all the MPOB staffs. All the knowledge and experience can be an
added value for myself and beneficial for future job scope after graduation. All this hands on
experience prove that this Structured Internship Program achieved all the objectives.

27

REFERENCES:
1. 8th Malaysia Genetic Congress 2009. Role of Genetics in Wealth Creation and Quality
of Life. Persatuan Genetik Malaysia, UKM, UPM and MOSTI
2. Brian A. Schumacher, Ph.D. April 2002. Methods for the determination of Total
Organic Carbon (TOC) in Soils and Sediments. Exposure Research Laboratory,
United States Environmental Protection Agency, Environmental Sciences Division
National. NCEA-C- 1282; EMASC-001. L.A. USA.
3. Nelson, D.W. and L.E. Sommers. 1996. Total carbon, organic carbon, and organic
matter. In: Methods of Soil Analysis, Part 2, 2nd ed., A.L. Page et al., Ed. Agronomy.
9:961-1010. Am. Soc. of Agron., Inc. Madison, WI.
4. B. De Vos et.al; 2005 Capability of Loss-on-Ignition as a Predictor of Total Organic
Carbon in Non-Calcareous Forest Soils. Communications in Soil Science and Plant
Analysis, 36: 28992921, Taylor & Francis, Inc. ISSN 0010-3624 print/1532-2416
online. DOI: 10.1080/00103620500306080
5. H.Zhang, T.Provin et.al; 2005. Soil Organic Matter: Loss On Ignition Methods.
6. Epstein. E. Mineral Nutrion of Plants: Principles and Prespective John Wiley &
Sons, Inc. USA. 1972.
7. Proceedings: Seminar on Fertilizers in Malaysian Agriculture. 28th March 1983.
Malaysian Society of Soil Science & Universiti Pertanian Malaysia.

28

CREDITS

A HEARTFELT GRATITUDE AND THANK FOR MPOB KLUANG RESEARCH

STATION AND THE STAFF ESPECIALLY:

DR. KHALID HARON (HEAD OF STATION)

CRYOPRESERVATION & MOLECULAR GENETICS

1. MDM NORZIHA BT ABDULLAH (RESEARCH OFFICER)
2. MS. SITI FADZILAH SABTU (RESEARCH OFFICER)
3. MDM. REZA JASMAN (RESEARCH ASSISSTANT)

BUNCH ANALYSIS
1. EN. ZUKI BIN MUSTAPHA (RESEARCH ASSISTANT)

AGRONOMY & GEOSPATIAL LABORATORY

1. MDM FARAWAHIDA MOHAMAD DARUS (RESEARCH OFFICER)
2. MDM SUHAIZA SUNGIT (RESEARCH ASSISTANT)
3. MS SITI ZABEDAH HASSAN (RESEARCH ASSISTANT)

FATTY ACID ANALYSIS

1. EN. ABU ZAKI BIN DELON (RESEARCH ASSISTANT)

29

LIST OF APPENDICES

APPENDIX 1: Phosphorus Content in Soil

APPENDIX 2: Magnesium, Calcium, Potassium Content in Soil

APPENDIX 3: Carbon Content in Soil

APPENDIX 4: Comparative Study of Carbon Determination in Soils using Loss

On Ignition, Total Organic Carbon Unit and Wet Chemistry (Walkley-Blake)

APPENDIX 5: Pottasium, Calcium, Magnesium Content in Leaf

APPENDIX 6: Boron Content in Leaf

APPENDIX 7: Moisture Content of Embryo

APPENDIX 8: DNA Quality (Optical Density)

APPENDIX 9: Process Flow Diagram of all the procedure with pictures

30