industrial training at mpob

8/10/2019 Industrial Training at MPOB

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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 ofstudent performance.


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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.


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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.


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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.


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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 andgeospatial 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.


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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


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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


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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


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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 fromthe 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 H 2SO 4

4. Add 0.3g Na 2S2O3

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

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.


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Results:

Samples Depth of soil

(cm)

Volume of HCL

(ml)

% N

1 0-15 2.71 0.152

2 15-30 2.47 0.138

3 30-45 2.88 0.161

4 0-15 2.78 0.156

5 15-30 2.28 0.128

6 30-45 2.83 0.158

( )

ii. 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-Vis9. Record all data.

* Extraction solution: Add 30ml 2M NH 4F, 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 H 2SO 4. Pour in 1L volumetric flask. Dissolves 0.2908g

Potassium Antimony in distilled water and add to the flask and make up tovolume with distilled water.


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

Preparation of standard solution for Calibration.

Extracting

solution

Reagent B Stock

solution

Water Total (ml)

Std 1 0.2 0.8 0.1 3.9 5

Std 2 0.2 0.8 0.3 3.7 5

Std 3 0.2 0.8 0.5 3.5 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 SrCl 2.

5ml of the mixture is added into the test tube.

8. For Mg analysis, 4ml SrCl 2 and 5ml filtrate is added to 25ml conical

flask and add up d.H 2O 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.



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iv. pH Determination.

Procedure:

1. Weight 10g soil samples in beaker and add 25ml d.H 2O.

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:

Sample pH

1 6.25

2 6.15

3 4.93

4 6.45

5 5.73

6 5.01

v. Carbon Determination.

Procedure:1. Weight 1g of soil samples in beaker.

2. Add 5ml Sodium Dichromate.

3. Add 10ml conc. H 2SO 4

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.


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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 H 2SO 4 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


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


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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 500 oC 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 25mlvolumetric 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 andused for calibration.


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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

OriginalSolution

(ml)

Vanadosolution

(ml)

Distilledwater (ml)

TotalVolume

(ml)All

samples1 1 3 5

Standard0.06%

1.5 1 2.5 5

Standard0.075% 1.5 1 2.5 5

Standard0.15%

1.5 1 2.5 5

Standard0.30%

1.5 1 2.5 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)


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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 H 2SO 4

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

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 Volume of HCL (ml) % N1 18.6 2.6042 8.35 1.1693 17.35 2.429

4 16.64 2.335 18.56 2.606 15.70 2.197 16.82 2.358 17.00 2.33


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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.


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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) FB / FC1 11.9 3 / 1

12 15.9 4 / 1 16 23.9 3 / 1 24 39.9 2 / 1

More than 40 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.


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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 forabout 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.


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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.


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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 60 oC 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.


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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 BREEDINGLABORATORY

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 healthyand fresh embryos)

3. Weight the container, embryo plus container

4. Put the samples in the oven at 105 oC for 16 hours (4.45pm 8.45am)

5. Take out the embryo from the oven.

6. Weight the dried embryo and record the data.



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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 thekernels.

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 -196 oC). 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.


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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 60 oC 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 25 oC/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 -20 oC for 1hour. [16ml x 0.6 = 9.6ml]

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

11. Decant the supernatant and tap at tissue paper. Wash the pallet in 5ml

wash buffer, incubate at 4 oC.

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 iswhite.

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 50 oC 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 4 oC. Continue the next day.


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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.


5. Transfer the supernatant into another tube by pipette using cut tips and

discard the pellet (the RNA)

6. Add 18.8mL of -20 oC 100%EtOH, invert tube slowly.

7. Incubate at -20 oC for 1hour.


9. Wash pellet with 6.3mL of chilled -20 oC 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 50 oC water bath. Before going back, incubate tube at 4 oC.

Continue next day.

iv. Day 4

1. Took out the corex tube and check the pellet.

2. Continue to incubate the tubes in 50 oC water bath until the pellet completely

dissolves.

3. Transfer the solution into 1.5mL tubes using pipette with cut off tips. Shortspin 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 4 oC.

Results of Optical Density: Refer Appendix 8


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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, anyenzymatic 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 -196 oC 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 quantityand 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.


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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.


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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 TotalOrganic 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 OrganicCarbon in Non-Calcareous Forest Soils. Communications in Soil Science and Plant

Analysis, 36: 2899 2921, 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.


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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)


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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

industrial training at mpob

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