KOK SAU YEE - Universiti Putra Malaysiapsasir.upm.edu.my/27222/1/FBSB 2011 34R.pdfENDOSPERM DURING...

UNIVERSITI PUTRA MALAYSIA

HISTOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF OIL PALM (ELAEIS GUINEENSIS JACQ.) ENDOSPERM DURING FRUIT

DEVELOPMENT

KOK SAU YEE

FBSB 2011 34

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HISTOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF OIL

PALM (ELAEIS GUINEENSIS JACQ.) ENDOSPERM DURING FRUIT

DEVELOPMENT

By

KOK SAU YEE

Thesis Submitted to the School of Graduate Studies,

Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of

Master of Science

June 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

HISTOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF OIL

PALM (ELAEIS GUINEENSIS JACQ.) ENDOSPERM DURING FRUIT

DEVELOPMENT

By

KOK SAU YEE

June 2011

Chairman : Parameswari a/p Namasivayam, PhD

Faculty : Biotechnology and Biomolecular Sciences

Oil palm (Elaeis guineensis) is commercially planted in Malaysia and Indonesia for

palm oil production. Palm oil has become the largest source of edible oil in the

world. With increasing global demand for vegetable oil coupled with the challenges

in production, there is an urgent need to improve palm oil productivity. Therefore, in

vitro propagation of oil palm has been in progress to regenerate desirable traits in

order to meet the world demand. However, the oil palm tissue culture technique is

still inefficient and there is also no universal culture medium for all genotypes. One

of the focus areas is the optimization of media formulation for enhanced somatic

embryogenesis through the understanding of endosperm development. Thus, the aim

of this study was to investigate changes in nutrient composition and morphology of

endosperm during fruit development.

Histological features of oil palm fruits were examined by histochemistry and light

microscopy. Oil palm endosperm formation started at 2 weeks after anthesis (WAA)

and it was in liquid form. Enlargement of endosperm occurred as the seed developed

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further. Cellularization occurred at 8 WAA and cells started grow around periphery

of the endocarp. The cells in endosperm continued to grow towards the centre of

endosperm. The mature endosperm contained numerous nutrient reserves which was

essential for nurturing embryo growth.

Biochemical compositions in developing oil palm fruits were analysed by

chromatographic and atomic spectroscopic techniques. Glutamic acid was the

predominant amino acid followed by aspartic acid and arginine throughout the

endosperm developmental stages of both planting materials, tenera and clonal. It was

suggested that the glutamic acid, aspartic acids and arginine were the main building

block in kernel protein. At early stages of seed development, the high hexose/sucrose

ratio in tenera (57.85) and clonal (250.53) was observed which is important to meet

the high metabolic demand required for cell division and differentiation. However,

the ratio was switched at 10WAA in both tenera and clonal (0.80 and 1.53,

respectively) which indicated the onset of endosperm maturation phase. Analysis of

fatty acids revealed that lauric acid was the most abundant fatty acid in mature oil

palm endosperm of tenera (54%) and clonal (51%).

The macro and micro mineral elements are essential for plant growth. High amount

of manganese and calcium were found at 5 WAA. Manganese content found in

tenera and clonal endosperm was 68 mg/100g dw and 158 mg/100g dw, respectively.

While, calcium content found in tenera and clonal endosperm was 797 mg/100g dw

and 6350 mg/100g dw, respectively. For both tenera and clonal, potassium (108 and

751 mg/100g dw, respectively) and phosphorus (653 and 695 mg/100g dw

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respectively) were accumulated in mature endosperm. Analyses of vitamins showed

that alpha-tocopherol and niacin were the predominant vitamins found in both oil

palm endosperms.

On the whole, the anatomical and nutritional study of oil palm endosperm has

provided information on cellular changes and nutrient requirements for zygotic

embryo development. This knowledge will be useful in optimizing the tissue culture

media of oil palm in order to improve the efficiency of somatic embryogenesis.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PENCIRIAN SIFAT HISTOLOGI DAN BIOKIMIA ENDOSPERMA

KELAPA SAWIT (ELAEIS GUINENSIS JACQ.) SEMASA

PERKEMBANGAN BUAH

Oleh

KOK SAU YEE

Jun 2011

Pengerusi : Parameswari a/p Namasivayam, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Kelapa sawit (Elaeis guineensis) ditanam secara komersial di Malaysia dan

Indonesia untuk pengeluaran minyak sawit. Minyak sawit telah menjadi sumber

minyak masak yang terbesar di dunia. Pengeluaran minyak sawit yang bertepatan

telah menjadi satu keperluan bersandaran dengan peningkatan permintaan dunia

terhadap minyak sawit serta cabaran dalam proses penghasilannya. Oleh sebab itu,

propagasi in vitro bagi kelapa sawit telah dijalankan bagi menghasilkan pokok kelapa

sawit yang mempunyai cirri-ciri yang diingini bagi memenuhi permintaan dunia.

Namun, teknik kultur tisu kelapa sawit masih lagi tidak cekap dan tiada media kultur

umum yang bersesuaian untuk semua genotip. Salah satu bidang yang perlu diberi

perhatian ialah pengoptimuman formulasi media demi meningkatkan embriogenesis

somatik, yang mana ianya dapat dicapai melalui pemahaman tentang perkembangan

endosperma. Oleh yang demikian, kajian ini dijalankan bertujuan untuk menyiasat

perubahan komposisi zat makanan dan morfologi endosperma semasa proses

perkembangan buah.

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Kajian mengenai ciri-ciri histology buah kelapa sawit telah dijalankan dengan teknik

histokimia dan mikroskopi cahaya. Pembentukan endosperma kelapa sawit bermula

pada 2 minggu selepas antesis (‘weeks after anthesis’, WAA) di mana endosperma

adalah dalam bentuk cecair. Pembesaran endosperma terus berlaku seiring dengan

perkembangan biji benih. Pengeselan berlaku pada tahap 8 WAA dan pertumbuhan

sel-sel mula dilihat pada bahagian bawah endokarp. Sel-sel di sekeliling endosperma

berkembang ke arah bahagian tengah endosperma. Endosperma yang matang

mengandungi banyak simpanan zat makanan yang sangat diperlukan untuk

pertumbuhan embrio.

Kajian mengenai komposisi biokimia dalam buah kelapa sawit yang sedang

berkembang telah dianalisis dengan teknik kromatografi dan spektroskopi atom.

Dalam kedua-dua bahan kajian yang digunakan iaitu ‘tenera’ and ‘clonal’, asid

glutamik merupakan asid amino yang dominan pada setiap tahap perkembangan

endosperma dan diikuti dengan asid aspartik dan arginina. Ini menunjukkan bahawa

asid glutamik, asid aspartik dan arginina ialah blok binaan utama dalam protin

isirong. Pada tahap awal perkembangan biji benih, nisbah heksosa/sukrosa yang

tinggi dalam ‘tenera’ (57.85) dan ‘clonal’ (250.23) telah dikenalpasti di mana ianya

adalah penting bagi memenuhi keperluan metabolik yang tinggi untuk proses

pembahagian dan pembezaan sel. Namun, nisbah tersebut telah berubah pada tahap

10 WAA bagi kedua-dua ‘tenera’ dan ‘clonal’ (0.80 dan 1.53, masing-masing), dan

ini menandakan bermulanya fasa pematangan endosperma. Analisis asid lemak pula

menunjukkan bahawa asid laurik adalah asid lemak yang paling banyak dijumpai

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dalam endosperma kelapa sawit yang matang bagi kedua-dua ‘tenera’ (54%) dan

‘clonal’ (51%).

Unsur-unsur mineral mikro dan makro adalah penting untuk pertumbuhan pokok.

Jumlah kalsium dan mangan yang tinggi telah didapati pada tahap 5 WAA.

Kandungan mangan dalam endosperma ‘tenera’ dan ‘clonal’ masing-masing ialah 68

mg/100g berat kering dan 158 mg/100g berat kering. Manakala, kandungan kalsium

dalam endosperma ‘tenera’ dan ‘clonal’ ialah 797 mg/100g berat kering dan 6350

mg/100g berat kering. Bagi kedua-dua ‘tenera’ dan ‘clonal’, kandungan kalium (108

dan 751 mg/100g berat kering, masing-masing) dan fosforus (653 dan 695 mg/100g

berat kering, masing-masing) telah berkumpul dalam endosperma yang matang.

Analisis vitamin pula menunjukkan bahawa alfa-tokoferol dan niasin adalah vitamin

utama yang ditemui dalam kedua-dua endosperma kelapa sawit.

Secara keseluruhannya, kajian anatomi and zat makanan endosperma kelapa sawit

telah membekalkan maklumat mengenai perubahan selular dan keperluan nutrien

untuk perkembangan embrio zigotik. Pengetahuan ini amatlah berguna dalam

pengoptimuman media kultur tisu kelapa sawit supaya kecekapan embriogenesis

somatik dapat ditingkatkan.

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ACKNOWLEDGEMENTS

I would like to acknowledge and thank to my supervisor, Dr. Parameswari for giving

me the opportunity to participate in this project and for her guidance and advice

throughout the course of this project. Sincere appreciation also goes to my

supervisory committee members, Prof. Gwendoline and Dr. Meilina for the use of

their facilities and I am deeply grateful to them for their valuable comments and

suggestions. Their patience and constant encouragements during the course of my

research was instrumental to the completion of this thesis.

Special thanks are given to the Advanced Biotechnology and Breeding Centre,

Malaysian Palm Oil Board (MPOB) for generously providing the oil palm fruit

samples, without which the project would not have made it this far. My deepest

appreciation goes to En. Halim for his patience in explaining the use of HPLC, and

also to Pn. Norlinawati and Kak Ija for guiding me in the use of the GC at the

Department of Food Science, UPM. My appreciation to Madam Choo for teaching

and guiding me on mineral analysis at the Department of Chemistry, UPM. I would

also like to thank Kak Ros (MPOB) for helping and sharing her knowledge on

histological techniques with me. I would also like to thank Dr. Lai (UPM) for kindly

sharing her knowledge on HPLC with me, Dr. Ooi (MPOB) for guiding me in the

histological study and Dr Lim (UM) for sharing her knowledge on seed anatomy

with me.

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I wish to thank En. Rizal for his assistance in freeze drying the samples at the

Institute of Bioscience, UPM. I also would like to express my gratitude to the staff at

the Department of Food Science (UPM), En. Azman, Abang Zul, Kak Wati, Kak Ija

and Pijar for all their help and advice that has enabled me to carry out my laboratory

work. Thanks to the staff of MPOB for helping in sampling the oil palm fruits.

Special thanks to Dr. Puad (UPM) for the use of the HPLC and Dr. Tan Yean Ping

(UPM) for the use of her lab.

I wish to thank the Ministry of Science, Technology and Innovation (MOSTI) for the

Agriculture eScience Grant that funded this project. I would like to acknowledge the

financial support given to me by University Putra Malaysia under the Graduate

Research Fellowship.

A special note to my friend, Yee Wen for her encouragement, care and contribution

of precious memories during my years of study. To Chong Boon, Sui Fong, Wendy

Yeo, Hui Yin, Swi See, Ikin, Seow Ling, Conie, Syin Ying, Vasagi, Jian Hou and

Lok, thanks a million for the friendship, assistance and support that you have

provided to me.

Lastly, I want to express my profound thanks to my beloved parents and siblings who

have always provided their continuous support and care throughout my study.

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I certify that a Thesis Examination Committee has met on 14 June 2011 to conduct

the final examination of Kok Sau Yee on her thesis entitled “Histological and

Biochemical Characterization of Oil Palm (Elaies guineensis Jacq.) Endosperm

During Fruit Development” in accordance with Universities and University College

Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15

March 1998. The committee recommends that the candidate be awarded Master of

Sciencce.

Members of the Examination Committee were as follows:

Lai Oi Ming, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Mohd Arif Syed, PhD

Professor



(Internal Examiner)

Janna Ong Abdullah, PhD

Associate Professor



(Internal Examiner)

Sharifah Shahrul Rabiah Syed Alwee, PhD

FELDA Biotechnology Centre

FELDA Agricultural Services SDN BHD

Malaysia

(External Examiner)

______________________________

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies


Date: 23 August 2011

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

PARAMESWARI a/p NAMASIVAYAM, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Science


(Chairman)

GWENDOLINE EE CHENG LIAN, PhD

Professor

Faculty of Science


(Member)

MEILINA-ONG ABDULLAH, PhD

Advanced Biotechnology and Breeding Centre

Malaysia Palm Oil Board

(Member)

________________________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies


Date:

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I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or other

institutions.

_____________________

KOK SAU YEE

Date: 14 June 2011

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL x

DECLARATION xii

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF ABBREVIATIONS xviii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1 Sexual Plant Reproduction 4

2.1.1 Embryo Development 7

2.1.2 Endosperm Development 10

2.2 Oil palm – An Introduction 13

2.2.1 Economic Importance of Oil Palm 17

2.2.2 The Inflorescences and Flowers of

Oil Palm 18

2.2.3 Oil Palm Fruit and Its Development 21

2.3 Vegetative Propagation of Oil Palm 23

2.4 Accumulation of Storage Reserves in Seed 25

2.5 Chemical Analytical Method for Seed Nutrient 28

2.5.1 Chromatographic Technique 28

2.5.2 Atomic Spectroscopic Technique 32

3 MATERIALS AND METHODS 33

3.1 Sample Collection 33

3.2 Sample Preparation 34

3.3 Proximate Analysis 34

3.3.1 Moisture Content 35

3.3.2 Ash Content 35

3.3.3 Crude Fat 36

3.3.4 Crude Protein 37

3.4 Amino Acid Analysis 38

3.4.1 Acid Hydrolysis 38

3.4.2 Derivatization 39

3.4.3 Chromatographic Condition 40

3.5 Sugar Analysis 41

3.5.1 Extraction Procedure 41

3.5.2 Cleanup Procedure 42


3.6 Fatty Acid Analysis 43

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3.6.1 FAME Preparation 43


3.7 Mineral Analysis 44

3.7.1 Wet Digestion 44

3.7.2 Analytical Method 45

3.8 Fat Soluble Vitamin Analysis 46

3.8.1 Extraction Procedure 46


3.9 Water Soluble Vitamin Analysis 48

3.9.1 Acid and Enzyme Hydrolysis 48


3.10 Histological Study 50

3.10.1 Fixation 50

3.10.2 Infiltration and Embedding 50

3.10.3 Sectioning and Staining 51

3.10.4 Microscopy 52

3.11 Statistical Method 52

4 RESULTS 53

4.1 Histological Characterization of Fruit

Development 53

4.2 Biochemical Characterization of Fruit

Development 66

4.3 Amino Acid Composition 70

4.4 Change of Sugar Content 75

4.5 Fatty Acid Distribution 80

4.6 Mineral Composition 82

4.6.1 Microelement 82

4.6.2 Macroelement 86

4.7 Vitamin Content 89

4.7.1 Fat Soluble Vitamin 89

4.7.2 Water Soluble Vitamin 92

5 DISCUSSION 94

5.1 Fruit Development 94

5.2 Amino Acid Metabolism 98

5.3 Soluble Carbohydrate Content during Fruit

Development 99

5.4 Fatty Acid Composition during Fruit

Development 102

5.5 Mineral Requirement in Fruit Development 104

5.6 Accumulation of Vitamin Content 109

6 CONCLUSIONS 113

REFERENCES 115

APPENDICES 139

BIODATA OF STUDENT 148

LIST OF PUBLICATION 149

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