UNIVERSITI PUTRA MALAYSIA

DEVELOPMENT AND APPLICATION OF EXPRESSED SEQUENCE TAGS AND DNA MICROARRAY FOR SOMATIC

EMBRYOGENESIS IN OIL PALM

LESLIE LOW ENG TI

FBSB 2009 20

DEVELOPMENT AND APPLICATION OF EXPRESSED SEQUENCE TAGS AND DNA MICROARRAY FOR SOMATIC

EMBRYOGENESIS IN OIL PALM

By

LESLIE LOW ENG TI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements

for the Degree of Doctor of Philosophy

February 2009

i

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirements for the degree of Doctor

of Philosophy

DEVELOPMENT AND APPLICATION OF EXPRESSED

SEQUENCE TAGS AND DNA MICROARRAY FOR SOMATIC EMBRYOGENESIS IN OIL PALM

By

LESLIE LOW ENG TI

February 2009

Chairman : Professor Raha Abdul Rahim, PhD Faculty : Biotechnology and Biomolecular Sciences Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing

crops in the world. However, genetic improvement of oil palm through

conventional breeding is extremely slow and costly, as the breeding cycle

can take up to 10 years. This has brought about interest in vegetative

propagation of oil palm. Since the introduction of oil palm tissue culture in

the 1970s, clonal propagation has proven to be useful in producing uniform

planting materials. However, despite considerable progress in improving the

tissue culture techniques, the callusing and embryogenesis rates from

proliferating callus cultures remain very low. Thus, understanding the gene

diversity and expression profiles during somatic embryogenesis is critical in

increasing the efficiency of these processes. To achieve this, a total of six

standard cDNA libraries, representing three developmental stages (non-

embryogenic callus, embryogenic callus and embryoids) in oil palm tissue

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culture, were generated in this study. Random sequencing of clones from

the embryogenic callus cDNA libraries generated 2,716 expressed sequence

tags (ESTs). These ESTs were combined with 14,883 ESTs available in

MPOB’s EST programme. The 17,599 ESTs were analysed, annotated and

assembled to generate 9,584 putative unigenes distributed in 3,268

consensi and 6,316 singletons. These unigenes were assigned putative

functions based on similarity and gene ontology annotations. A subset of

these ESTs were selected and spotted on cDNA microarrays. Both the EST

and microarray data analysis were able to identify expression profiles that

could differentiate non-embryogenic callus from embryogenic samples. The

in silico EST data analysis identified 52 unigenes that showed potential to be

developed as candidate markers for embryogenesis. The microarray

experiment identified 76 unigenes that could differentiate non-embryogenic

callus from embryogenic callus, embryoids and shoots from polyembyoids.

The EST and microarray data analysis revealed that lipid transfer proteins

were highly expressed in embryogenic tissues. The results also showed that

glutathione S-transferases were highly expressed in non-embryogenic

callus. This study has provided an overview of genes expressed during oil

palm tissue culture and real-time PCR analysis identified four genes

(pOP-EA00703, pOP-EA01249, pOP-EA01117, pOP-SFB01045) that had

the potential to be developed as molecular markers for embryogenesis.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PEMBANGUNAN DAN APLIKASI PENANDA JUJUKAN TERUNGKAP AND DNA MIKROATUR DALAM KAJIAN

EMBRIOGENESIS SOMATIK KELAPA SAWIT

Oleh

LESLIE LOW ENG TI

February 2009

Pengerusi : Profesor Raha Abdul Rahim, PhD Fakulti : Bioteknologi dan Sains Biomolekul

Kelapa sawit (Elaeis guineensis Jacq.) merupakan salah satu tanaman

penghasil minyak yang terpenting di dunia. Proses pembaikan genetik kelapa

sawit melalui kaedah konvensional mengambil masa yang lama dan

memerlukan kos yang tinggi kerana satu kitaran pembiak-bakaan mengambil

masa sehingga 10 tahun. Faktor ini telah mengalih perhatian industri kelapa

sawit kepada proses propagasi vegetatif. Semenjak penggunaan proses

kultur tisu kelapa sawit pada tahun 1970an, propagasi klonal didapati

berkesan dalam penghasilan pokok kelapa sawit yang seragam. Walaupun

pelbagai penyelidikan telah dijalankan dalam pembaikan teknik proses kultur

tisu kelapa sawit, kadar penghasilan kalus dan embriogenesis masih rendah.

Oleh itu, pemahaman terhadap kepelbagaian dan profil pengekspresan gen

semasa embriogenesis somatik adalah kritikal untuk meningkatkan

kecekapan proses tersebut. Untuk mencapai objektif ini, sebanyak enam

perpustakaan cDNA daripada tiga peringkat perkembangan (kalus tidak

embriogenik, kalus embriogenik dan embriod) proses kultur tisu telah

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dihasilkan. Penjujukan klon secara rawak daripada perpustakaan cDNA

kalus embriogenik telah menghasilkan 2,716 penanda jujukan terungkap

(EST). Jujukan-jujukan EST ini telah digabungkan dengan 14,883 jujukan

EST yang terdapat di bawah program EST MPOB. Analisa pergabungan

17,599 jujukan EST menemui 3,268 jujukan konsensi dan 6,316 singleton.

Penentuan fungsi putatif ke atas unigen tersebut adalah berdasarkan anotasi

kesamaan dan ontologi gen. Sebahagian daripada koleksi EST ini juga telah

dicetak di atas mikroatur DNA. Analisa data EST dan mikroatur telah

mengenalpasti profil pengekspresan gen yang dapat membezakan kalus

tidak embriogenik daripada sampel-sampel embriogenik. Bardasarkan

analisa data ‘in silico’ EST, sebanyak 52 gen mempunyai potensi untuk

dibangunkan sebagai penanda molekul bagi proses embriogenesis.

Eksperimen mikroatur pula menemui 76 gen yang dapat membezakan kalus

tidak embriogenik daripada kalus embriogenik, embriod dan pucuk daripada

poliembriod. Analisa ‘in silico’ EST dan DNA mikroatur menunjukkan bahawa

pengekspresan protein pemindah lipid adalah tinggi di dalam tisu

embriogenik. Manakala glutation S-transferase menunjukkan pengekspresan

yang tinggi di dalam kalus tidak embriogenik. Kajian ini telah memberikan

gambaran menyeluruh mengenai gen-gen yang diekspres semasa kultur tisu

kelapa sawit. Teknik ‘real-time PCR’ telah mengenalpasti empat gen

(pOP-EA00703, pOP-EA01249, pOP-EA01117, pOP-SFB01045) yang

mempunyai potensi untuk dibangunkan sebagai penanda molekul bagi

proses embriogenesis.

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ACKNOWLEDGEMENTS

I wish to express my sincere appreciation and heartfelt gratitude to

Prof. Dr. Raha Abdul Rahim, Assoc. Prof. Dr. Ho Chai Ling, Dr. Cheah Suan

Choo and Dr. Sharifah Shahrul for their invaluable advice and guidance

throughout the course of this project as my supervisors. I am truly indebted

and thankful to Rajinder for his ideas, support and help during the course of

my research in MPOB. I am also forever grateful to Dr. Meilina Ong, Leslie

Ooi and Chan Pek Lan for the long winded and often confusing discussions,

as well as their ability to survive the process of reviewing my thesis. I will

‘bug’ you no more. My deepest gratitude is also extended to Dr. Ravigadevi,

Dr. Umi, Dr. Arif, Dr. Maizura, Dr. Maria, Dr. Tarmizi, En. Zamzuri, Dr. Ooi

Siew Eng, Ms. Rohani, Rozana, Boon Soo Heong, Ma Lay Sun, Wong Yin

Ling, Halimah, Elyana, Chua Kia Ling, Maslinda and Lee Weng Wah for

contributing in many different ways. To Mdm Wooi (formerly of Ebor

Laboratories), Pn. Suzaini (Ebor Laboratories), Mr. Aw Khoo Teng (Felda)

and En Zamzuri (MPOB), your support in this research programme by

supplying the tissue culture material is truly appreciated. I also wish to thank

the Ministry of Science, Technology and the Environment (MOSTE) for

funding the EST portion of the research, under the Malaysia-MIT

Biotechnology Partnership Programme (MMBPP) and the Board of MPOB for

funding the DNA microarray experiments under the DNA Chip Programme.

A personal note of thanks to Dr. Cheah, Rajinder and Leslie Ooi for their

commitment to educate me on the finer points of research and for not

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causing me bodily harm when I was learning the tricks of the trade by doing

everything I was not supposed to do. Also to all the wonderful people

(Rahimah, Hilda, En. Jamil, Shikin, Faizun, Razali, Zul, Mat Nor, Mat Shari,

Norjihan and etc.) that keep the veins of research alive, your contributions

are truly amazing. Special thanks to Amos, Wee Teng, Grace, Iza, Jay,

Pek Lei, Siew, Mel, Yun Ting, Elyana, Rahaya, Halimah, Ooi, Jane, Leow,

Teh, Mei, Siew Eng, Chee Meng, Chua, Zana, Kia Ling, Soo Heong and

Pek Lan for the wonderful memories and laughter that helped me maintain

my sanity during the trials and tribulations of research. I am glad to have

known all of you. I am also indebted to my ex-UPM mates (Teck Soon, Sia,

Chen, Nyiam, Ah Hee, David, Lilian, Wing Hong) and friends (Teck Yee,

Teck Heng, Tsuey Fen) for their support and understanding. Last but not

least, I am truly grateful and indebted to my family for their understanding

and support, and for helping shape me to become the person I am today.

Their unconditional love will always be in my heart.

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I certify that a Thesis Examination Committee has met on 22th February 2009 to conduct the final examination of Leslie Low Eng Ti on his thesis entitled "Development and Application of Expressed Sequence Tags (ESTs) and DNA Microarray for Somatic Embryogenesis in Oil Palm" in accordance with the Universities and Universiti Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Thesis Examination Committee are as follows:

Tan Soon Guan, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Parameswari A/P Namasivayam, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Puad Abdullah, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Roohaida Othman, PhD Associate Professor Faculty Of Science and Technology Universiti Kebangsaan Malaysia (External Examiner)

_______________________ BUJANG KIM HUAT, PhD Professor/Deputy Dean School of Graduate Studies, Universiti Putra Malaysia

Date: 28 April 2009

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows. Raha Abdul Rahim, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Ho Chai Ling, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Cheah Suan Choo, PhD Chief Scientific Officer Asiatic Centre for Genome Technology (ACGT) (Member) Sharifah Sharul Rabiah Syed Alwi, PhD Senior Manager (Biotechnology) Felda Agricultural Services Sdn. Bhd. (Member)

____________________________ HASANAH MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 14 May 2009

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DECLARATION I hereby declare that the thesis is based on my original work except for quotations, and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

___________________ LESLIE LOW ENG TI

Date: 9 June 2008

x

TABLE OF CONTENTS

Page ABSTRACT ii ABSTRAK iv ACKNOWLEDGEMENTS vi APPROVAL viii DECLARATION x LIST OF TABLES xv LIST OF FIGURES xvii LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS xix

CHAPTER

1 INTRODUCTION 1 2 LITERATURE REVIEW 6

2.1 The Oil Palm 6

2.2 Plant Tissue Culture 9

2.3 Somatic Embryogenesis 10

2.4 Oil Palm Tissue Culture 13

2.5 Plant RNA Extraction 18

2.6 Expressed Sequence Tags (ESTs) 20

2.7 Bioinformatics Tools 22

2.8 Gene Expression Profiling 25

2.9 Microarray Technology 27

2.10 RNA Amplification 33

2.11 Real-time PCR 35

3 ESTABLISHMENT OF ESTS FROM OIL PALM TISSUE CULTURES

40

3.1 INTRODUCTION 40 3.2 MATERIALS AND METHODS 43

xi

3.2.1 Plant Materials 43 3.2.2 RNA Extraction 43 3.2.3 Determination of RNA Quality 46 3.2.4 Poly(A)+ RNA Isolation 47 3.2.5 In vitro Translation 49 3.2.6 SDS-Polyacylamide Gel Electrophoresis (SDS-

PAGE) 50

3.2.7 Construction of cDNA Library 52 3.2.8 Preparation of EST Sequencing Templates 56 3.2.9 EST Sequencing 58 3.2.10 Sequence Analysis 59 3.2.11 In silico Identification Of Simple Sequence

Repeat (SSR) Markers 61

3.2.12 Assignment of GO terms 61 3.3 RESULTS 63

3.3.1 RNA Extraction and mRNA Isolation 63 3.3.2 In vitro Translation 65 3.3.3 cDNA Library Construction 68 3.3.4 Generation of Expressed Sequence Tags (ESTs) 71 3.3.5 Features of ESTs 75 3.3.6 Protein Coding Regions 79 3.3.7 EST-derived Simple Sequence Repeat (SSR)

Markers 84

3.3.8 Gene Ontology Annotation 87 3.3.9 In silico Screening of EST Data 93

3.4 DISCUSSION 98 3.4.1 Characteristics of the Oil Palm Transcriptome 98 3.4.2 Identification of Somatic Embryogenesis-related

Genes 102

4 PROTOCOL OPTIMIZATIONS FOR DNA MICROARRAY

EXPERIMENTS 108

4.1 INTRODUCTION 108 4.2 MATERIALS AND METHODS 110

4.2.1 Plant materials 110 4.2.2 Evaluation of RNA Extraction Protocols 110

xii

4.2.3 Determination of RNA Quality 116 4.2.4 Microarray Experiments 117 4.2.5 Real Time PCR 124 4.2.6 Hybridization of Nucleic Acids 126

4.3 RESULTS 130 4.3.1 RNA Extraction Protocol Evaluation 130 4.3.2 Confirmation Of Modified Palm Protocol In Oil

Palm Tissues 134

4.3.3 RNA Amplification 135 4.3.4 Microarray Validation of Modified Palm Protocol 140 4.3.5 Evaluation of Microarray Data and Normalization

Technique 145

4.3.6 Differential Gene Expression (DGE) 156 4.3.7 Identification of Reference Genes (RGs) 159

4.4 DISCUSSION 165 4.4.1 RNA Extraction Protocol Evaluation 165 4.4.2 Evaluation and Quality Assessment of Modified

Palm Protocol in Microarray Experiments 167

4.4.3 Microarray Data Analysis 169 4.4.4 Identification of Reference Genes for Real-time

PCR Experiments 170

5 DNA MICROARRAY ANALYSIS OF GENE EXPRESSION IN

OIL PALM TISSUE CULTURE 173

5.1 INTRODUCTION 173 5.2 MATERIALS AND METHODS 176

5.2.1 Plant Material 176 5.2.2 Preparation of Total RNA 176 5.2.3 Preparation of Cy3- and Cy5-labeled Targets 177 5.2.4 Microarray Hybridization 177 5.2.5 Microarray Analysis 178 5.2.6 Sequence Analysis 179 5.2.7 Real Time PCR 179

5.3 RESULTS 181 5.3.1 RNA Extraction and Amplification 181 5.3.2 Microarray Experiment 181

xiii

5.3.3 Cluster Analysis and Functional Classifications 187 5.3.4 Real-time PCR Analysis 195

5.4 DISCUSSION 204 5.4.1 Identification of Genes Associated with

Embryogenesis 205

6 GENERAL DISCUSSION AND CONCLUSION 215 REFERENCES 220 APPENDIXES 246 BIODATA OF STUDENT 249 LIST OF PUBLICATIONS 250

xiv

LIST OF TABLES

Table Page

3.1 RNA yields from callus and embryoid tissues 64

3.2 Poly(A)+ RNA yield from callus and embryoid tissues 64

3.3 3H-leucine incorporated into polypeptides after in vitro translation reaction

66

3.4 Characteristics of the cDNA libraries 70

3.5 Sequence analysis of embryogenic callus cDNA libraries 74

3.6 Putative identification of clusters with at least 10 ESTs in the group

76

3.7 Cluster analysis of 17,599 ESTs 78

3.8 Identification of sequences specific to a single tissue 80

3.9 Putative identity of the 20 most abundant sequences present in all three tissues

80

3.10 Codon usage in Elaeis guineensis 83

3.11 Frequency of non-redundant gene-derived SSRs 85

3.12 Distribution of SSRs in putative full-length ORFs 88

3.13 Gene ontology (GO) functional classification for ESTs generated from NEC, EC and EMB cDNA libraries

90

3.14 Distribution of ESTs in NEC, EC and EMB tissue at a significance threshold of 0.001

94

4.1 Total RNA yield and purity of RNA extraction protocols not suitable for LEAF samples

132

4.2 Total RNA yield and purity of RNA extraction protocols suitable for LEAF samples

132

4.3 Total RNA yield from 9 oil palm tissues 136

4.4. aRNA yield amplified from the total RNA of 9 oil palm tissues

138

4.5 Number of probes that hybridized to cDNA from various oil palm tissues

144

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4.6 Evaluation of normalization technique using regression analysis

154

4.7 Comparisons of microarray and reverse northern results 155

4.8 Reproducibility of the microarray data in eight tissues versus LEAF experiments

157

4.9 Number of differentially and non-differentially expressed genes in the microarray experiments

157

4.10 Blastx analysis of eight candidate oil palm reference genes 162

4.11 Primer and probe sequences of eight candidate oil palm reference genes

162

5.1 Total RNA yields and purity of the tissue culture samples 182

5.2 aRNA yields and purity of the tissue culture samples 182

5.3 Expression profile of pOP-EB03029 spotted in replicate on the cDNA microarray

188

5.4 Expression profile of gene clones in 13 consensus sequences

190

5.5 Gene ontology (GO) functional classification of unigenes identified in the SOTA analysis

194

5.6 Blast analysis of gene clones selected for qRT-PCR analysis

197

5.7 Primer and probe sequences of gene clones selected for qRT-PCR analysis

197

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LIST OF FIGURES

Figure Page

2.1 History and developments of the Deli dura in Indonesia and Malaysia till 1979

8

2.2 A schematic overview of the stages in somatic embryo development

12

2.3 Flow chart of oil palm tissue culture process 16

2.4 Overview of DNA microarray experiment 30

3.1 Gel analysis of total RNA from callus and embryoid samples

64

3.2 In vitro translation products of mRNA isolated from EC, NEC and EMB analyzed by SDS-PAGE

67

3.3 Insert size distribution of clones isolated from the CEO and CEM cDNA libraries

72

3.4 Distribution of ESTs in the EC library clusters 76

3.5 Distribution of consensi in cDNA libraries 81

3.6 The percentage distribution of the different SSR motifs (mono-, di-, tri-, tetra- and pentanucleotide)

86

3.7 Hierarchical clustering of normalized EST distribution in a set of 52 consensi

96

4.1 Gel electrophoresis of RNA samples 133

4.2 The gel image and electropherogram of RNA from 9 oil palm tissues

137

4.3 The gel image and electropherogram of aRNA from 9 oil palm tissues

139

4.4 Scan image of an EC versus LEAF dye-swap experiment 141

4.5 cDNA microarrays hybridized with Cy5-labeled targets of nine oil palm tissues

142

4.6 Estimation of limit of detection for the oil palm microarray 146

4.7 Screenshots of the Input, Output and Quality Assessment worksheets in MEV Converter

149

xvii

4.8 Scatter plots of log expression ratio versus log intensity of datasets from a dye-swap experiment

151

4.9 Reverse northern analysis in dot blot format of 35 gene clones selected from the microarray experiments

154

4.10 Box plot of Ct values of the eight candidate oil palm reference genes

163

4.11 Average expression stability values of eight candidate reference genes

164

4.12 Determination of the optimal number of RGs for normalization

164

5.1 Interactive plot of the SAM analysis in TMeV 185

5.2 Hierarchical clustering of tissue culture samples 186

5.3 SOTA analysis of 118 gene clones 189

5.4a Microarray and qRT-PCR expression profile of pOP-EA00703 and pOP-EA01249 in three sets of tissue culture samples

198

5.4b Microarray and qRT-PCR expression profile of pOP-EA01637 and pOP-EA01117 in three sets of tissue culture samples

199

5.4c Microarray and qRT-PCR expression profile of pOP-EA02220 and pOP-EA03463 in three sets of tissue culture samples

200

5.4d Microarray and qRT-PCR expression profile of pOP-G00052 and pOP-SFB01045 in three sets of tissue culture samples

201

xviii

ABBREVIATIONS % Percentage

α Alpha

β Beta

λ Lambda

°C Degree Celsius

μg Microgram

μl Microliter

μM Micromolar

A Adenine

ABA Abscisic Acid

AFLP Amplified Fragment Length Polymorphism

AGL15 Agamous-like 15

ANOVA Analysis of Variance

aRNA antisense RNA

ASP Automated Slide Processor

BBM Baby Boom

BLAST Basic Local Alignment Search Tool

bp Base Pair

C Cytosine

cDNA complementary DNA

Ci Curie

cm Centimetre

CTAB Cetyltrimethylammonium Bromide

cps Counts Per Second

xix

Ct Threshold Cycle

Cy3 Cyanine 3

Cy5 Cyanine 5

D x P Dura x Pisifera

dATP 2'-deoxy-adenosine-5'-triphosphate

dCTP 2'-deoxy-cytidine-5'-triphosphate

DEPC Diethyl Pyrocarbonate

DGE Differential Gene Expression

dGTP 2'-deoxy-guanosine-5'-triphosphate

dH2O Deionized Water

DMSO Dimethylsulphonyl Oxide

DNA Deoxyribonucleic Acid

DNase 1 Deoxyribonuclease 1

dNTP Deoxynucleotide Triphosphates

dTTP 2'-deoxy-thymidine-5'-triphosphate

EC Embryogenic Callus

EDTA Ethylenediaminetetraacetatic Acid

EGTA Ethylene glycol bis-(β-aminoethylene ether)

EMB Embryoid

EC/EMB Embryogenic Cultures (Embryogenic Callus and

Embryoid)

ERE Ethylene Responsive Element

ESTs Expressed Sequence Tags

EtBr Ethidium Bromide

FDR False-Discovery Rate

xx

Flourophores Fluorescent Dyes

FRET Fluorescence Resonance Energy Transfer

FUS3 Fusca3

g Gram

G Guanine

GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase

GO Gene Ontology

GSH Tripeptide glutathione

GST Glutathione S-transferase

GUI Graphical User Interface

H2O2 Hydrogen Perokside

HCl Hydrochloride Acid

hr Hours

i.e. that is

INF Inflorescence

IPTG Isopropyl-β-D-thiogalactoside

Jacq. Jacquin

K Potassium

k Kilo

kb Kilobase

kDA Kilodalton

KOH Potassium Hydroxide

L Liter

LB Luria Bertani

LEA Late Embryogenesis Abundant

xxi

LEAF Spear Leaf

LEC1 Leafy Cotyledon 1

LEC2 Leafy Cotyledon 2

LiCl Lithium Chloride

LOWESS Locally Weighted Scatterplot Smoothing

LUS Lucidea™ Universal ScoreCard™

M Molar

Mb Megabase

MES Mesocarp

MgCl2 Magnesium Chloride

MgSO4 Magnesium Sulphate

MIDAS TIGR Microarray Data Analysis System

min Minute

mL Milliliter

mm Millimeter

mM Millimolar

MMLV-RT Maurine Moloney Leukemia Virus Reverse Transcriptase

mmol Millimole

MPOB Malaysia Palm Oil Board

mRNA Messenger RNA

MT Metallothionein

mW Milliwatt

NaCl Sodium Chloride

NaOAc Sodium Acetate

NaOH Sodium Hydroxide

xxii

NEC Non-Embryogenic Callus

ng Nanogram

nt Nucleotide

nr Non-Redundant Protein

O2 Oxygen

OD Optical density

ORF Open Reading Frame

PAGE Polyacrylamide Agarose Gel Electrophoresis

PAS p-aminosalicylic acid

PCR Polymerase Chain Reaction

PEG Polyethylene Glycol

pfu Plaque Forming Unit

pmol Picomole

Poly(A)+ RNA Polyadenylated Rna

PORIM Palm Oil Research Institute Of Malaysia

PPO 2,5-Diphenyloxazole

PVP Polyvinylpyrrolidone

PVPP Polyvinylpolypyrrolidone

QTL Quantitative Trait Loci

qRT-PCR Quantitative Real-Time PCR

R-I plot Ratio-Intensity Plot

RFLP Restriction Fragment Length Polymorphism

RFU Relative Fluorescent Units

RGs Reference Genes

RN Reverse Northern

xxiii

RNA Ribonucleic Acid

RNase Ribonuclease

ROS Reactive O2 Species

rRNA Ribosomal RNA

RT Room Temperature

SAGE Serial Analysis Of Gene Expression

SAM Significance Analysis of Microarray

SAP Shrimp Alkaline Phosphatase

sarkosyl Sodium N-lauroyl sarcosine

SD Standard Deviation

SDS Sodium Dodecyl Sulphate

sec Seconds

SERK Somatic Embryogenesis Receptor Kinase

SNPs Single Nucleotide Polymorphisms

SOD Superoxide Dismutase

SOTA Self Organisation Tree Algorithm

SSR Simple Sequence Repeat

LEAF Spear Leaf

SSC Sodium Saline Citrate

SSPE Saline Sodium Phosphate EDTA

SSR Simple Sequence Repeat

ST Shoot from polyembryoids

STE Sodium-Tris-EDTA

T Thiamine

TAE Tris-Acetate-EDTA

xxiv