universiti putra malaysia development and …psasir.upm.edu.my/5665/1/a_fbsb_2009_20.pdf ·...
Post on 03-Mar-2019
225 Views
Preview:
TRANSCRIPT
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
ii
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.
iii
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
iv
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.
v
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
vi
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.
vii
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
viii
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
ix
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
xv
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
xvi
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
top related