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UNIVERSITI PUTRA MALAYSIA
GENERATION OF EXPRESSED SEQUENCE TAGS (ESTs) FROM NON-EMBRYOGENIC CALLUS cDNA LIBRARIES OF OIL PALM
(ELAEIS GUINEENSIS JACQ.)
AMOSTAN CHI YEE
FSMB 2003 10
GENERATION OF EXPRESSED SEQUENCE TAGS (ESTs) FROM NON-EMBRYOGENIC CALLUS cDNA LmRARIES OF OIL PALM
(ELAEIS GUINEENSIS JACQ.)
By
AMOS TAN CHI YEE
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of
Master of Science
July 2003
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science
GENERATION OF EXPRESSED SEQUENCE TAGS (ESTs) FROM NONEMBRYOGENIC CALLUS cDNA LIBRARIES OF OIL PALM
(ELAEIS GUlNEENSIS JACQ.)
By
AMOS TAN CHI VEE
Chairman: Dr. Tan Siang Hee
July 2003
Faculty Food Science and Biotechnology
Due to the economic importance of tissue culture in oil palm, it is desirable to
understand the chemical characteristics and molecular changes associated with
callogenesis and embryogenesis. Therefore partial sequencing of randomly selected
complementary DNA (cDNA) clones was carried out to investigate and understand the
gene expression pattern during non-embryogenic callus in oil palm. A total of 2,296
clones from cDNA libraries constructed using messenger RNA (mRNA) from non-
embryogenic callus tissues have been partially sequenced to generate expressed sequence
tags (ESTs). The comparison of the coding capacity of these 1 ,832 ESTs with the non-
redundant sequences in the National Center for Biotechnology Information (NCBI)
databases resulted in assignment of putative function to 971 (53.0%) of the ESTs. The
remaining 861 (47.0%) were considered as novel genes. Hybridization of the 80 selected
cDNA clones to 32P-Iabelled first strand cDNA probes synthesized from the total mRNA
of NEC was compared with probes prepared from mRNA of leaves. A total of 12 clones,
which hybridized predominantly to the NEC but not to the leaf probe were selected for
Northern analysis. The Northern analysis showed that 9 out of the 1 2 ESTs clones
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selected were expressed predominantly in the callus tissue. However, carrying out a
sequence comparison using the BLAST algorithm, only l out of the 9 clones had
significant homology to a known gene, glutathione-S-transferase (GST) gene. The
remaining 8 unidentifiable ESTs were then subjected to a motif search using the Blocks
IMProved Searcher (BLIMPS) program. Based on the search results, 4 different motifs
were identified to be related to the 8 ESTs and they are either involved in developmental
regulation or protein storage function. The results presented here therefore demonstrate
the effectiveness and efficiency of the EST strategy in gene discovery in the oil palm
callus tissue.
iv
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
PENJANAAN PENANDA JUJUKAN TEREKSPRES (ESTs) DARIP ADA KALUS TAK-EMBRIOGENIK PERPUSTAKAAN eDNA POKOK SAWIT
(ELAEIS GUlNEENSIS JACQ.)
Oleh
AMOS TAN CHI YEE
Pengerusi : Dr. Tan Siang Hee
Julai 2003
Fakulti Sains Makanan dan Bioteknologi
Berdasarkan kepada kepentingan ekonomi kultur tisu pokok sawit, adalah
menjadi satu keperluan untuk memahami pertukaran eiri-eiri kimia dan molekul yang
berkaitan dengan proses kalogenesis dan embriogenesis. Justeru itu, penjujukan-separa
klon-klon DNA pelengkap (eDNA) yang dipilih seeara rawak telah dijalankan bagi
memahami paten pengekspresan gen-gen pada kalus tak embriogenik (NEe) pokok
sawit. Perpustakaan eDNA telah dibina menggunakan RNA pengutus (mRNA) daripada
kalus tak embriogenik. Sebanyak 2,296 klon daripada perpustakaan eDNA telah
dijujukkan untuk mengenalpasti penanda jujukan terekspres (EST). Perbandingan
kapasiti pengkodan 1,832 EST dengan jujukan tak redundan di pangkalan data National
Center for Biotechnology Information (NCBI) telah mrngenalpasti 917 (53.0%) EST
mempunyai fungsi putatif Baki 861 (47.0%) EST dianggap sebagai gen-gen novel.
Sebanyak 80 eDNA klon yang telah terpilih dihibridisasi kepada prob eDNA dimana
bebenang pertamanya dilabel dengan 32p. Prob eDNA disintesis daripada mRNA NEC
dan dibandingkan dengan prob-prob yang dihasilkan daripada mRNA daun. Sejurnlah 12
klon telah menghibridisasi seeara dominan kepada NEC tetapi tidak kepada prob-prob
daun yang telah dipilih untuk analisis Northern. Analisis Northern menunjukkan 9
v
daripada 12 klon EST mengekspres secara dominan dalam tisu kalus. Bagaimanapun,
perbandingan jujukan menggunakan algoritma BLAST menunjukkan hanya 1 daripada 9
klon menpunyai homologi yang signifikan kepada gen yang dikenali iaitu gen
glutathione-S-transferase. Lapan EST yang tidak dapat dikenalpasti telah dianalisis
menerusi pencarian motif menggunakan program BLIMPS (Blocks IMProved Searcher).
Berdasarkan kepada keputusan yang diperolehi, 4 motif yang berlainan telah dikenalpasti
mempunyai kaitan dengan 8 EST tersebut. Motif-motif tersebut terlibat sarna ada dalam
fungsi regulasi pengembangan atau penyimpanan protein. Keputusan-keputusan yang
diperolehi menunjukkan keberkesanan strategi EST bagi penemuan gen-gen dalam kalus
pokok sawit.
vi
ACKNOWLEDGEMENTS
I wish to express my sincere appreciation and heartfelt gratitude to Dr. Tan Siang
Hee, Dr. Cheah Suan Choo, Dr. Sharifah Shahrul and Dr. Suhaimi Napis for their
invaluable advice and guidance throughout the course of this project as my supervisors.
I'm indebted to Mr. Rajinder for his ideas, support and unconditional help that surpass
his responsibility as a group leader. Furthermore I'm blessed to have him as a friend who
shares the same 'highly evolved' and 'God-forbid' sense humour as mine. I'm also
forever grateful to Dr. Meilina Ong (fondly known as Mei) for literally handing over to
me 'her bible of knowledge' that was actually the nucleus of this 'Pulp Fiction' of mine.
My appreciation also extends to Dr. Ravigadevi, Dr. Arif, Dr. Maizura, Dr. Siti, Dr.
Maria and Dr. Tarmizi for contributing in many different ways. To Mdm Wooi (formerly
at Ebor Laboratories) and Pn. Suzaini (Ebor Laboratories), the deepest appreciation for
their constant supply of oil palm callus and embryoid tissue, without which, this project
would not be progressing as smoothly as it did. I also wish to thank the Ministry of
Science, Technology and the Environment (MOSTE) for funding this research, under the
Malaysia-MIT Biotechnology Partnership Programme (MMBPP).
An extra thanks also goes out to Dr. Cheah for employing me in the first place
under the MMBPP programme. It was there that I spent a memorable 2 'l2 years of my
life with a bunch of outstanding 'rebels', who I now consider my "Band of Brothers"
(Kia Ling, Ah Ti, Andy, CK, Tuck Fatt, Jihan) and "Sisters" (Halimah, Elyana, Soo
Heong, Pek Lan, Mel, Shah, Jane, Nina, Emily & Noni). Many of you shared your life's
colourful portraits, technical expertise, and resources that I'll be indebted to forever. I'm
also fortunate to have the initial 'jump-start' provided by the lovely tissue culturist
upstairs (Mei, Dr. Sharifah, Siew Eng, Parames & Ayu) with the classification of my
Vll
EST clones. I'm also blessed to have friends like Weng Wah (bioinformatics guru), Kia
Ling and Chieh Wen (blotting wizards) who provided me FOC with their highly sought
after skills and advice, without which my project would have taken virtually forever to
complete.
The unsung heroes, the ARO's (Ms. Leslie Ooi, Ms. Rahimah, En. Jamil" &
Faizun) the RA's under the genomics group (particularly, Siti, Abang Cat, Abang Zul,
Mat Nor, Mat Shari, Kak. Jabariah, Ayu, Shikin, and etc.) and the administrative staff
(Kak. Wai, Rahaya and etc) who helped me in more ways than one that would be almost
impossible to catalog them all. Thank you all for also sharing some colourful moments of
your life with me while I was constantly waiting for the PCR, centrifuge or the vacuum
concentrator to finish processing my samples. Special thanks also to my housemates
(Chen, Leslie, Andy) for safe-guarding my earthly processions and biological organisms
(my feng-shui goldfishes) to the best of their abilities while I was living out of my
Samsonite cabin luggage (55x40x20cm) for the last 11 months. Thank you guys also for
making sure that I'm always well fed and handling the administrative matters associated
with the 'twilight zone policies' of our graduate school. What success that I have today I
owe a great deal to my family and my friend, Yee Joo who had to sacrifice her many
weekends to accompany me to the laboratory for which the boredom would almost be
unbearable without a good-spirited person like her. Last but not the least, I would like to
thank my better half, Yoke Fah for being there when I needed it most and sacrificing so
much for so little. Thank you all for your support and unconditional love.
viii
I certify that an Examination Committee met on 18th June 2003 to conduct the final examination of Amos Tan Chi Yee on his Master of Science thesis entitled "Generation of Expressed Sequence Tags (ESTs) from Non-embryogenic Callus cDNA Libraries of Oil Palm (Elaeis guineensis Jacq.)" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:
MICHAEL WONG VUI LING, Ph.D., Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Chairman)
TAN SIANG BEE, Ph.D., Lecturer Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)
SUHAlMI NAPIS, Ph.D., Assoc. Professor/ Deputy Director Institute Multimedia and Software Universiti Putra Malaysia (Member)
CHEAH SUAN CHOO, Ph.D., Head Advanced Breeding & Biotechnology Centre Biology Division Malaysia Palm Oil Board (Member)
SHARIFAH SHARUL RABIAH SYED AL WI, Ph.D., Senior Research Officer Advanced Breeding & Biotechnology Centre Biology Division Malaysia Palm Oil Board (Member)
.-rJCL.LI."LC1I..T ALI, Ph.D. ProfessorlDeputy an School of Graduate Studies, Universiti Putra Malaysia
4 SEP 2003 Date:
ix
This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as partial fulfillment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follows.
TAN SIANG HEE, Ph.D., Lecturer Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Chairman)
SUHAIMI NAPIS, Ph.D., Assoc. Professor/ Deputy Director Institute Multimedia and Software Universiti Putra Malaysia (Member)
CHEAH SUAN CHOO, Ph.D., Head Advanced Breeding & Biotechnology Centre Biology Division Malaysia Palm Oil Board (Member)
SHARIFAH SHARUL RABIAH SYED ALWI, Ph.D., Senior Research Officer Advanced Breeding & Biotechnology Centre Biology Division Malaysia Palm Oil Board (Member)
x
,e �r J�' #
AINI IDERIS, Ph.D., Professor/Dean, School of Graduate Studies, Universiti Putra Malaysia
Date: :1 6 SEP 2003
DE CLARA TION
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
AMOS TAN em VEE
xi
DEDICATION ABSTRACT
TABLE OF CONTENTS
ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS
CHAPTER
1 INTRODUCTION
Page 11
111
V Vll IX Xl
XIV XV XVI
1
2 LITERATURE REVIEW 3 2.1 The Oil Palm 3
2.1.1 Origin and Distribution 3 2.1.2 Botany 4
2.1.2.1 Classification 4 2.1.2.1 Morphology 5
2.3 The Significance of Clonal Propagation of Oil Palm 6 2.4 Tissue Culture of Oil Palm 7
2.4.1 Tissue Culture Process 7 2.4.2 Ortet Selection 8 2.4.3 Stages of Oil Palm Tissue Culture 10 2.4.4 Morphogenesis in Oil Palm Tissue Culture 12
2.4.4.1 Embryogenic Callus (Ee) vs Non-embryogenic Callus (NEC) 14
2.4.4.2 Histology of EC and NEC 15 2.4.4.3 Scanning Electron Microscopical Examination of EC and
NEC 17 2.4.4.4 Physiological and Protein Profiles of EC and NEC 17
2.5 Gene Expression Analysis of Oil Palm 19 2.5.1 Complementary DNA (cDNA) Library 20 2.5.2 Strategies in Library Construction 21
2.6 Expressed Sequence Tags (ESTs) 21 2.6.1 Oil Palm ESTs 23
2.7 Potential Applications of Oil Palm ESTs 23 2.7.1 Genetic Linkage Mapping 23 2.7.2 DNA Microarray 24
2.8 Bioinformatics 25
3 MATERIALS AND METHODS 28 3.1 Plant Materials 28 3.2 RNA Extraction 28
3.2.1 Poly (At RNA Isolation 30 3.2.2 In vitro Translation of Poly (Af RNA 33 3.2.3 SDS-P AGE Gel Electrophoresis (One Dimensional) 34
xii
3.3 Construction of Non-embryogenic Callus cDNA Library 3.4 Preparation of EST Sequencing Templates
3.4.1 Screening by PCR Amplification 3.4.2 Purification by EXO-SAP 3.4.3 Sequencing of ESTs 3.4.4 Sequence Analysis
3.5 Hybridization of Nucleic Acids 3.5.l Reverse Transcription (RT) 3.5.2 Random Prime Labeling 3.5.3 Reverse Northern! Dot-Blot Hybridization 3.5.4 Purification of Probes for Northern Hybridization 3.5.5 Northern Hybridization
4 RESULTS AND DISCUSSIONS 4.1 RNA Extraction and mRNA Isolation
4.1.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-P AGE) 4.2 Characteristics of the cDNA Libraries Constructed 4.3 General Analysis of ESTs
4.3.1 Single-Pass Sequencing of Random cDNAs 4.3.2 Sequence Analysis and Database Comparisons 4.3.3 Quality of the cDNA Libraries
4.4 ESTs with Similarity to Oil Palm Genes 4.5 The Classifcation of Non-embryogenic Calli ESTs of Oil Palm 4.6 Gene Expression Studies
4.6.1 Reverse Northern Analysis by DNA Dot Blot Analysis 4.6.2 Northern Blot Analysis of the 12 Selected ESTs Clones
4.7 Motif Analysis of the 8 Selected ESTs Clones
5 CONCLUSION
REFERENCES APPENDICES Appendix A: Formulation for Media and Solutions Appendix B: The 957 ESTs with significant amino acid homology VITA
xiii
35 39 39 40 40 41 42 42 43 44 46 47
51 51 54 57 59 59 60 62 63 65 69 69 79 86
91
93 108 108 111 132
LIST OF TABLES
Table Page
1 The results for the RNA extraction of various tissues 51
2 The scintillation count results for the mRNA isolated from NEC, 54 EC & embryo ids
3 Characteristics of non-embryogenic callus cDNA libraries 57
4 Analysis results of the non-embryogenic callus cDNA library 61
5 ESTs matches to E.coli genome at the nucleotide level 63
6 Representation of each EST and its putative identity on the dot- 70
blot membranes
7a Dot Blot Arrays (10 Scan) of the eighty selected cDNA clones 73 hybridized with 32P-Iabeled first-strand cDNA probes prepared from total RNA of NEC and quantitated using the Gel Doc 2000 system (BIO-RAD) & Quantity One (Version 4.2.2 Build 009) software
7b Dot Blot Arrays (10 Scan) of the eighty selected cDNA clones 75 hybridized with 32P-labeled first-strand cDNA probes prepared from total RNA of spear leaf and quantitated using the Gel Doc 2000 system (BIO-RAD) & Quantity One (Version 4.2.2 Build 009) software
8a Similarity search results (BLASTX) for the 14 putative clones 77
8b Similarity search results (BLASTN) for the 14 putative clones 78
9 Characteristics of the 12 cDNA clones obtained in the Northern 83 blot analysis
10 BLIMPS results 87
xiv
Figure
1
2
3
4
5
6
7
8a
8b
9
10a-1
lla
llb
LIST OF FIGURES
The oil palm tissue culture process (Loiret, 1981)
The hypothesized patterns of morphogenesis in oil palm tissue culture (Duval et al., 1995)
Embryogenic callus (EC) (a-c). Non-embryogenic callus (NEC) (d-f)
dbEST: database of "Expressed Sequence Tags" release 071103 (Summary of top 30 organisms in the dBEST)
A typical gel analysis of total RNA extracted from either tissue culture materials or vegetative tissues of oil palm
In vitro translation products of mRNA isolated from nonembryogenic callus and embryogenic callus analyzed by SDSPAGE
Functional classification of NEC ESTs of oil palm, showing the proportions of predicted genes according to their putative biological functions
Duplicate membranes prepared with the 80 selected cDNA clones from the NEC cDNA library were hybridized with 32p_ labeled first-strand cDNA probes prepared from NEC RNA
Duplicate membranes prepared with the 80 selected cDNA clones from the NEC cDNA library were hybridized with 32p_ labeled first-strand cDNA probes prepared from spear leaf RNA
The multiple alignment results for the 14 suspected clones
Northern blot analysis of selected cDNAs suspected of specifically expressed during callus development
Integrity and equal loading of RNA (for Northern blot analysis) was checked by rRNAs visualization with ethidium bromide
A 18S rRNA probe was used as a hybridization control
xv
Page
9
13
16
26
53
55
66
71
71
77
80
85
85
ABBREVIATIONS
% percentage
a alpha
� beta
A, lambda
°C degree Celsius
J.!g lIDcrogram
J.!l microliter
J.!M micromolar
A Adenine
BIS N'N'-methylenebisacrylamide
BLAST Basic Local Alignment Search Tool
bp base pair
C Cytosine
CaCh Calcium Chloride
cDNA copy DNA
cfu colony forming unit
Ci Curie
cm centimeter
cps counts per second
CsCI Cesium Chloride
C-terminal Carboxyl terminal
D x P Dura x Pisifera
dATP 2'-deoxy-adenosine-5'-triphosphate
dCTP 2'-deoxy-cytidine-5'-triphosphate
xvi
DEPC
dGTP
dH20
DMF
DMSO
DNA
Dnasel
dNTP
dTTP
E.coli
EC
e.g.
EDTA
EGTA
Em
ESTs
EtBr
g
G
HCI
hr
IPTG
Jacq.
k
kb
Diethyl Pyrocarbonate
2'-deoxy-guanosine-5'-triphosphate
deionized water
Dimethyl Fluoride
Dimethylsulphonyl oxide
Deoxyribonucleic acid
Deoxyribonuclease 1
deoxynuc1eotide triphosphates
2'-deoxy-thymidine-5'-triphosphate
Esherichia coli
Embryogenic Callus
example
Ethylenediarninetetraacetatic acid
Ethylene glycol bis- (�-aminoethylene ether)
Erythromycin
Expressed Sequences Tags
Ethidium Bromide
gram
Guanine
Hydrochloride Acid
hours
isopropyl-�-D-thiogalactoside
Jacquin
kilo
kilobase
XVll
KCl Potassium Chloride
kDA Kilo dalton
L Liter
LB Luria Bertani
LiCI Lithium Chloride
M Molar
mA milliampere
MgCh Magnesium Chloride
MgS04 Magnesium Sulphate
mm minute
ml milliliter
mm millimeter
mM millimolar
MMLV Maurine Moloney Leukemia Virus
mmol millimole
MPOB Malaysia Palm Oil Board
mRNA messenger RNA
IDS millisecond
MW Molecular Weight
N Nonality
NaCI Sodium Chloride
NaOAc Sodium Acetate
NaOH Natrium Hydroxide
NEC Non-Embryogenic Callus
ng nanogram
xviii
nm
nM
nt
N-terminal
OD
ORF
PAGE
PCR
PE
pfu
Poly A+RNA
PPO
RE
RNA
RNase
rpm
rRNA
RT
SDS
sec
STE
T
TAB
TBE
TCA
nanometer
nanomolar
nucleotide
Amino terminal
Optical density
Open Reading Frame
Polyacrylamide Agarose Gel Electrophoresis
Polymerase Chain Reaction
Pro embryo
plaque forming unit
Polyadenylated RNA
2,5-Diphenyloxazole
Restriction enzyme
Ribonucleic Acid
ribonuclease
revolution per minute
ribosomal RNA
Room Temperature
Sodium Dodecyl Sulphate
seconds
Sodium-Tris-EDTA
Thiamine
Tris-Acetate-EDT A
Tris-Borate-EDTA
Trichloroacetic Acid
xix
TE
TEMED
Tm
U.P.M
UV
v/v
w/v
X-gal
XLI-B
Tris-EDTA
N, N, N', N'-Tetramethylethylenediamine
Annealing temperature
University Putra Malaysia
ultraviolet
volume per volume
weight per volume
5 -bromo-4-chloro-3-indolyl-J3-D-galactopyranose
XLI-Blue MRF'
xx
CHAPTER I
INTRODUCTION
Oil palm (Elaeis guineensis Jacq.) was first introduced into Malaysia from
Central and Western Africa in the early 1870' s (Hartley, 1977). It is currently the second
major source of vegetable oil in the world after soybean (Mielke, 1996). The oil palm
belongs to the family Palmaceae and the genus Eiaeis. There are two important species in
the genus Eiaeis, they are E. guineensis and E. oleifera. The former is of African origin
and the latter is of South American origin. This diploid monocotyledon is persistent in
the dominance of the single vegetative apex, hence producing no adventitious or
auxiliary shoots.
Prior to the advent of tissue culture, there was no known reliable method of
vegetative propagation of oil palm (Wong et al., 1996). Therefore tissue culture is an
important aspect in the development of the oil palm industry (Cheah & Wooi, 1993),
especially in the generation of superior and uniform oil palm planting materials.
Pioneering work in oil palm tissue culture was carried out by Staritsky (1970) and
Rabechault et ai., (1970), and the first successful cloning of mature palms was reported
by Rabechault et aI. , (1972) and Corley et aI. , (1977). However, tissue culture has its
limitation. The current success rate of callogenesis of oil palm explants remains low, at
about 20 percent, while the average rate of embryogenesis is only 6 percent as reported
by most tissue culture laboratories (W ooi, 1995). Despite the economic importance of oil
palm tissue culture, little is known about the chemical characteristics and molecular
changes associated with caliogenesis and embryogenesis.
1
In plants, a number of approaches have been used to understand the complexity of
gene expression and interaction. The generation of large numbers of partial cDNA
sequences, or expressed sequence tags (ESTs), has provided a method to sample a large
number of genes from an organism. Since its introduction in 1991, the generation of
expressed sequence tags (ESTs) by single-run partial sequencing has proven to be a rapid
and efficient way of obtaining information on mRNA expression patterns from a wide
variety of tissues, cell types, or developmental stages (Adam et ai. , 1995; Kawamoto et
aI., 1996). It was found that as little as 150 to 400 bases is sufficient for a match to
sequences resident in the databases (Adams et aI., 1991). The results of these
comparisons are used as a guide to assign putative identification to the ESTs.
With respect to tissue culture, it has been suggested that many of the functional
genes necessary for embryogenesis are expressed at an early stage from an unorganized
growth state (Sung & Okimoto, 1983; Giuliano & Terzi, 1985; Wilde et aI., 1988;
Zimmerman, 1993). Thus a comparison of gene expression patterns in embryogenic
callus and non-embryogenic callus using statistical analysis of ESTs would give us an
insight of not only the embryogenic potency but also the regeneration mechanism of the
plant. Therefore this study is aimed at understanding the gene expression pattern in non
embryogenic callus of oil palm. This is carried out by the generation and statistical
analysis of ESTs from non-embryogenic callus of oil palm. This study hopes to generate
some insights into the complexity of genes expressed in non-embryogenic callus of oil
palm.
2
CHAPTER II
LITERATURE REVIEW
2.1 The Oil Palm
2.1.1 Origin and Distribution
It is thought that the centre of origin of the oil palm is the tropical rain forest
region of West and Central Africa. The plant later spread from Senegal to Angola in a
wide belt along the coast and interior of the Congo River (Hartley, 1977). The oil palm
grows and reproduces readily in a wild and semi-wild state and its successful distribution
was due to the influence of human activities (Ascenco, 1966). The sporadic occurrence
on the East Mrican coast was most likely due to Arab slave traders.
The palm fruits were taken by the early slave traders in the 16th Century to the
New World, where it became established first in Bahia, Brazi1. Favorable climate and
shifting of cultivation practices in Brazil resulted in extensive areas of semi-wild palm
groves. Oil palm seedlings including Eiaeis guineensis were grown in European
conservatories in the 18th Century, and, in the following century, it was brought to
Calcutta as ornamental plants. The original introduction to the East was from the island
of Mauritius. Four seedling trees were then established in 1848 in the Botanic Garden at
Bogor in Java, which were until the 1950s the original parent to all the oil palms in
Indonesia and Malaya (Whitmore, 1979).
3
Oil palm was first introduced into Malaya in 1875 (Iman, 1984) and the first
commercial plantings for production of edible oil were in 1 917. By 1938, there were 3 0,
000 ha of land in Malaya already planted with oil palm. After W orId War II, production
of palm oil increased considerable due to favourable economics. Prices of both oil and
kernels reached four or five times their pre-war level and this remained comparatively
steady for the next 20 years (Hartley, 1977). Due to the higher profitability and demands
caused by the oil's wide use in industrial applications, rubber plantations were slowly but
steadily being replaced by oil palm plantation in addition to new plantation areas
developed (Moll, 1987).
2.1.2 Botany
2.1.2.1 Classification
The genus Eiaeis belongs to the subfamily Cocoideae and to the palm family,
Palmae or Arecaceae, which is an important member of the monocotyledonous group. It
has a basic chromosome number of n=16 and classified in the order Spadiflorae. The
word, Elaeis is derived from the Greek word 'elaion', oil, while the specific name
guineensis shows the palm origin, which is the Guinea coast. The fruits of the oil palm
vary widely and the classifications are based on variations of the internal structure of the
fruits. The examples of these classifications are Dura (shell 2 to 8 mm thick), Pisifera
(shell-less), and Tenera (Shell 0.5 to 4 mm thick). The tenera fruit form is a hybrid of the
dura and pisifera forms, often designated as D x P. Since the late 1 950s most
commercially planted palms in Malaysia are Tenera (Whitmore, 1979). The South
American oil palm, Elaeis oleifera (syn. Elaeis melanococca) is another species in the
genus Elaeis. However due to its low oil yield, it is of little economic importance except
4