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

Dedicated to Dad, Mom, Audrey, Adrian and Yoke Fah

ii

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

iii

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