Molecular Analysis of Oil Palm (Elaeis guineensis Jacq) Flowering Associated Genes and their Potential Application in Breeding Programmes
MOLECULAR ANALYSIS OF OIL PALM (Elaeis guineensis Jacq.)
FLOWERING ASSOCIATED GENES AND THEIR POTENTIAL
APPLICATION IN BREEDING PROGRAMMES
WALTER AJAMBANG NCHU
GRADUATE SCHOOL
BOGOR AGRICUTURAL UNIVERSITY
BOGOR
2015
DECLARATION OF ORIGINALITY AND AUTHENTICITY
INCLUDING TRANSFER OF COPYRIGHT*
This is to declare that the dissertation titled “Molecular Analysis of Oil Palm
(Elaeis guineensis Jacq.) Flowering Associated Genes and their Potential Application in
Breeding Programmes” is the result of my personal research under the direction of the
supervising committee and has never been presented in any form wherever. Any other
sources of information that have been mentioned in this dissertation from published or
unpublished works of other authors have been acknowledged in the text and included in
the reference chapter.
Based on this assertion, I therefore transfer the copyrights of this dissertation to the
Bogor Agricultural University.
Bogor, February 2015
Walter Ajambang Nchu
NIM A263118081
SUMMARY
WALTER AJAMBANG NCHU. Molecular Analysis of Oil Palm (Elaeis guineensis
Jacq.) Flowering Associated Genes and their Potential Application in Breeding
Programmes. Under the supervision of SUDARSONO as head, SINTHO
WAHYUNING ARDIE and HUGO VOLKAERT as members of the committee.
The oil palm (Elaeis guineensis Jacq.) is an important economic crop that is used
in the food, chemical and bio-diesel industries. Breeding to increase yields in oil palm
has been focused on improving the sex ratio of more female to male inflorescences.
Environmental and genetic factors largely influence oil palm growth and yields. Soil
water availability, especially in Sub Saharan Africa and source-sink balances have been
the main factors affecting sex ratio and inflorescence production in oil palm. Male
inflorescences have been induced in the highly feminine Pisifera race of oil palm by
carbohydrate depletion through complete defoliation. However, the efficiency of this
process was still a major problem especially in this era of climatic instability. Also, the
molecular genetic mechanisms underlying the process of male inflorescence induction
by complete defoliation are yet to be uncovered. The objective of this study was to
investigate the phenotypic responses and identify the molecular genetic mechanisms
responding to complete defoliation of oil palm in order to efficiently increase oil palm
seed production through improved pollen production and expand existing breeding
programmes. In this dissertation, we present the quantity of environmental factors and
the periods of treatment for which male inflorescence induction or sex determination is
effective and determine the exact moment at which sex differentiation genes are
initiated in response to complete defoliation treatment. Knowledge of this moment
enabled us to accurately isolate the genes and study the molecular mechanisms
underlying male inflorescence induction in oil palm caused by complete defoliation
using RNA-seq.
Complete defoliation significantly induced male inflorescence of up to 104%. An
acute soil water deficit of 16.8 mm between the 30th and 60th days after complete
defoliation (DAD) had a positive effect on male inflorescence induction. Generally,
complete defoliation treatments carried out during the dry seasons produced more male
inflorescences than during the wet season. Unlike inflorescence induction, inflorescence
emergence was highest during the wet season irrespective of the date of its induction.
Regression analysis on 18 time-specific, climate related variables indicated that oil palm
initiates response mechanisms between day 30 and day 60 after complete defoliation
stress. Coincidentally, total soluble sugar measured on completely defoliated trees and
control trees at 45 DAD, showed a sugar depletion of 55% in leaf tissues and 21% in
inflorescence tissues. Preferential sex induction should be a way of acclimation in oil
palm under carbohydrate depletion stress caused by complete defoliation.
Knowing that the oil palm initiates its responses during the second month after
complete defoliation stress, we extracted tissues from both control and completely
defoliated trees for RNA isolation and RNA-seq at 45 DAD. Samples were collected
from three early developmental and distinct consequential stages of the inflorescence.
Next Generation Sequencing (NGS) of 18 transcriptomes was performed on an Illumina
Hiseq2000 platform producing more than 96 000 transcripts of which about 50% were
up regulated. Difference in gene expression was significant between treatment and
between tissues. Gene Ontology analysis showed that 45% of differentially expressed
genes (DEG) responding to complete defoliation stress were located in the chloroplast
and mitochondrion. The chloroplasts and mitochondria are the two powerhouses of the
cell, one regulating carbohydrate production through photosynthesis and the other
regulating cell energy availability in the form of ATP. Similarly, the biological
processes that were functionally enriched from the DEG data set were photosynthesis
and carbohydrate related processes. Gene network analysis showed that abiotic stress
response DEGs were co-expressed with vegetative to reproductive phase transition of
the meristem, gibberellin, auxin, jasmonic acid and photoperiodism. This co-expression
confirmed that abiotic stress caused by complete defoliation was involved in male
inflorescence induction in oil palm, regulated by sugar status and plant growth
hormones. Genes linked to carbohydrate metabolism, flower development, stress
response, circadian rhythm, PGR and vegetative to reproductive phase transition of the
meristem may be responsible for the regulation of inflorescence emergence in oil palm.
Sequence data generated from this study should be used to compare the expression from
other races of the oil palm especially the commercial race, Tenera and Dura. These
sequences should be used to quantify gene expression on breeding populations in the
process of selecting parents for seed production
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Keywords: complete defoliation, carbohydrate depletion, inflorescence induction, oil
palm, NGS, RNA-seq, genomics
RINGKASAN
WALTER AJAMBANG NCHU. Analisis Molekuler Gen-Gen yang Berkaitan dengan
Pembungaan dan Aplikasi dalam Pemuliaan Tanaman Kelapa Sawit. Dibimbing oleh
SUDARSONO sebagai ketua, SINTHO WAHYUNING ARDIE dan HUGO
VOLKAERT sebagai anggota komisi pembimbing.
Kelapa sawit (Elaeis guineensis Jacq.) adalah tanaman tropis yang digunakan
dalam industri makanan, kosmetik dan bio-diesel. Tujuan utama dalam pemuliaan
kelapa sawit adalah menaikan sex ratio bunga betina terhadap bunga jantan. Faktor
lingkungan dan genetik mempunyai peran besar dalam produktivitas kelapa sawit.
Ketersediaan air, terutuma di Afrika, dan ketersediaan kandungan kabohidrat
mempengaruhi sex ratio kelapa sawit. Pemangkasan daun sawit adalah salah satu cara
untuk menginduksi bunga jantan di kelapa sawit namun masih ada beberapa hal terkait
efisiensi teknik tersebut, apalagi gen-gen yang berperan dalam proses ini belum
diketahui. Penelitian ini bertujuan untuk menyelidiki perubahan fenotipe dan
menentukan gen-gen yang terekspresi akibat pemangkasan daun sawit. Dalam disertasi
ini, diberikan informasi tentang berapa jumlah faktor lingkungan yang dibutuhkan
untuk menginduksi bunga jantan pada kelapa sawit secara efisien. Selain itu,
diberitahukan juga berapa waktu yang dibutuhkan oleh kelapa sawit sebelum merespon
terhadap stres pemangkasan daun. Informasi ini digunakan untuk pelajari ekspresi gen
terkait pembungaan dalam kelapa sawit yang diakibatkan oleh stres pemangkasan daun.
Data menunjukan bahwa teknik pemangkasan daun bisa menaikan produksi bunga
jantan kelapa sawit sampai 104%. Teknik ini paling efisien di musim kering dan
optimum saat cekaman air sama dengan 16.8 mm dalam waktu 60 hari pertama setelah
pemangkasan daun. Analisis regresi menunjukan bahwa tanaman kelapa sawit akan
memberikan respon terhadap cekaman kabohidrat yang diakibatkan oleh pemangkasan
daun merupakan ekspresi gen dalam waktu 30 sampai 60 hari setelah pemangkasan.
Kadar gula yang diambil dari jaringan bunga dan daun kelapa sawit 45 hari setelah
pemangkasan menunjukan bahwa kadar gula menurun 21% di jaringan bunga dan 55%
di jaringan daun dibanding kontrol.
Ekspresi gen di jaringan bunga kelapa sawit mengunakan Next Generation
Sequencing (NGS) menujukan bahwa mayoritas gen gen yang berperan dalam regulasi
cekaman kabohidrat di kelapa sawit terletak di kloroplas dan mitokondria. Analisis
fungsi menggunakan Gene Ontology, menujukan bahwa gen gen tersebut berperan
dalam metabolisme kabohidrat dan fotosintesis. Analisis ko-ekspresi menujukan
keterkaitan antara cekaman kabohidrat dan induksi bunga serta peran zat pertumbuhan
tanaman seperti auxin, gibberellin dan jasmonic acid. Ini membuktikan bahwa gen gen
dalam lintasan cekaman kabihidrat yang diakibatkan oleh pemangkasan daun berperan
dalam induksi bunga di jantan tanaman kelapa sawit.
Kata kunci: pemangkasan daun, cekaman kabohidrat, induksi bunga, NGS, kelapa
sawit.
© Copyright IPB, 2015
Copyright protected under the law.
No part or all of this work may be reproduced without citing the source. Copying may
be done only on the basis of education, research, scientific writing, reports or review;
and which should not cause any prejudice to IPB.
No part or all of this work may be reproduced in any form or by any means without
permission from IPB.
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MOLECULAR ANALYSIS OF OIL PALM (Elaeis guineensis Jacq.)
FLOWERING ASSOCIATED GENES AND THEIR POTENTIAL
APPLICATION IN BREEDING PROGRAMMES
WALTER AJAMBANG NCHU
Dissertation
Submitted in partial fulfilment of the requirements for the degree
Doctor of Philosophy
in
Plant Breeding and Biotechnology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
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Examiners during Closed Exam:
Prof. Dr. Ir. Iskandar Z. Siregar M.Sc
Dr. Desta Wirnas, SP, M.Si
Examiners during Public Defence:
Dr. Ir. Sudrajat, MS
Dr. Adi Pancoro
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Dissertation Title
Name
NIM
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: Molecular Analysis of Oil Palm (Elaeis guineensis Jacq.)
Flowering Associated Genes and their Potential Application
in Breeding Programmes
: Walter Ajambang Nchu
: A263118081
Approved by
Supervisory committee
Prof. Dr.Ir.Sudarsono, M.Sc.
Head
Dr.Sintho W Ardie, SP, M.Si.
Member
Dr. Hugo Volkaert
Member
Endorsed by
Head of Mayor
Plant Breeding and Biotechnology
Dr. Ir. Yudiwanti Wahyu, E.K, MS.
Date of Public Defence:
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Dean Graduate School
Dr. Ir. Dahrul Syah, M.Sc. Agr.
Date Graduated:
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FOREWORD
Glory be to God the Father Almighty for his ever increasing guidance and
protection especially during the difficult moments the author went through while
undertaking studies in Bogor, without which all human efforts wouldn’t have borne any
fruits.
The author wishes to convey his sincere and special thanks to the head of the
supervisory committee Prof. Dr. Ir. Sudarsono, MSc. for his advice and remarks during
the preparation, execution and presentation of this dissertation. The author pays
gratitude to members of the supervising committee, Dr. Sintho W. Ardie, SP, M.Si and
Dr. Hugo Volkaert, for their resourceful instructions and comments on this work. The
author is grateful to Prof. Dr. Ir. Iskandar Z. Siregar M.Sc, Dr. Desta Wirnas, SP, M.Si,
Dr. Ir. Sudrajat, MS and Dr. Adi Pancoro who gave their precious time to respectively
serve as closed examiners and open examiners of this dissertation.
This project would never have existed without the vision and wisdom of Dr. Zok
Simon and Bapak Widya Wiryawan, former General Manager of IRAD Cameroon and
present CEO of ASTRA Agro Lestari Indonesia respectively. Special thanks go to
Bapak Bambang Palgoenadi, Dr. Ngeve Mbua Jacob and Dr. Woin Noe for bravely
executing the training aspect of this project. The author is indebted to Bapak Satyoso
Hartodedjo of ASTRA Agro, Dr. Koona and Dr. Ngando of CEREPAH for the smooth
management of this project.
The Late Joseph Asende Nchu has a special place in the author’s mind for
initiating and accompanying him in this long and exciting journey. The author is
grateful to Mama Comfort Namondo for her constant support during his stay abroad.
The author presents special gratitude to his family members especially the wife
Lylie Michele Ajambang and children Jeff, Megane and Naomi for their endurance
during his long absence from home. High appreciation is extended to the colleagues of
PMB and ASTRA labs for their technical and scientific assistance during the execution
of this project.
The author hopes to receive public criticisms that may improve the quality of
this dissertation. The author wishes that this dissertation makes an impact in science and
development especially in the field of oil palm breeding and genomics.
Bogor, February 2015
Walter Ajambang Nchu
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TABLE OF CONTENTS
TABLE OF TABLES
xiii
TABLE OF FIGURES
TABLE OF APPENDICES
xiii
xv
1 INTRODUCTION
Background
Problem statement
Objectives of research
Importance of research
Novelty
Scope, framework and limitations
2 LITERATURE REVIEW
Origin and botany of oil palm
Major changes caused by carbohydrate depletion
Hormones associated with flowering
Genes involved in floral induction and initiation in oil palm
The role of environmental factors and assimilates
The oil palm trunk as a carbohydrate reserve
Previous work on gene expression in oil palm with molecular methods
3 MASSIVE CARBOHYDRATE RESERVES BUFFERS AND DELAYS
RESPONSE TO STRESS CAUSED BY COMPLETE DEFOLIATION IN
OIL PALM (Elaeis guineensis Jacq.)
Abstract
Introduction
Materials and Methods
Results and discussions
Conclusion
4 INFLORESCENCE EMISSION TIME IN OIL PALM (Elaeis guineensis
Jacq) IS SEASONAL, IRRESPECTIVE OF INDUCTION DATE
Abstract
Introduction
Materials and Methods
Results and discussions
Conclusion
5 COMPARATIVE EXPRESSION PROFILING OF THREE EARLY
INFLORESCENCE STAGES OF OIL PALM RESPONDING TO
COMPLETE DEFOLIATION SHOWS THAT VEGETATIVE TO
REPRODUCTIVE PHASE TRANSITION OF MERISTEM IS CAUSED BY
SUGAR DEPLETION
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Abstract
Introduction
Materials and Methods
Results and discussions
Conclusion
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6 GENERAL DISCUSSIONS
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7 GENERAL CONCLUSIONS AND RECOMMENDATIONS
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REFERENCES
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APENDICES
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AUTOBIOGRAPHY
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TABLE OF TABLES
2.1
3.1
4.1
4.2
4.3
4.4
5.1
Cell and physiological responses of some major phyto-hormones
involved in flower sex determination and development, including
related references
Correlation coefficients for selected variables that had a significant
relationship with male inflorescence number
Number of inflorescences produced before and after defoliation
T-test for number of inflorescence emission between the wet and dry
season
Functional networks involving down regulated and up regulated
DEGs
Ten top DEGs based to FC and p-value
Summary data from the different libraries
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TABLE OF FIGURES
1.1
Flow chart of research activities
2.1
Un-emerged female inflorescence extracted from leaf axil number 14 and
un-emerged male inflorescences extracted from leaf number 13
Possible interactions resulting from the severe pruning of oil palm as
proposed by Adam et al. (2011). The circles represent the biological
processes while the squares represent the products
Apical meristem of oil palm tree showing undetermined flower bud and
future leaves
Stages of the developmental processes between sex differentiation and
flower maturity in oil palm as reproduced by Durand-Gasselin et al. (1999).
The top most row represents the leaf numbers. Leaf number 0 is the middle
unopened leaf commonly known as the spear. Leaf number +1 is the first
opened leaf outwards from leaf number 0. Leaf number -1 is the next leaf
that will succeed the spear. The leaf numbers carrying minus signs are unemerged leaves. It is assumed that leaf initiation takes place at leaf position
number -40 inside the apical meristem of the oil palm
Complete defoliation on mature pisifera trees. (a) Mature non defoliated
pisifera palm (b) completely defoliated pisifera palm. (c) A recovering
pisifera palm 45 DAD. The arrow shows the number of leaves that have
been produced after complete defoliation. (d) A mature male inflorescence
at anthesis. Anthesis is when pollen on the flowers has stained maturity and
is capable of fertilising female flowers. The white bars on (a) and (b) are
drawn to 1 m scale while that of d is 10 cm scale. Arrow points to newly
2.2
2.3
2.4
3.1
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3.2
3.3
3.4
3.5
3.6
3.7
3.8
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
5.1
5.2
5.3
5.4
5.5
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developed oil palm leaves after complete defoliation.
Leaf counting during inflorescence extraction
Monthly soil water deficit (curve) and monthly precipitation (bars)
recorded between 2007 and 2013 at La Dibamba Cameroon
Male inflorescence production and mean annual precipitation between 2001
and 2012 at La Dibamba before and after treatment. The broken line
between year 2006 and 2007 represents the start of defoliation treatment.
The number of male inflorescences produced is represented by bars
corresponding to the primary vertical axis while precipitation curve is
represented by the secondary vertical axis
The relationship between precipitation, rainy days and male inflorescence
induction on plants subjected to complete defoliation stress
Stages leading to tissue extraction for total soluble sugar analysis. (a) a
mature pisifera being felled 45 DAD, (b) transportation of the reduced
crown to the laboratory for tissue extraction, (c) leaf and inflorescence
extraction (d) inflorescence located at leaf axil + 5 (on red background)
placed on the leaf petiole
Total soluble sugar content in inflorescences and leaves from non
defoliated (control) plants and previously defoliated plants 45 days after
defoliation
The effect of increasing soil water deficit on male inflorescence production
Distribution of inflorescence emission in months after complete defoliation
Average monthly emission of male inflorescences based on the season of
defoliation treatment
Distribution of gene expression level in non-defoliated (blue) and
defoliated tissues (red)
Venn diagram of DEGs on stress and control tissues
Volcano plot for DEGs between non-defoliated and defoliated samples.
The y-axis represents the level of significance of the expression change
between samples measured on –log10 p-value (pval = 0.05), while the xaxis represents the fold change of DEGs. DEGs located above the red line
are significant based on the p-value while those below are not significant.
The DEGs located between the two blue lines are non DEGs based |FC| ≥ 2
Functional categoristaion of DEGs based on biological processes
Average monthly male inflorescence production and mean monthly
precipitation for the period between 2007 and 2012 recorded at La
Dibamba Oil Palm Research Centre
Average monthly FFB production and mean monthly precipitation between
2007 and 2012 at La Dibamba Oil Palm Research Centre
Extracted inflorescences from axils of palm leaves
A dissected oil palm showing a series of inflorescence buds enclosed in the
axils of future leaves
Distribution of expression levels based on FC between the non defoliated
(blue) and defoliated (red) tissues
Volcano plots for DEGs for the three different comparisons (a) axil -20/axil
-27 (b) axil +5/axil -27 (c) axil +5/axil -20
Differential gene expression at FC ≥ 2 and p ≤ 0.05 between inflorescence
stages under stress
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5.6
5.7
5.8
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5.10
5.11
5.12
5.13
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Gene bar chart for (a) up regulated DEGs (b) down regulated DEGs from
three inflorescence stages based on cellular component
Gene bar chart for (a) up regulated DEGs and (b) down regulated DEGs
from three inflorescence stages based on molecular functions
Gene bar chart for (a) up regulated DEGS and (b) down regulated DEGs
from three inflorescence stages based on biological process
Gene set enrichment score from the comparison between inflorescence at
leaf axil +5 against inflorescence of leaf axil -27
Gene set enrichment score from the comparison between inflorescence
located at leaf axil -20 against inflorescence of leaf axil -27
Gene set enrichment score from the comparison between inflorescence at
leaf axil +5 against inflorescence located at leaf axil -20
Network of related genes from the abiotic stress process analysed on Gene
MANIA
Network of related genes from the reproductive process analysed on Gene
MANIA
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TABLE OF APPENDICES
1
2
3
4
5
6.
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Male flower and pollen production before and after complete defoliation
List of RNA samples extracted on both leaves and flowers for RNA-seq
Box plot for normalised transcripts
Tree diagram for samples based on inter sample correlation
Differential expression of genes between treatments
Differential expression of genes between samples under stress
Functional annotation chart from DAVID
RNA Integrity Number (RIN)
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CHAPTER I
INTRODUCTION
Background
Oil palm (Elaeis guineensis Jacq.) is the world’s leading oil crop with 35% of
total world consumption of vegetable oils (Soy stats 2014). Oil palm is cultivated on
approximately 15 million hectares across the world and consumption is expected to
double by the year 2020 (FAO 2009). Apart from its traditional use as a source of food,
oil palm is used in the manufacture of margarine, pharmaceuticals, soap and cosmetics,
animal feed and organic manure, building material, furniture and biofuel. Production of
biodiesel from oil palm has been increasing in recent years, particularly in Africa and
Latin America (FAO 2010; Mitchell 2011).
Socio-economic benefits of a sustainable oil palm plantation could include
poverty alleviation and long-term employment opportunities (Badrun 2010; Kurniawan
2010; Norrochmat and Hadianto 2010; Pahan 2011). Profit sharing may provide a
further incentive, attracting more workers to the palm oil sector, along with better living
and working conditions (Albán and Cárdenas 2007). Indonesia’s palm oil export was
the major force behind the stabilisation of its economy during the 2008-2009 global
financial crisis. Oil palm production has favoured the development of upstream
companies such as the seed industry, fertiliser, agrochemicals, agro mechanicals and
financial services; side stream companies and downstream companies in Cameroon.
Problem statement
The commercial cultivation of oil palm is faced with many problems ranging
from land availability, environmental issues, social issues, agronomy, diseases, climate
change and the availability of quality seeds. Seed availability is the foundation for any
agricultural project. There are three different races of the cultivated oil palm species,
categorised basically by the thickness of the shell, which is controlled by a co-dominant
monogenic inheritance (Beinaert and Vanderweyen 1941). Moretzsohn et al. (2000)
identified the allele pairs of the gene as sh+/sh+ for dura race, sh+/sh- for tenera race
and sh-/sh- for the pisifera race. The commercial oil palm cultivar is a hybrid called
Tenera denoted as T or DP derived from a cross of two homozygous parents based on
shell thickness known as the Dura or female parent represented by DD and the Pisifera
or male parent represented by PP. In the process of seed production in oil palm, a
trained pollinator artificially carries out pollination. Pollen is harvested from the Pisifera
parent and then manually pollinated on to the Dura inflorescences. The quantity and
quality of pollen used in this pollination process is highly related to the quantity and
quality of total seed produced.
The Pisifera is very feminine under optimal plantation environmental conditions.
Pisifera rarely produces male inflorescences that can be used as pollen. Also, the
Pisifera used in seed production are descendants of the group B sub population of oil
palm, which are known to inherit a high sex ratio of female flowers to male flowers.
Breeding for yield traits has favoured the improvement of female flowers than male
flower. This is because the main parameters breeders have been evaluating in breeding
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programmes are the number of bunches per tree (BNO) and the total bunch weight
(BW), which are both heritable (Hardon et al. 1985; Rafii et al. 2002). Under normal
environmental conditions, seed producers may have to import pollen from countries
with sub optimal environmental conditions such as the arid zones of Africa. In addition
to high expenditure, pollen importation has to undergo the tight regulations concerning
the transfer of biological organisms. In Cameroon, the seed production unit spends 50%
of its production cost on pollen importation. Thus it was imperative for each seed
production centre to be able to produce its own pollen locally in order to reduce seed
production costs and also to save time and energy in handling complex international
regulations regarding international transfer of biological material.
Hardon and Corley (1976) reported that oil palm produces male inflorescence
after it has undergone a high production of fruit bunches. When the plant produces
Fresh Fruit Bunches (FFB), it losses energy as bunches are continuously harvested from
the plant. In contrast, the Pisifera race aborts its fruits immediately after fertilisation and
hence does not produce bunches. Therefore most of its energy is stored in the trunk and
a lesser part of it is used for plant maintenance. This means that the Pisifera plant shall
never loss enough energy to let it undergo the required depletion of stored energy that
will enable it to produce male inflorescences. Corley (1976) and Durand Gasselin et al.
(1999) reported that severe defoliation of Pisifera leaves induces male inflorescence in
oil palm, and that the treatment was most effective during the dry season in Ivory Coast.
This is an indication that oil palm sex differentiation is strongly affected by climatic
factors, with male inflorescence being influenced by carbon depletion and water deficit.
Although phenological studies have been carried out which suggests points of
interaction between genetic and the environment factors in the induction of male
inflorescences on oil palm (Corley 1976; Uhl 1988; Van Heel et al. 1987; Tomlinson
1990; Durand-Gasselin et al. 1999; Adam et al. 2011), nothing is yet known about the
nominal values of climatic factors, the exact amount of time necessary for the oil palm
to respond and initiate the mechanism of male inflorescence induction, genetic basis of
sex determination, nor the molecular mechanisms by which these processes are
regulated in oil palm. A number of questions concerning the mode of interaction
between molecular-genetic processes and sex differentiation still remain unanswered
(Adam et al. 2011; Walter et al. 2012).
The development of molecular biology, supported by sophisticated
bioinformatics, can be used to understand the genetic basis and molecular mechanisms
underlying the process of sex differentiation in oil palm. Molecular biology methods
such as Next Generation Sequencing (NGS) have been used on genomic DNA and RNA
transcripts by several researchers in order to assign putative functions to genes
(Jouannic et al. 2005; Adam et al. 2005; Ho et al. 2007; Adam et al. 2007; Low et al.
2008; Adam et al. 2011; Jen et al. 2013).
RNA-seq is the sequencing of cDNA derived from cellular RNA by using
massively parallel sequencing technologies such as Sanger, Roche 454 and Illumina.
RNA-seq can show a repertoire of the genes expressed in a particular tissue such as
flowers and meristem tissues of oil palm and at a specific time point. In this way, RNAseq can reproduce a nearly complete picture of transcriptomic events taking place in a
biological sample and its data can be used to characterise and quantify expressed genes
(Wang et al. 2009; Riggins et al. 2010; Brautigam et al. 2011). RNA-seq is practical in
non model species which do not yet have a reference genome. It is efficient in cases
where resources may be limited because sequencing will target only coding regions. Jen
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et al. (2013) reported that among the three commonly RNA-seq used technologies,
Illumina showed better gene coverage than Roche 454 and Sanger on oil palm tissues
because of its higher sequencing depth.
Objective of research
Investigate the phenotypic responses and identify the molecular genetic mechanisms
responding to complete defoliation of oil palm in order to efficiently increase oil palm
seed production and also expand existing breeding programs.
Specific objectives
1. Explore the effects of complete defoliation of Pisifera on male inflorescence
induction.
2. Estimate the critical moment in which genes are expressed in response to
complete defoliation in order to be able to isolate and clone these genes for
further molecular and downstream research.
3. Determine the effects of voluntary inflorescence induction by complete
defoliation on the seasonal trend of inflorescence emergence.
4. Identify the molecular mechanisms associated to complete defoliation of oil
palm.
Importance of research
1. This research shall permit us to know the exact time at which genes are switched
on in response to complete defoliation treatment.
2. Understand the precise period during the year for which complete defoliation is
effective in producing male flowers.
3. The specific period of gene expression will permit a successful isolation of
target genes expressed as a result of complete defoliation.
4. The molecular processes regulating male inflorescence induction and emergence
in oil palm shall be identified.
Novelty
Durand-Gasselin et al. (1999) initiated work on oil palm defoliation in the Ivory
Coast as a method of male inflorescence induction in oil palm. They based their study
on the phenotypic response to severe pruning of oil palm leaves. Some work has been
done on oil palm gene expression by researchers such as Jouannic et al. (2005), Adam
et al. (2007), Ho et al. (2007), Tranbarger et al. (2011) and Morcillo et al. (2013)
focusing on different oil palm tissues using techniques such as EST, micro array and
other hybridisation. An incomplete oil palm genome is available from the Malaysian Oil
Palm Board (Singh et al. 2013) and is stored in the domain
http://www.genomsawit.mpob.gov.my.
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4!
Previous research has not indicated the moment at which genes are expressed in
response to complete defoliation. ESTs collected from oil palm flower tissues were not
based on any previous flower induction treatment. The collection of ESTs were not time
specific nor tissue specific. This present research is unique in the sense that;
i.
ii.
iii.
iv.
v.
vi.
Samples were collected from tissues that have been stressed and compared with
tissues in normal conditions.
Samples were collected with respect to the spatial-temporal expression of genes
responsible for sex differentiation.
Flowering was induced by the application of a treatment whereas the other
researchers did not induce flowering.
NGS through RNA-seq was used to identify genes associated with male floral
induction. Specific primers from MADS box genes were not used as was done
by other authors.
No prior hybridisation such as Suppression Subtractive Hybridisation (SSH) or
Fluorescence in situ Hybridisation (FISH) techniques in order to identify only
previously identified genes but carried out sequencing, assembly and annotation
with bioinformatics enabling us to identify new transcripts.
An attempt was made to identify genes associated with floral induction and sex
differentiation rather than studying genes associated with floral morphology.
Scope, framework and limitations of the research
This research is based on four inter-linked objectives aiming at contributing to
the development of oil palm, specifically oil palm genomics, breeding and seed
production. These activities were carried out in different locations because of the
perennial nature of the plant and also because of the availability of laboratory facilities.
The four activities were:
1. The complete defoliation of Pisifera parent plants.
2. RNA-seq on flower related tissues extracted on Pisifera parents after
complete defoliation.
3. Analysis of gene expression levels associated to male flower induction.
4. The quantification of gene expression levels associated to male inflorescence
induction
The first activity was carried out on selected parents used in oil palm seed
production in Cameroon and Indonesia. They were of the LM2T origin and were self
pollinated at La Me Ivory Coast in 1990 and later planted at the oil palm research centre
in Cameroon in 1993. Part of this population has been transferred to Indonesia in South
East Asia for collaborative research and breeding purpose. Under the conditions of the
research station where these Pisifera are planted (i.e. La Dibamba of Cameroon with
rainfall of between 2 500 - 3 000 mm and an annual average temperature of 28 °C), it
has been difficult for these plants to produce male flowers. La Dibamba had to carry out
some trials in order to reduce seed production cost by inducing the Pisifera plants to
produce male inflorescences and hence pollen. Data collected on the variables is
presented in Chapter three of this dissertation.
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5!
Experiment two shall be developed based on the first results obtained in
experiment one. By using mathematical models, we shall be able to predict the time and
amount of environmental conditions necessary for switching gene expression in
response to complete defoliation. Samples of tissue shall be extracted from the
completely defoliated tree and unstressed tree to identify the group of Differentially
Expressed Genes (DEG) associated with this stress and male inflorescence induction in
Pisifera. Next Generation Sequencing will be used to sequence transcripts expressed in
response to the complete defoliation of the Pisifera. A simulated defoliation activity,
tissue extraction and RNA isolation shall be carried out on Pisifera at the PT ASTRA
Research Centre in Kumai Central Kalimantan. NGS shall be carried out in the
Macrogen laboratories in South Korea.
Experiment three and four shall be based on the results of experiment two. The
level of expression of different transcripts found in the different tissues shall be
compared with the normal level of expression contained in the oil palm reference
genome.
Oil palm is a perennial crop and enters production only at the 4th year after
planting. This makes it difficult to plan and execute a field research within time limits
for a dissertation. The blocks used for experiment 1 are thus carved out after the crop
had been planted because these are plants that are 20 years of age. The impossibility to
extract tissues from Cameroon and carry them to Indonesia for RNA isolation led us to
simulate the experiment in Kalimantan taking into consideration the result of the trial in
Cameroon. This permitted us to extract the tissues from a similar plant in Kalimantan
Indonesia where the laboratory facilities are available. The flow chart of research
activities is presented in Figure 1.1.
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6!
Exp 1. Complete defoliation of Pisifera
Phenotypic and physiological responses
Analysis of climatic and time factors
Exp 2. Extraction of flower related tissues for mRNA isolation
mRNA isolation
Quality control
NGS
Sequence processing
Quality control
Transcriptome mapping to reference genome
Transcriptome
characterisation/ gene
annotation
Differential gene
expression analysis
Gene ontology
Exp 3. Transcript quantification/expression levels
Figure 1.1 Flow chart of research activities
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7!
CHAPTER II
LITERATURE REVIEW
Origin and botany of oil palm
The oil palm presently exists in the cultivated and wild states in the equatorial
and tropical regions of Africa, South East Asia and South and Central America. Many
authors have put forward various arguments on the origin of the oil palm (Zeven 1964;
Hartley 1988; Soh et al. 2003). There is no doubt that the cultivated African oil palm
Elaeis guineensis Jacq. is of African origin because there is supporting pre historic
evidence that it originated from the area around Nigeria, Cameroon (Gulf of Guinea)
and the Congo (Zeven 1964; Hartley 1988). Early European traders collected oil palm
seeds from Africa and planted them in The Amsterdam Botanic Gardens, Netherlands
and later transferred it to the Bogor Botanic Gardens Indonesia, then a Dutch colony in
the year 1848. The four palms planted in Bogor then became the foundation stock for
the development of the world oil palm industry (Hunger 1917).
The oil palm is a diploid (2n = 2x = 32) classified under the family Arecaceae
(Dransfield et al. 2005). The crown of a mature palm consists of between 30 to 50
leaves. The number of leaves produced annually by a plantation palm decreases from 30
to 40 after 2 years of age to an average of 20 to 25 per annum after 8 years of age. The
roots of the oil palm can reach a horizontal distance of 16 m and a vertical distance of 8
m in well drained soils. Secondary roots develop on primary roots and tertiary roots
develop on secondary roots forming a network of dense root system (Corley and Tinker
2003). The oil palm is monoecious, with male and female inflorescences occurring
separately on the same tree (Dransfield et al. 2008). The female and male inflorescences
are shown in Figure 2.1.
The oil palm is commonly divided into three races based on their shell thickness
(sh). The sh gene had been discovered by Beinaert and Vanderweyen (1941). The Dura
has a shell thickness between 2 to 8 mm; the Tenera 0.2 to 2 mm and the Pisifera has no
shell. The fleshy mesocarp of Dura yields between 15 to 17%, oil that of Tenera yields
between 21 to 23% oil and Pisifera more than 23% oil. The Pisifera mostly aborts its
fruits and thereby produces virtually empty bunches, thus it is not cultivated on large
scale for commercial purpose.
Major changes caused by carbohydrate depletion in plants
Several factors are responsible for source sink imbalances in plants. Instability in
climatic and environmental factors such as rainfall, soil water availability, light and
temperature and attack by pathogens may greatly reduce carbohydrate supply to sink
tissues (Ho et al. 2001; Rolland et al. 2002; Xu and Zhou 2006; Xue et al. 2008;
Janecek and Klimesova 2014). Drought caused significant reduction in starch ranging
from 54% to 60% on all the organs of P. radiata (Mitchell et al. 2012), defoliation
reduced water soluble carbohydrates concentration by 43% in Lolium perenne (Lee et
al. 2010), while defoliation reduced carbohydrate production in the baobab (Lee et al.
2010; Simbo et al. 2013).
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8!
Figure 2.1 Un-emerged female inflorescence extracted from leaf axil number 14 (left)
and un-emerged male inflorescences extracted from leaf number 13 (right).
The inflorescence from leaf axil number 14 is two weeks older than the
inflorescence from leaf axil number 13. Female inflorescences are always
longer and larger than their corresponding male inflorescence of similar age
and from the same plant.
Sugar deprivation can cause substantial physiological and biochemical changes in
plants leading to potato tuber induction (Simko 1994), changes root system and crown
morphology in tobacco (Paul and Stitt 1993) and changes in sex ratio in oil palm
(Durand-Gasselin et al. 1999). Primary response to stress involves the accumulation of
water-soluble carbohydrates such as sucrose, glucose, fructose and fructans (Xu and
Zhou 2006). Sugar responsive genes coordinate information on sugar status between the
source and sink locations. Morphological changes resulting from sugar level alterations
can be explained by sugar’s interaction with plant growth hormones such as gibberellin
in germinating seeds, cytokinin and auxin (Vincentz et al. 1993).
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9!
Hormones associated with flowering
Concerning hormones associated with oil palm sex differentiation, Corley
(1976) observed that the growth regulator auxin NAA (naphthalene acetic acid),
favoured female flower production in palms grown in polythene bags. The
masculinising effect of GA3 has also been reported in cucumber (Fuchs et al. 1977;
Pimenta Lange et al. 2012). GA, ABA, IAA and Cytokinin content were found to be
higher in the developmental stages of male flower than in female flowers in poplar
(Song et al. 2013). In the much-studied Arabidopsis, individual pathways that include
photoperiod (Srikanth and Schmid 2011), vernalisation (Kim et al. 2009), autonomous
(Mouradov et al. 2002), ageing (Fornara and Coupland 2009) and GA (MutasaGottgens Hedden 2009), regulate flowering time. The GA4 which is most likely
produced in the leaves and transported to the meristem up regulates one or both of the
LFY genes, a floral meristem identity gene and the SUPRESSOR OF
OVEREXPRESSION OF CONSTANS 1 (SOC1), a floral integrator leading to flowering
(Bernier and Perilleux 2005).
Table 2.1 Cell and physiological responses of some major phyto-hormones involved in
flower sex determination and development, including related references
Hormones
Abscisic acid
Auxin
Brassinosteroid
Cytokinin
Ethylene
Gibberellic acid
Jasmonic acid
Cell and physiological responses
Flowering initiation, flower, leaf
and
fruit
senescence
and
abscission,
stomata
closing,
concentration increases during
drought
Increases cell elongation and
division, initiation of floral
primordia, specifies the number
and identity of floral organs
Slows abscission, stimulates cell
division
Regulates cell division and delays
senescence, affect meristem size,
required for pollen development
Species
Populus
tomentosa
Reference
Song et al.
(2013)
Oil
palm, Corley
Populus
(1976);
tomentosa
Song et al.
(2013)
Maize
Hartwig et
al. (2011)
Soybean,
Wong et al.
Nicotiana
(2013);
plumbaginifolia Vincentz et
al. (1993)
Floral transition, feminising role Arabidopsis
Achard et
in flower development, induces
al. (2007)
fruit ripening, synthesis increases
as a result of stress
Masculinising
effect,
induce Lolium
King
and
phase transitions and stimulates temulentum
Evans 1991
fruit development
Developing
inflorescences, Arabidopsis
Reinbothe et
defensive responses to stress
al. 2009
ABA, jasmonates and stress inducible genes are co-regulated by sugars in plant
tissues (Reinbothe et al. 2009; Sadka et al. 1994;). GA had been suggested to be
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responsible for stamen abortion in maize inflorescences (DeLong et al. 1993; Dellaporta
and Calderon-Urrea 1993) although it has the opposite effect on cucumber. The role of
phytohormones in flower development and sex determination is summarised in Table
2.1.
Genes involved in floral induction and initiation in oil palm
Flower initiation includes all of the development processes necessary for the
irreversible commitment by the meristem to produce a flower or inflorescence.
Vegetative to reproductive phase transition of meristem results from consequential
activities of floral meristem identity genes that specify floral meristem fate and of floral
organ identity genes that determine the pattern of whorl establishment. Floral meristem
identity genes stimulate floral organ identity genes to promote floral development. The
floral organ identity genes are designated as ABC classes of homeotic genes. The ABC
model includes the APETALA 1,2,3 (AP); PISTILLATA 1 (P1) and AGAMOUS (AG).
The class A genes (AP1 and AP2) are responsible for sepals identity, the class B (AP3
and P1) plus the class A genes are responsible for petal identity, the class C (AG) plus
the class B genes are responsible for stamens identity and the class C genes alone
specifies the carpel identity. The SUPERMAN genes maintain whorl boundaries.
Superman mutants in Arabidopsis develop extra stamens at the expense of carpels,
rendering carpels defective or under developed (Schultz et al. 1991; Nibau et al. 2011).
It is believed that in unisexual flowers, sex is determined by the selective
repression of growth or abortion of either the male or female reproductive organs. It is
known that the identity of reproductive organs is controlled by homeotic genes
belonging to the MADS box gene family. Kater et al. (2001) reported that the arrest of
either male or female organ development is dependent on their positions in the plant.
Although sex determination in animals is well studied, little is known about the
molecular processes involved in plant sex determination. Only few genes that influence
sex have been cloned. The TASSELSEED2 gene in maize which is responsible for the
abortion of pistil primordia in the tassel (DeLong et al. 1993) and the ANTHER EAR1
gene (Bensen et al. 1995) which encodes an enzyme in the gibberellin biosynthesis
pathway responsible for stamen abortion in maize ear. The class B and C members of
the MADS box family encode putative transcription factors with a highly conserved
DNA binding domain and may influence the sex of plants. They suggest a role of the
class C genes in controlling the arrest of the reproductive organs in cucumber.
The role of environmental factors and assimilates
Vegetative to reproductive phase transition of the meristem is very important for
fitness. Carbohydrates, day length, hormonal status and temperature are involved in
meristem switching from vegetative to reproductive phase in plant. Thus it is imperative
to understand how plants cope with stress during their reproductive phase, a condition
that affects agricultural productivity in this era of global warming and climate change
(Boyer and McLaughlin 2007; Barnabas et al. 2008; Thakur et al. 2010). Three major
factors can be said to control sex determination in oil palm. These factors include
abiotic factors (e.g. water stress), metabolic factors (e.g. carbon reserves), and genetic
factors. The effect of carbohydrates on Arabidopsis floral transition has been mentioned
in certain reports (Roldan et al. 1999; Ohto et al. 2001). Sugar supply can rescue late
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11!
flowering phenotypes and promote dark phase flowering (Roldan et al. 1999) while in
some cases, it was shown to significantly reduce flowering (Zhou et al. 2008). Sugar
concentration changes are associated to plant tissue senescence and abortion (Rolland et
al. 2002; Yoshida et al. 2002; Kakumanu et al. 2012). The expression of hexokinases
was linked to senescence in transgenic plants (Xiao et al. 2000). Carbohydrate related
genes have been linked to embryo abortion in maize (Zhuang et al. 2007). Adam et al.
(2011) proposed a mechanism of sex differentiation in oil palm that is presented in
Figure 2.2.
Photosynthesis
Water
Glucose
Starch
Hormone status
Genetic
factors
Female inflorescence
Indeterminate
inflorescences
Male inflorescence
Figure 2.2 Possible interactions resulting from the severe defoliation of oil palm as
proposed by Adam et al. (2011). The circles represent the biological
processes while the squares represent the products.
Adam et al. (2005) noticed a small group of cells at the base of oil palm leaves
and suspected them to be precursor cells for inflorescence meristem and suggested that
these cells should be identified to detect mRNA transcripts of putative genes. Figure 2.3
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12!
shows inflorescence buds at the base of leaves in a dissected meristem of a mature
pisifera palm.
Undetermined!flower!buds!
Shown!in!leaf!axil!beyond!@20!
Figure 1.3 Apical meristem of oil palm tree showing undetermined flower bud and
future leaves. The meristem was obtained from a dissected cross section of a
mature pisifera plant in the PT ASTRA Agro Lestari oil palm plantation in
Kalimantan.
!
The results of Adam et al. (2005; 2011) arise from the conclusions of previous
researchers such as Corley (1976) and Durand-Gasselin et al. (1999) who indicated that
the period between flower sex differentiation and flower maturity takes about 22
months. A diagrammatic representation of the activities between flower initiation and
flower maturity is shown in Figure 2.4.
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Figure 2.4 Stages of the developmental processes between sex differentiation and
flower maturity in oil palm as reproduced by Durand-Gasselin et al. (1999).
The top most row represents the leaf numbers. Leaf number 0 is the middle
unopened leaf commonly known as the spear. Leaf number +1 is the first
opened leaf outwards from leaf number 0. Leaf number -1 is the next leaf
that will succeed the spear. The leaf numbers carrying minus signs are unemerged leaves. It is assumed that leaf initiation takes place at leaf position
number -40 inside the apical meristem of the oil palm. Inflorescences are
initiated on every leaf axil from which it will later develop through to leaf
number +20 where it obtains maturity. The second row indicates the number
of months between inflorescence initiation and inflorescence maturity in an
adult oil palm tree. Adult oil palm trees emit two leaves every month while
younger trees emit 3 leaves every month. Younger trees obviously take
shorter time between inflorescence initiation and flower maturity that adult
trees. The period between inflorescence sex determination and inflorescence
maturity in younger palms is estimated to be 14.5 months. Inflorescence
maturity is attained when the flowers are in anthesis or receptive for
pollination.
The oil palm trunk as a carbohydrate reserve
Oil palm trunks contain considerable energy reserves that are used to supply
assimilate to sinks during periods of high-energy demand or during reduced
photosynthetic activities. The total extractable carbohydrate in the trunk of an eightyear-old palm amounted to 37 kg (Henson et al. 1999), 47.9 kg (Gray 1969) and 65%
(Legros et al. 2006) of the total carbohydrate of the plant. Sugars make up 68.6% of
t
FLOWERING ASSOCIATED GENES AND THEIR POTENTIAL
APPLICATION IN BREEDING PROGRAMMES
WALTER AJAMBANG NCHU
GRADUATE SCHOOL
BOGOR AGRICUTURAL UNIVERSITY
BOGOR
2015
DECLARATION OF ORIGINALITY AND AUTHENTICITY
INCLUDING TRANSFER OF COPYRIGHT*
This is to declare that the dissertation titled “Molecular Analysis of Oil Palm
(Elaeis guineensis Jacq.) Flowering Associated Genes and their Potential Application in
Breeding Programmes” is the result of my personal research under the direction of the
supervising committee and has never been presented in any form wherever. Any other
sources of information that have been mentioned in this dissertation from published or
unpublished works of other authors have been acknowledged in the text and included in
the reference chapter.
Based on this assertion, I therefore transfer the copyrights of this dissertation to the
Bogor Agricultural University.
Bogor, February 2015
Walter Ajambang Nchu
NIM A263118081
SUMMARY
WALTER AJAMBANG NCHU. Molecular Analysis of Oil Palm (Elaeis guineensis
Jacq.) Flowering Associated Genes and their Potential Application in Breeding
Programmes. Under the supervision of SUDARSONO as head, SINTHO
WAHYUNING ARDIE and HUGO VOLKAERT as members of the committee.
The oil palm (Elaeis guineensis Jacq.) is an important economic crop that is used
in the food, chemical and bio-diesel industries. Breeding to increase yields in oil palm
has been focused on improving the sex ratio of more female to male inflorescences.
Environmental and genetic factors largely influence oil palm growth and yields. Soil
water availability, especially in Sub Saharan Africa and source-sink balances have been
the main factors affecting sex ratio and inflorescence production in oil palm. Male
inflorescences have been induced in the highly feminine Pisifera race of oil palm by
carbohydrate depletion through complete defoliation. However, the efficiency of this
process was still a major problem especially in this era of climatic instability. Also, the
molecular genetic mechanisms underlying the process of male inflorescence induction
by complete defoliation are yet to be uncovered. The objective of this study was to
investigate the phenotypic responses and identify the molecular genetic mechanisms
responding to complete defoliation of oil palm in order to efficiently increase oil palm
seed production through improved pollen production and expand existing breeding
programmes. In this dissertation, we present the quantity of environmental factors and
the periods of treatment for which male inflorescence induction or sex determination is
effective and determine the exact moment at which sex differentiation genes are
initiated in response to complete defoliation treatment. Knowledge of this moment
enabled us to accurately isolate the genes and study the molecular mechanisms
underlying male inflorescence induction in oil palm caused by complete defoliation
using RNA-seq.
Complete defoliation significantly induced male inflorescence of up to 104%. An
acute soil water deficit of 16.8 mm between the 30th and 60th days after complete
defoliation (DAD) had a positive effect on male inflorescence induction. Generally,
complete defoliation treatments carried out during the dry seasons produced more male
inflorescences than during the wet season. Unlike inflorescence induction, inflorescence
emergence was highest during the wet season irrespective of the date of its induction.
Regression analysis on 18 time-specific, climate related variables indicated that oil palm
initiates response mechanisms between day 30 and day 60 after complete defoliation
stress. Coincidentally, total soluble sugar measured on completely defoliated trees and
control trees at 45 DAD, showed a sugar depletion of 55% in leaf tissues and 21% in
inflorescence tissues. Preferential sex induction should be a way of acclimation in oil
palm under carbohydrate depletion stress caused by complete defoliation.
Knowing that the oil palm initiates its responses during the second month after
complete defoliation stress, we extracted tissues from both control and completely
defoliated trees for RNA isolation and RNA-seq at 45 DAD. Samples were collected
from three early developmental and distinct consequential stages of the inflorescence.
Next Generation Sequencing (NGS) of 18 transcriptomes was performed on an Illumina
Hiseq2000 platform producing more than 96 000 transcripts of which about 50% were
up regulated. Difference in gene expression was significant between treatment and
between tissues. Gene Ontology analysis showed that 45% of differentially expressed
genes (DEG) responding to complete defoliation stress were located in the chloroplast
and mitochondrion. The chloroplasts and mitochondria are the two powerhouses of the
cell, one regulating carbohydrate production through photosynthesis and the other
regulating cell energy availability in the form of ATP. Similarly, the biological
processes that were functionally enriched from the DEG data set were photosynthesis
and carbohydrate related processes. Gene network analysis showed that abiotic stress
response DEGs were co-expressed with vegetative to reproductive phase transition of
the meristem, gibberellin, auxin, jasmonic acid and photoperiodism. This co-expression
confirmed that abiotic stress caused by complete defoliation was involved in male
inflorescence induction in oil palm, regulated by sugar status and plant growth
hormones. Genes linked to carbohydrate metabolism, flower development, stress
response, circadian rhythm, PGR and vegetative to reproductive phase transition of the
meristem may be responsible for the regulation of inflorescence emergence in oil palm.
Sequence data generated from this study should be used to compare the expression from
other races of the oil palm especially the commercial race, Tenera and Dura. These
sequences should be used to quantify gene expression on breeding populations in the
process of selecting parents for seed production
!
Keywords: complete defoliation, carbohydrate depletion, inflorescence induction, oil
palm, NGS, RNA-seq, genomics
RINGKASAN
WALTER AJAMBANG NCHU. Analisis Molekuler Gen-Gen yang Berkaitan dengan
Pembungaan dan Aplikasi dalam Pemuliaan Tanaman Kelapa Sawit. Dibimbing oleh
SUDARSONO sebagai ketua, SINTHO WAHYUNING ARDIE dan HUGO
VOLKAERT sebagai anggota komisi pembimbing.
Kelapa sawit (Elaeis guineensis Jacq.) adalah tanaman tropis yang digunakan
dalam industri makanan, kosmetik dan bio-diesel. Tujuan utama dalam pemuliaan
kelapa sawit adalah menaikan sex ratio bunga betina terhadap bunga jantan. Faktor
lingkungan dan genetik mempunyai peran besar dalam produktivitas kelapa sawit.
Ketersediaan air, terutuma di Afrika, dan ketersediaan kandungan kabohidrat
mempengaruhi sex ratio kelapa sawit. Pemangkasan daun sawit adalah salah satu cara
untuk menginduksi bunga jantan di kelapa sawit namun masih ada beberapa hal terkait
efisiensi teknik tersebut, apalagi gen-gen yang berperan dalam proses ini belum
diketahui. Penelitian ini bertujuan untuk menyelidiki perubahan fenotipe dan
menentukan gen-gen yang terekspresi akibat pemangkasan daun sawit. Dalam disertasi
ini, diberikan informasi tentang berapa jumlah faktor lingkungan yang dibutuhkan
untuk menginduksi bunga jantan pada kelapa sawit secara efisien. Selain itu,
diberitahukan juga berapa waktu yang dibutuhkan oleh kelapa sawit sebelum merespon
terhadap stres pemangkasan daun. Informasi ini digunakan untuk pelajari ekspresi gen
terkait pembungaan dalam kelapa sawit yang diakibatkan oleh stres pemangkasan daun.
Data menunjukan bahwa teknik pemangkasan daun bisa menaikan produksi bunga
jantan kelapa sawit sampai 104%. Teknik ini paling efisien di musim kering dan
optimum saat cekaman air sama dengan 16.8 mm dalam waktu 60 hari pertama setelah
pemangkasan daun. Analisis regresi menunjukan bahwa tanaman kelapa sawit akan
memberikan respon terhadap cekaman kabohidrat yang diakibatkan oleh pemangkasan
daun merupakan ekspresi gen dalam waktu 30 sampai 60 hari setelah pemangkasan.
Kadar gula yang diambil dari jaringan bunga dan daun kelapa sawit 45 hari setelah
pemangkasan menunjukan bahwa kadar gula menurun 21% di jaringan bunga dan 55%
di jaringan daun dibanding kontrol.
Ekspresi gen di jaringan bunga kelapa sawit mengunakan Next Generation
Sequencing (NGS) menujukan bahwa mayoritas gen gen yang berperan dalam regulasi
cekaman kabohidrat di kelapa sawit terletak di kloroplas dan mitokondria. Analisis
fungsi menggunakan Gene Ontology, menujukan bahwa gen gen tersebut berperan
dalam metabolisme kabohidrat dan fotosintesis. Analisis ko-ekspresi menujukan
keterkaitan antara cekaman kabohidrat dan induksi bunga serta peran zat pertumbuhan
tanaman seperti auxin, gibberellin dan jasmonic acid. Ini membuktikan bahwa gen gen
dalam lintasan cekaman kabihidrat yang diakibatkan oleh pemangkasan daun berperan
dalam induksi bunga di jantan tanaman kelapa sawit.
Kata kunci: pemangkasan daun, cekaman kabohidrat, induksi bunga, NGS, kelapa
sawit.
© Copyright IPB, 2015
Copyright protected under the law.
No part or all of this work may be reproduced without citing the source. Copying may
be done only on the basis of education, research, scientific writing, reports or review;
and which should not cause any prejudice to IPB.
No part or all of this work may be reproduced in any form or by any means without
permission from IPB.
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MOLECULAR ANALYSIS OF OIL PALM (Elaeis guineensis Jacq.)
FLOWERING ASSOCIATED GENES AND THEIR POTENTIAL
APPLICATION IN BREEDING PROGRAMMES
WALTER AJAMBANG NCHU
Dissertation
Submitted in partial fulfilment of the requirements for the degree
Doctor of Philosophy
in
Plant Breeding and Biotechnology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
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Examiners during Closed Exam:
Prof. Dr. Ir. Iskandar Z. Siregar M.Sc
Dr. Desta Wirnas, SP, M.Si
Examiners during Public Defence:
Dr. Ir. Sudrajat, MS
Dr. Adi Pancoro
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Dissertation Title
Name
NIM
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: Molecular Analysis of Oil Palm (Elaeis guineensis Jacq.)
Flowering Associated Genes and their Potential Application
in Breeding Programmes
: Walter Ajambang Nchu
: A263118081
Approved by
Supervisory committee
Prof. Dr.Ir.Sudarsono, M.Sc.
Head
Dr.Sintho W Ardie, SP, M.Si.
Member
Dr. Hugo Volkaert
Member
Endorsed by
Head of Mayor
Plant Breeding and Biotechnology
Dr. Ir. Yudiwanti Wahyu, E.K, MS.
Date of Public Defence:
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Dean Graduate School
Dr. Ir. Dahrul Syah, M.Sc. Agr.
Date Graduated:
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FOREWORD
Glory be to God the Father Almighty for his ever increasing guidance and
protection especially during the difficult moments the author went through while
undertaking studies in Bogor, without which all human efforts wouldn’t have borne any
fruits.
The author wishes to convey his sincere and special thanks to the head of the
supervisory committee Prof. Dr. Ir. Sudarsono, MSc. for his advice and remarks during
the preparation, execution and presentation of this dissertation. The author pays
gratitude to members of the supervising committee, Dr. Sintho W. Ardie, SP, M.Si and
Dr. Hugo Volkaert, for their resourceful instructions and comments on this work. The
author is grateful to Prof. Dr. Ir. Iskandar Z. Siregar M.Sc, Dr. Desta Wirnas, SP, M.Si,
Dr. Ir. Sudrajat, MS and Dr. Adi Pancoro who gave their precious time to respectively
serve as closed examiners and open examiners of this dissertation.
This project would never have existed without the vision and wisdom of Dr. Zok
Simon and Bapak Widya Wiryawan, former General Manager of IRAD Cameroon and
present CEO of ASTRA Agro Lestari Indonesia respectively. Special thanks go to
Bapak Bambang Palgoenadi, Dr. Ngeve Mbua Jacob and Dr. Woin Noe for bravely
executing the training aspect of this project. The author is indebted to Bapak Satyoso
Hartodedjo of ASTRA Agro, Dr. Koona and Dr. Ngando of CEREPAH for the smooth
management of this project.
The Late Joseph Asende Nchu has a special place in the author’s mind for
initiating and accompanying him in this long and exciting journey. The author is
grateful to Mama Comfort Namondo for her constant support during his stay abroad.
The author presents special gratitude to his family members especially the wife
Lylie Michele Ajambang and children Jeff, Megane and Naomi for their endurance
during his long absence from home. High appreciation is extended to the colleagues of
PMB and ASTRA labs for their technical and scientific assistance during the execution
of this project.
The author hopes to receive public criticisms that may improve the quality of
this dissertation. The author wishes that this dissertation makes an impact in science and
development especially in the field of oil palm breeding and genomics.
Bogor, February 2015
Walter Ajambang Nchu
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TABLE OF CONTENTS
TABLE OF TABLES
xiii
TABLE OF FIGURES
TABLE OF APPENDICES
xiii
xv
1 INTRODUCTION
Background
Problem statement
Objectives of research
Importance of research
Novelty
Scope, framework and limitations
2 LITERATURE REVIEW
Origin and botany of oil palm
Major changes caused by carbohydrate depletion
Hormones associated with flowering
Genes involved in floral induction and initiation in oil palm
The role of environmental factors and assimilates
The oil palm trunk as a carbohydrate reserve
Previous work on gene expression in oil palm with molecular methods
3 MASSIVE CARBOHYDRATE RESERVES BUFFERS AND DELAYS
RESPONSE TO STRESS CAUSED BY COMPLETE DEFOLIATION IN
OIL PALM (Elaeis guineensis Jacq.)
Abstract
Introduction
Materials and Methods
Results and discussions
Conclusion
4 INFLORESCENCE EMISSION TIME IN OIL PALM (Elaeis guineensis
Jacq) IS SEASONAL, IRRESPECTIVE OF INDUCTION DATE
Abstract
Introduction
Materials and Methods
Results and discussions
Conclusion
5 COMPARATIVE EXPRESSION PROFILING OF THREE EARLY
INFLORESCENCE STAGES OF OIL PALM RESPONDING TO
COMPLETE DEFOLIATION SHOWS THAT VEGETATIVE TO
REPRODUCTIVE PHASE TRANSITION OF MERISTEM IS CAUSED BY
SUGAR DEPLETION
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Abstract
Introduction
Materials and Methods
Results and discussions
Conclusion
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6 GENERAL DISCUSSIONS
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7 GENERAL CONCLUSIONS AND RECOMMENDATIONS
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REFERENCES
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APENDICES
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AUTOBIOGRAPHY
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TABLE OF TABLES
2.1
3.1
4.1
4.2
4.3
4.4
5.1
Cell and physiological responses of some major phyto-hormones
involved in flower sex determination and development, including
related references
Correlation coefficients for selected variables that had a significant
relationship with male inflorescence number
Number of inflorescences produced before and after defoliation
T-test for number of inflorescence emission between the wet and dry
season
Functional networks involving down regulated and up regulated
DEGs
Ten top DEGs based to FC and p-value
Summary data from the different libraries
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TABLE OF FIGURES
1.1
Flow chart of research activities
2.1
Un-emerged female inflorescence extracted from leaf axil number 14 and
un-emerged male inflorescences extracted from leaf number 13
Possible interactions resulting from the severe pruning of oil palm as
proposed by Adam et al. (2011). The circles represent the biological
processes while the squares represent the products
Apical meristem of oil palm tree showing undetermined flower bud and
future leaves
Stages of the developmental processes between sex differentiation and
flower maturity in oil palm as reproduced by Durand-Gasselin et al. (1999).
The top most row represents the leaf numbers. Leaf number 0 is the middle
unopened leaf commonly known as the spear. Leaf number +1 is the first
opened leaf outwards from leaf number 0. Leaf number -1 is the next leaf
that will succeed the spear. The leaf numbers carrying minus signs are unemerged leaves. It is assumed that leaf initiation takes place at leaf position
number -40 inside the apical meristem of the oil palm
Complete defoliation on mature pisifera trees. (a) Mature non defoliated
pisifera palm (b) completely defoliated pisifera palm. (c) A recovering
pisifera palm 45 DAD. The arrow shows the number of leaves that have
been produced after complete defoliation. (d) A mature male inflorescence
at anthesis. Anthesis is when pollen on the flowers has stained maturity and
is capable of fertilising female flowers. The white bars on (a) and (b) are
drawn to 1 m scale while that of d is 10 cm scale. Arrow points to newly
2.2
2.3
2.4
3.1
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3.2
3.3
3.4
3.5
3.6
3.7
3.8
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
5.1
5.2
5.3
5.4
5.5
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developed oil palm leaves after complete defoliation.
Leaf counting during inflorescence extraction
Monthly soil water deficit (curve) and monthly precipitation (bars)
recorded between 2007 and 2013 at La Dibamba Cameroon
Male inflorescence production and mean annual precipitation between 2001
and 2012 at La Dibamba before and after treatment. The broken line
between year 2006 and 2007 represents the start of defoliation treatment.
The number of male inflorescences produced is represented by bars
corresponding to the primary vertical axis while precipitation curve is
represented by the secondary vertical axis
The relationship between precipitation, rainy days and male inflorescence
induction on plants subjected to complete defoliation stress
Stages leading to tissue extraction for total soluble sugar analysis. (a) a
mature pisifera being felled 45 DAD, (b) transportation of the reduced
crown to the laboratory for tissue extraction, (c) leaf and inflorescence
extraction (d) inflorescence located at leaf axil + 5 (on red background)
placed on the leaf petiole
Total soluble sugar content in inflorescences and leaves from non
defoliated (control) plants and previously defoliated plants 45 days after
defoliation
The effect of increasing soil water deficit on male inflorescence production
Distribution of inflorescence emission in months after complete defoliation
Average monthly emission of male inflorescences based on the season of
defoliation treatment
Distribution of gene expression level in non-defoliated (blue) and
defoliated tissues (red)
Venn diagram of DEGs on stress and control tissues
Volcano plot for DEGs between non-defoliated and defoliated samples.
The y-axis represents the level of significance of the expression change
between samples measured on –log10 p-value (pval = 0.05), while the xaxis represents the fold change of DEGs. DEGs located above the red line
are significant based on the p-value while those below are not significant.
The DEGs located between the two blue lines are non DEGs based |FC| ≥ 2
Functional categoristaion of DEGs based on biological processes
Average monthly male inflorescence production and mean monthly
precipitation for the period between 2007 and 2012 recorded at La
Dibamba Oil Palm Research Centre
Average monthly FFB production and mean monthly precipitation between
2007 and 2012 at La Dibamba Oil Palm Research Centre
Extracted inflorescences from axils of palm leaves
A dissected oil palm showing a series of inflorescence buds enclosed in the
axils of future leaves
Distribution of expression levels based on FC between the non defoliated
(blue) and defoliated (red) tissues
Volcano plots for DEGs for the three different comparisons (a) axil -20/axil
-27 (b) axil +5/axil -27 (c) axil +5/axil -20
Differential gene expression at FC ≥ 2 and p ≤ 0.05 between inflorescence
stages under stress
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5.7
5.8
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Gene bar chart for (a) up regulated DEGs (b) down regulated DEGs from
three inflorescence stages based on cellular component
Gene bar chart for (a) up regulated DEGs and (b) down regulated DEGs
from three inflorescence stages based on molecular functions
Gene bar chart for (a) up regulated DEGS and (b) down regulated DEGs
from three inflorescence stages based on biological process
Gene set enrichment score from the comparison between inflorescence at
leaf axil +5 against inflorescence of leaf axil -27
Gene set enrichment score from the comparison between inflorescence
located at leaf axil -20 against inflorescence of leaf axil -27
Gene set enrichment score from the comparison between inflorescence at
leaf axil +5 against inflorescence located at leaf axil -20
Network of related genes from the abiotic stress process analysed on Gene
MANIA
Network of related genes from the reproductive process analysed on Gene
MANIA
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TABLE OF APPENDICES
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6.
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Male flower and pollen production before and after complete defoliation
List of RNA samples extracted on both leaves and flowers for RNA-seq
Box plot for normalised transcripts
Tree diagram for samples based on inter sample correlation
Differential expression of genes between treatments
Differential expression of genes between samples under stress
Functional annotation chart from DAVID
RNA Integrity Number (RIN)
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CHAPTER I
INTRODUCTION
Background
Oil palm (Elaeis guineensis Jacq.) is the world’s leading oil crop with 35% of
total world consumption of vegetable oils (Soy stats 2014). Oil palm is cultivated on
approximately 15 million hectares across the world and consumption is expected to
double by the year 2020 (FAO 2009). Apart from its traditional use as a source of food,
oil palm is used in the manufacture of margarine, pharmaceuticals, soap and cosmetics,
animal feed and organic manure, building material, furniture and biofuel. Production of
biodiesel from oil palm has been increasing in recent years, particularly in Africa and
Latin America (FAO 2010; Mitchell 2011).
Socio-economic benefits of a sustainable oil palm plantation could include
poverty alleviation and long-term employment opportunities (Badrun 2010; Kurniawan
2010; Norrochmat and Hadianto 2010; Pahan 2011). Profit sharing may provide a
further incentive, attracting more workers to the palm oil sector, along with better living
and working conditions (Albán and Cárdenas 2007). Indonesia’s palm oil export was
the major force behind the stabilisation of its economy during the 2008-2009 global
financial crisis. Oil palm production has favoured the development of upstream
companies such as the seed industry, fertiliser, agrochemicals, agro mechanicals and
financial services; side stream companies and downstream companies in Cameroon.
Problem statement
The commercial cultivation of oil palm is faced with many problems ranging
from land availability, environmental issues, social issues, agronomy, diseases, climate
change and the availability of quality seeds. Seed availability is the foundation for any
agricultural project. There are three different races of the cultivated oil palm species,
categorised basically by the thickness of the shell, which is controlled by a co-dominant
monogenic inheritance (Beinaert and Vanderweyen 1941). Moretzsohn et al. (2000)
identified the allele pairs of the gene as sh+/sh+ for dura race, sh+/sh- for tenera race
and sh-/sh- for the pisifera race. The commercial oil palm cultivar is a hybrid called
Tenera denoted as T or DP derived from a cross of two homozygous parents based on
shell thickness known as the Dura or female parent represented by DD and the Pisifera
or male parent represented by PP. In the process of seed production in oil palm, a
trained pollinator artificially carries out pollination. Pollen is harvested from the Pisifera
parent and then manually pollinated on to the Dura inflorescences. The quantity and
quality of pollen used in this pollination process is highly related to the quantity and
quality of total seed produced.
The Pisifera is very feminine under optimal plantation environmental conditions.
Pisifera rarely produces male inflorescences that can be used as pollen. Also, the
Pisifera used in seed production are descendants of the group B sub population of oil
palm, which are known to inherit a high sex ratio of female flowers to male flowers.
Breeding for yield traits has favoured the improvement of female flowers than male
flower. This is because the main parameters breeders have been evaluating in breeding
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programmes are the number of bunches per tree (BNO) and the total bunch weight
(BW), which are both heritable (Hardon et al. 1985; Rafii et al. 2002). Under normal
environmental conditions, seed producers may have to import pollen from countries
with sub optimal environmental conditions such as the arid zones of Africa. In addition
to high expenditure, pollen importation has to undergo the tight regulations concerning
the transfer of biological organisms. In Cameroon, the seed production unit spends 50%
of its production cost on pollen importation. Thus it was imperative for each seed
production centre to be able to produce its own pollen locally in order to reduce seed
production costs and also to save time and energy in handling complex international
regulations regarding international transfer of biological material.
Hardon and Corley (1976) reported that oil palm produces male inflorescence
after it has undergone a high production of fruit bunches. When the plant produces
Fresh Fruit Bunches (FFB), it losses energy as bunches are continuously harvested from
the plant. In contrast, the Pisifera race aborts its fruits immediately after fertilisation and
hence does not produce bunches. Therefore most of its energy is stored in the trunk and
a lesser part of it is used for plant maintenance. This means that the Pisifera plant shall
never loss enough energy to let it undergo the required depletion of stored energy that
will enable it to produce male inflorescences. Corley (1976) and Durand Gasselin et al.
(1999) reported that severe defoliation of Pisifera leaves induces male inflorescence in
oil palm, and that the treatment was most effective during the dry season in Ivory Coast.
This is an indication that oil palm sex differentiation is strongly affected by climatic
factors, with male inflorescence being influenced by carbon depletion and water deficit.
Although phenological studies have been carried out which suggests points of
interaction between genetic and the environment factors in the induction of male
inflorescences on oil palm (Corley 1976; Uhl 1988; Van Heel et al. 1987; Tomlinson
1990; Durand-Gasselin et al. 1999; Adam et al. 2011), nothing is yet known about the
nominal values of climatic factors, the exact amount of time necessary for the oil palm
to respond and initiate the mechanism of male inflorescence induction, genetic basis of
sex determination, nor the molecular mechanisms by which these processes are
regulated in oil palm. A number of questions concerning the mode of interaction
between molecular-genetic processes and sex differentiation still remain unanswered
(Adam et al. 2011; Walter et al. 2012).
The development of molecular biology, supported by sophisticated
bioinformatics, can be used to understand the genetic basis and molecular mechanisms
underlying the process of sex differentiation in oil palm. Molecular biology methods
such as Next Generation Sequencing (NGS) have been used on genomic DNA and RNA
transcripts by several researchers in order to assign putative functions to genes
(Jouannic et al. 2005; Adam et al. 2005; Ho et al. 2007; Adam et al. 2007; Low et al.
2008; Adam et al. 2011; Jen et al. 2013).
RNA-seq is the sequencing of cDNA derived from cellular RNA by using
massively parallel sequencing technologies such as Sanger, Roche 454 and Illumina.
RNA-seq can show a repertoire of the genes expressed in a particular tissue such as
flowers and meristem tissues of oil palm and at a specific time point. In this way, RNAseq can reproduce a nearly complete picture of transcriptomic events taking place in a
biological sample and its data can be used to characterise and quantify expressed genes
(Wang et al. 2009; Riggins et al. 2010; Brautigam et al. 2011). RNA-seq is practical in
non model species which do not yet have a reference genome. It is efficient in cases
where resources may be limited because sequencing will target only coding regions. Jen
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et al. (2013) reported that among the three commonly RNA-seq used technologies,
Illumina showed better gene coverage than Roche 454 and Sanger on oil palm tissues
because of its higher sequencing depth.
Objective of research
Investigate the phenotypic responses and identify the molecular genetic mechanisms
responding to complete defoliation of oil palm in order to efficiently increase oil palm
seed production and also expand existing breeding programs.
Specific objectives
1. Explore the effects of complete defoliation of Pisifera on male inflorescence
induction.
2. Estimate the critical moment in which genes are expressed in response to
complete defoliation in order to be able to isolate and clone these genes for
further molecular and downstream research.
3. Determine the effects of voluntary inflorescence induction by complete
defoliation on the seasonal trend of inflorescence emergence.
4. Identify the molecular mechanisms associated to complete defoliation of oil
palm.
Importance of research
1. This research shall permit us to know the exact time at which genes are switched
on in response to complete defoliation treatment.
2. Understand the precise period during the year for which complete defoliation is
effective in producing male flowers.
3. The specific period of gene expression will permit a successful isolation of
target genes expressed as a result of complete defoliation.
4. The molecular processes regulating male inflorescence induction and emergence
in oil palm shall be identified.
Novelty
Durand-Gasselin et al. (1999) initiated work on oil palm defoliation in the Ivory
Coast as a method of male inflorescence induction in oil palm. They based their study
on the phenotypic response to severe pruning of oil palm leaves. Some work has been
done on oil palm gene expression by researchers such as Jouannic et al. (2005), Adam
et al. (2007), Ho et al. (2007), Tranbarger et al. (2011) and Morcillo et al. (2013)
focusing on different oil palm tissues using techniques such as EST, micro array and
other hybridisation. An incomplete oil palm genome is available from the Malaysian Oil
Palm Board (Singh et al. 2013) and is stored in the domain
http://www.genomsawit.mpob.gov.my.
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Previous research has not indicated the moment at which genes are expressed in
response to complete defoliation. ESTs collected from oil palm flower tissues were not
based on any previous flower induction treatment. The collection of ESTs were not time
specific nor tissue specific. This present research is unique in the sense that;
i.
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iii.
iv.
v.
vi.
Samples were collected from tissues that have been stressed and compared with
tissues in normal conditions.
Samples were collected with respect to the spatial-temporal expression of genes
responsible for sex differentiation.
Flowering was induced by the application of a treatment whereas the other
researchers did not induce flowering.
NGS through RNA-seq was used to identify genes associated with male floral
induction. Specific primers from MADS box genes were not used as was done
by other authors.
No prior hybridisation such as Suppression Subtractive Hybridisation (SSH) or
Fluorescence in situ Hybridisation (FISH) techniques in order to identify only
previously identified genes but carried out sequencing, assembly and annotation
with bioinformatics enabling us to identify new transcripts.
An attempt was made to identify genes associated with floral induction and sex
differentiation rather than studying genes associated with floral morphology.
Scope, framework and limitations of the research
This research is based on four inter-linked objectives aiming at contributing to
the development of oil palm, specifically oil palm genomics, breeding and seed
production. These activities were carried out in different locations because of the
perennial nature of the plant and also because of the availability of laboratory facilities.
The four activities were:
1. The complete defoliation of Pisifera parent plants.
2. RNA-seq on flower related tissues extracted on Pisifera parents after
complete defoliation.
3. Analysis of gene expression levels associated to male flower induction.
4. The quantification of gene expression levels associated to male inflorescence
induction
The first activity was carried out on selected parents used in oil palm seed
production in Cameroon and Indonesia. They were of the LM2T origin and were self
pollinated at La Me Ivory Coast in 1990 and later planted at the oil palm research centre
in Cameroon in 1993. Part of this population has been transferred to Indonesia in South
East Asia for collaborative research and breeding purpose. Under the conditions of the
research station where these Pisifera are planted (i.e. La Dibamba of Cameroon with
rainfall of between 2 500 - 3 000 mm and an annual average temperature of 28 °C), it
has been difficult for these plants to produce male flowers. La Dibamba had to carry out
some trials in order to reduce seed production cost by inducing the Pisifera plants to
produce male inflorescences and hence pollen. Data collected on the variables is
presented in Chapter three of this dissertation.
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Experiment two shall be developed based on the first results obtained in
experiment one. By using mathematical models, we shall be able to predict the time and
amount of environmental conditions necessary for switching gene expression in
response to complete defoliation. Samples of tissue shall be extracted from the
completely defoliated tree and unstressed tree to identify the group of Differentially
Expressed Genes (DEG) associated with this stress and male inflorescence induction in
Pisifera. Next Generation Sequencing will be used to sequence transcripts expressed in
response to the complete defoliation of the Pisifera. A simulated defoliation activity,
tissue extraction and RNA isolation shall be carried out on Pisifera at the PT ASTRA
Research Centre in Kumai Central Kalimantan. NGS shall be carried out in the
Macrogen laboratories in South Korea.
Experiment three and four shall be based on the results of experiment two. The
level of expression of different transcripts found in the different tissues shall be
compared with the normal level of expression contained in the oil palm reference
genome.
Oil palm is a perennial crop and enters production only at the 4th year after
planting. This makes it difficult to plan and execute a field research within time limits
for a dissertation. The blocks used for experiment 1 are thus carved out after the crop
had been planted because these are plants that are 20 years of age. The impossibility to
extract tissues from Cameroon and carry them to Indonesia for RNA isolation led us to
simulate the experiment in Kalimantan taking into consideration the result of the trial in
Cameroon. This permitted us to extract the tissues from a similar plant in Kalimantan
Indonesia where the laboratory facilities are available. The flow chart of research
activities is presented in Figure 1.1.
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Exp 1. Complete defoliation of Pisifera
Phenotypic and physiological responses
Analysis of climatic and time factors
Exp 2. Extraction of flower related tissues for mRNA isolation
mRNA isolation
Quality control
NGS
Sequence processing
Quality control
Transcriptome mapping to reference genome
Transcriptome
characterisation/ gene
annotation
Differential gene
expression analysis
Gene ontology
Exp 3. Transcript quantification/expression levels
Figure 1.1 Flow chart of research activities
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CHAPTER II
LITERATURE REVIEW
Origin and botany of oil palm
The oil palm presently exists in the cultivated and wild states in the equatorial
and tropical regions of Africa, South East Asia and South and Central America. Many
authors have put forward various arguments on the origin of the oil palm (Zeven 1964;
Hartley 1988; Soh et al. 2003). There is no doubt that the cultivated African oil palm
Elaeis guineensis Jacq. is of African origin because there is supporting pre historic
evidence that it originated from the area around Nigeria, Cameroon (Gulf of Guinea)
and the Congo (Zeven 1964; Hartley 1988). Early European traders collected oil palm
seeds from Africa and planted them in The Amsterdam Botanic Gardens, Netherlands
and later transferred it to the Bogor Botanic Gardens Indonesia, then a Dutch colony in
the year 1848. The four palms planted in Bogor then became the foundation stock for
the development of the world oil palm industry (Hunger 1917).
The oil palm is a diploid (2n = 2x = 32) classified under the family Arecaceae
(Dransfield et al. 2005). The crown of a mature palm consists of between 30 to 50
leaves. The number of leaves produced annually by a plantation palm decreases from 30
to 40 after 2 years of age to an average of 20 to 25 per annum after 8 years of age. The
roots of the oil palm can reach a horizontal distance of 16 m and a vertical distance of 8
m in well drained soils. Secondary roots develop on primary roots and tertiary roots
develop on secondary roots forming a network of dense root system (Corley and Tinker
2003). The oil palm is monoecious, with male and female inflorescences occurring
separately on the same tree (Dransfield et al. 2008). The female and male inflorescences
are shown in Figure 2.1.
The oil palm is commonly divided into three races based on their shell thickness
(sh). The sh gene had been discovered by Beinaert and Vanderweyen (1941). The Dura
has a shell thickness between 2 to 8 mm; the Tenera 0.2 to 2 mm and the Pisifera has no
shell. The fleshy mesocarp of Dura yields between 15 to 17%, oil that of Tenera yields
between 21 to 23% oil and Pisifera more than 23% oil. The Pisifera mostly aborts its
fruits and thereby produces virtually empty bunches, thus it is not cultivated on large
scale for commercial purpose.
Major changes caused by carbohydrate depletion in plants
Several factors are responsible for source sink imbalances in plants. Instability in
climatic and environmental factors such as rainfall, soil water availability, light and
temperature and attack by pathogens may greatly reduce carbohydrate supply to sink
tissues (Ho et al. 2001; Rolland et al. 2002; Xu and Zhou 2006; Xue et al. 2008;
Janecek and Klimesova 2014). Drought caused significant reduction in starch ranging
from 54% to 60% on all the organs of P. radiata (Mitchell et al. 2012), defoliation
reduced water soluble carbohydrates concentration by 43% in Lolium perenne (Lee et
al. 2010), while defoliation reduced carbohydrate production in the baobab (Lee et al.
2010; Simbo et al. 2013).
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Figure 2.1 Un-emerged female inflorescence extracted from leaf axil number 14 (left)
and un-emerged male inflorescences extracted from leaf number 13 (right).
The inflorescence from leaf axil number 14 is two weeks older than the
inflorescence from leaf axil number 13. Female inflorescences are always
longer and larger than their corresponding male inflorescence of similar age
and from the same plant.
Sugar deprivation can cause substantial physiological and biochemical changes in
plants leading to potato tuber induction (Simko 1994), changes root system and crown
morphology in tobacco (Paul and Stitt 1993) and changes in sex ratio in oil palm
(Durand-Gasselin et al. 1999). Primary response to stress involves the accumulation of
water-soluble carbohydrates such as sucrose, glucose, fructose and fructans (Xu and
Zhou 2006). Sugar responsive genes coordinate information on sugar status between the
source and sink locations. Morphological changes resulting from sugar level alterations
can be explained by sugar’s interaction with plant growth hormones such as gibberellin
in germinating seeds, cytokinin and auxin (Vincentz et al. 1993).
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Hormones associated with flowering
Concerning hormones associated with oil palm sex differentiation, Corley
(1976) observed that the growth regulator auxin NAA (naphthalene acetic acid),
favoured female flower production in palms grown in polythene bags. The
masculinising effect of GA3 has also been reported in cucumber (Fuchs et al. 1977;
Pimenta Lange et al. 2012). GA, ABA, IAA and Cytokinin content were found to be
higher in the developmental stages of male flower than in female flowers in poplar
(Song et al. 2013). In the much-studied Arabidopsis, individual pathways that include
photoperiod (Srikanth and Schmid 2011), vernalisation (Kim et al. 2009), autonomous
(Mouradov et al. 2002), ageing (Fornara and Coupland 2009) and GA (MutasaGottgens Hedden 2009), regulate flowering time. The GA4 which is most likely
produced in the leaves and transported to the meristem up regulates one or both of the
LFY genes, a floral meristem identity gene and the SUPRESSOR OF
OVEREXPRESSION OF CONSTANS 1 (SOC1), a floral integrator leading to flowering
(Bernier and Perilleux 2005).
Table 2.1 Cell and physiological responses of some major phyto-hormones involved in
flower sex determination and development, including related references
Hormones
Abscisic acid
Auxin
Brassinosteroid
Cytokinin
Ethylene
Gibberellic acid
Jasmonic acid
Cell and physiological responses
Flowering initiation, flower, leaf
and
fruit
senescence
and
abscission,
stomata
closing,
concentration increases during
drought
Increases cell elongation and
division, initiation of floral
primordia, specifies the number
and identity of floral organs
Slows abscission, stimulates cell
division
Regulates cell division and delays
senescence, affect meristem size,
required for pollen development
Species
Populus
tomentosa
Reference
Song et al.
(2013)
Oil
palm, Corley
Populus
(1976);
tomentosa
Song et al.
(2013)
Maize
Hartwig et
al. (2011)
Soybean,
Wong et al.
Nicotiana
(2013);
plumbaginifolia Vincentz et
al. (1993)
Floral transition, feminising role Arabidopsis
Achard et
in flower development, induces
al. (2007)
fruit ripening, synthesis increases
as a result of stress
Masculinising
effect,
induce Lolium
King
and
phase transitions and stimulates temulentum
Evans 1991
fruit development
Developing
inflorescences, Arabidopsis
Reinbothe et
defensive responses to stress
al. 2009
ABA, jasmonates and stress inducible genes are co-regulated by sugars in plant
tissues (Reinbothe et al. 2009; Sadka et al. 1994;). GA had been suggested to be
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10!
responsible for stamen abortion in maize inflorescences (DeLong et al. 1993; Dellaporta
and Calderon-Urrea 1993) although it has the opposite effect on cucumber. The role of
phytohormones in flower development and sex determination is summarised in Table
2.1.
Genes involved in floral induction and initiation in oil palm
Flower initiation includes all of the development processes necessary for the
irreversible commitment by the meristem to produce a flower or inflorescence.
Vegetative to reproductive phase transition of meristem results from consequential
activities of floral meristem identity genes that specify floral meristem fate and of floral
organ identity genes that determine the pattern of whorl establishment. Floral meristem
identity genes stimulate floral organ identity genes to promote floral development. The
floral organ identity genes are designated as ABC classes of homeotic genes. The ABC
model includes the APETALA 1,2,3 (AP); PISTILLATA 1 (P1) and AGAMOUS (AG).
The class A genes (AP1 and AP2) are responsible for sepals identity, the class B (AP3
and P1) plus the class A genes are responsible for petal identity, the class C (AG) plus
the class B genes are responsible for stamens identity and the class C genes alone
specifies the carpel identity. The SUPERMAN genes maintain whorl boundaries.
Superman mutants in Arabidopsis develop extra stamens at the expense of carpels,
rendering carpels defective or under developed (Schultz et al. 1991; Nibau et al. 2011).
It is believed that in unisexual flowers, sex is determined by the selective
repression of growth or abortion of either the male or female reproductive organs. It is
known that the identity of reproductive organs is controlled by homeotic genes
belonging to the MADS box gene family. Kater et al. (2001) reported that the arrest of
either male or female organ development is dependent on their positions in the plant.
Although sex determination in animals is well studied, little is known about the
molecular processes involved in plant sex determination. Only few genes that influence
sex have been cloned. The TASSELSEED2 gene in maize which is responsible for the
abortion of pistil primordia in the tassel (DeLong et al. 1993) and the ANTHER EAR1
gene (Bensen et al. 1995) which encodes an enzyme in the gibberellin biosynthesis
pathway responsible for stamen abortion in maize ear. The class B and C members of
the MADS box family encode putative transcription factors with a highly conserved
DNA binding domain and may influence the sex of plants. They suggest a role of the
class C genes in controlling the arrest of the reproductive organs in cucumber.
The role of environmental factors and assimilates
Vegetative to reproductive phase transition of the meristem is very important for
fitness. Carbohydrates, day length, hormonal status and temperature are involved in
meristem switching from vegetative to reproductive phase in plant. Thus it is imperative
to understand how plants cope with stress during their reproductive phase, a condition
that affects agricultural productivity in this era of global warming and climate change
(Boyer and McLaughlin 2007; Barnabas et al. 2008; Thakur et al. 2010). Three major
factors can be said to control sex determination in oil palm. These factors include
abiotic factors (e.g. water stress), metabolic factors (e.g. carbon reserves), and genetic
factors. The effect of carbohydrates on Arabidopsis floral transition has been mentioned
in certain reports (Roldan et al. 1999; Ohto et al. 2001). Sugar supply can rescue late
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11!
flowering phenotypes and promote dark phase flowering (Roldan et al. 1999) while in
some cases, it was shown to significantly reduce flowering (Zhou et al. 2008). Sugar
concentration changes are associated to plant tissue senescence and abortion (Rolland et
al. 2002; Yoshida et al. 2002; Kakumanu et al. 2012). The expression of hexokinases
was linked to senescence in transgenic plants (Xiao et al. 2000). Carbohydrate related
genes have been linked to embryo abortion in maize (Zhuang et al. 2007). Adam et al.
(2011) proposed a mechanism of sex differentiation in oil palm that is presented in
Figure 2.2.
Photosynthesis
Water
Glucose
Starch
Hormone status
Genetic
factors
Female inflorescence
Indeterminate
inflorescences
Male inflorescence
Figure 2.2 Possible interactions resulting from the severe defoliation of oil palm as
proposed by Adam et al. (2011). The circles represent the biological
processes while the squares represent the products.
Adam et al. (2005) noticed a small group of cells at the base of oil palm leaves
and suspected them to be precursor cells for inflorescence meristem and suggested that
these cells should be identified to detect mRNA transcripts of putative genes. Figure 2.3
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12!
shows inflorescence buds at the base of leaves in a dissected meristem of a mature
pisifera palm.
Undetermined!flower!buds!
Shown!in!leaf!axil!beyond!@20!
Figure 1.3 Apical meristem of oil palm tree showing undetermined flower bud and
future leaves. The meristem was obtained from a dissected cross section of a
mature pisifera plant in the PT ASTRA Agro Lestari oil palm plantation in
Kalimantan.
!
The results of Adam et al. (2005; 2011) arise from the conclusions of previous
researchers such as Corley (1976) and Durand-Gasselin et al. (1999) who indicated that
the period between flower sex differentiation and flower maturity takes about 22
months. A diagrammatic representation of the activities between flower initiation and
flower maturity is shown in Figure 2.4.
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13!
Figure 2.4 Stages of the developmental processes between sex differentiation and
flower maturity in oil palm as reproduced by Durand-Gasselin et al. (1999).
The top most row represents the leaf numbers. Leaf number 0 is the middle
unopened leaf commonly known as the spear. Leaf number +1 is the first
opened leaf outwards from leaf number 0. Leaf number -1 is the next leaf
that will succeed the spear. The leaf numbers carrying minus signs are unemerged leaves. It is assumed that leaf initiation takes place at leaf position
number -40 inside the apical meristem of the oil palm. Inflorescences are
initiated on every leaf axil from which it will later develop through to leaf
number +20 where it obtains maturity. The second row indicates the number
of months between inflorescence initiation and inflorescence maturity in an
adult oil palm tree. Adult oil palm trees emit two leaves every month while
younger trees emit 3 leaves every month. Younger trees obviously take
shorter time between inflorescence initiation and flower maturity that adult
trees. The period between inflorescence sex determination and inflorescence
maturity in younger palms is estimated to be 14.5 months. Inflorescence
maturity is attained when the flowers are in anthesis or receptive for
pollination.
The oil palm trunk as a carbohydrate reserve
Oil palm trunks contain considerable energy reserves that are used to supply
assimilate to sinks during periods of high-energy demand or during reduced
photosynthetic activities. The total extractable carbohydrate in the trunk of an eightyear-old palm amounted to 37 kg (Henson et al. 1999), 47.9 kg (Gray 1969) and 65%
(Legros et al. 2006) of the total carbohydrate of the plant. Sugars make up 68.6% of
t