Oil palm (Elaeis guineensis Jacq.) var. tenera in vitro embryogenesis effectiveness from young leaves, mature zygotic embryo and immature female flower explants
OIL PALM (Elaeis guineensis Jacq.) var. Tenera in Vitro
EMBRYOGENESIS EFFECTIVENESS FROM YOUNG
LEAVES, MATURE ZYGOTIC EMBRYO AND IMMATURE
FEMALE FLOWER EXPLANTS
MONDJELI CONSTANTIN
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
2012
DECLARATION OF ORIGINALITY AND AUTHENTICITY
This is to declare that the thesis titled « Oil Palm (Elaeis guineensis Jacq.) var.
Tenera In Vitro Embryogenesis Effectiveness from Young Leaves, Mature Zygotic
Embryo and Immature Female Flower Explants » is the result of my personal work under
the direction of the supervising committee and has never been presented in any form where
ever. Any other source of information that has been mentioned in this thesis from published or
unpublished works of other authors has been acknowledged in the text and included in the
reference chapter.
Bogor, December 2011
Mondjeli Constantin
NRP: A253098021
ABSTRAK
MONDJELI CONSTANTIN. 2011. Keefektifan Embriogenesis in Vitro Kelapa Sawit
(Elaeis guineensis Jacq. var. Tenera) dari Eksplan Daun Muda, Embrio Zigotik Matang
dan Bunga Betina Immature. Dibawah bimbingan NI MADE ARMINI WIENDI dan ADE
WACHJAR.
Tiga percobaan terpisah terkait sumber eksplan kelapa sawit varietas Tenera meliputi
daun muda, embrio zigotik matang dan bunga betina immature, yang diambil dari pohon
induk berumur 9 dan 13 tahun, telah dilakukan untuk menguji responnya terhadap produksi
embrio somatik. Pada berbagai komposisi media, eksplan-eksplan ini ditanam pada media
dasar MS dan Euwens yang mengandung kombinasi ZPT NAA (107.41µM) dan 2,4-D (0, 22.62,
45.24 atau 67.86 µM) dan media dasar MS dengan konsentrasi ZPT tinggi picloram 450µM
atau 2,4-D 450µM mengunakan 0,03% (w/v) arang aktif untuk menginduksi kalus embriogenik.
Persentase oksidasi berkisar antara 24 – 100% untuk eksplan daun dan antara 0 – 46.67%
pada eksplan embrio zigotik, sementara pada eksplan bunga 100%. Tingkat kontaminasi
berkisar antara 1.11 sampai 100 % pada eksplan daun, 0 – 35.56% pada eksplan embrio
zigotik dan 8.33 - 41.67% pada eksplan bunga betina. Pada 28 minggu setelah penanaman,
didapatkan kalus globular yang kompak dan putih dari daun nomor 5 (L5). Media MS dengan
kombinasi ZPT NAA (107.41µM) dan 2,4-D (67.86 µM) merupakan media yang optimal
untuk induksi kalus embriogenik (3.63%), sementara media MS dengan NAA (107.41µM)
menghasilkan persentase pembentukan kalus tertinggi (30.56%). Pada 36 minggu setelah
penanaman, didapatkan embrioid langsung dari daun nomor 6 (L6) pada media MS yang
mengandung kombinasi ZPT NAA (107.41µM) dan 2,4-D (45.24 µM). Pada eksplan embrio
zigotik dewasa, hasil kalus embriogenik terbaik (7,90%) didapatkan pada media Euwens
yang mengandung NAA (107.41µM) setelah 28 minggu. Persentase pertumbuhan tunas
tertinggi (38.33%) juga didapatkan pada media yang sama (NAA 107.41µM). Pada eksplan
bunga betina immature, tidak dijumpai kalogenesis dalam kurun waktu penelitian ini. Setelah
4 kali subkultur berturut turut pada media yang sama dengan konsentrasi zat pengatur
tumbuh yang menurun bertahap, kalus embriogenik dan embrioid tidak dapat berkembang
dalam kurun waktu penelitian ini, dari eksplan daun muda dan embrio zygotic matang.
Kata kunci: Embriogenesis somatik, kelapa sawit, daun muda, bunga betina muda, embrio
zigotik
SUMMARY
MONDJELI CONSTANTIN. 2011. Oil Palm (Elaeis guineensis Jacq.) var. Tenera in
Vitro Embryogenesis Effectiveness from Young Leaves, Mature Zygotic Embryo and
Immature Female Flower Explants. Under the supervision of NI MADE ARMINI
WIENDI and ADE WACHJAR.
Oil palm (Elaeis guineensis Jacq.) is an important commercial crop for all the
producer countries. It is the world‟s leading vegetable oil crop. In the last decade, the interest
in palm oil as biofuel was eventually caused constraints on worldwide supply for edible palm
oil. In addition, low production and lack of conventional seeds and seedlings are always
observed from the potential producer countries. More than one hundred million of tissue
culture plantlets are needed per year. Indonesia is the largest world producer country with
about 23 million tons of palm oil per year. The study was carried out to find in short term the
optimum medium for callus induction and somatic embryo formation from young leaves,
mature zygotic embryo and immature female flower explants of oil palm (E.guineensis Jacq.)
var.Tenera. Three different independent experiments related to explant sources of oil palm
(Elaeis guineensis Jacq.) var. Tenera include young leaf, mature zygotic embryo and
immature female flower explants isolated from 9 and 13 years old trees were investigated on
somatic embryos production. These explants were inoculated onto solid modified MS and
Eeuwens basal medium containing 107.41µM NAA and associated with 0, 22.62, 45.24 or
67.86 µM 2,4-D and onto another MS basal medium with high concentration 450µM picloram
or 450 µM 2,4-D in the presence of 0,03% (w/v) activated charcoal to induce embryogenic
calli. The percentage of oxidation was ranged from 25 to 100% for leaf explant and from 0 to
46.67% for zygotic embryo explant, while it was 100% for female flower explant. The level
of contamination was varied from 1.11 to 100 % for leaf explant, from 0 to 35.56% for
zygotic embryo explant, and 8.33 to 41.67% for female flower explant. After 28 weeks of
culture, compact and pearly-white, globular callus was obtained from the leaf number five
(L5). The media 2,4-D and NAA concentration (67.86 and 107.41µM) respectively
combination was the optimal media for embryogenic callus induction, while the media
containing only NAA (107.41µM) induced the highest percentage of calli formation
(30.56%). After 36 weeks of culture, direct embryoids was obtained from leaf number six
(L6) onto the media containing the combination of NAA (107.41µM) and 2,4-D (45.24 µM).
According to zygotic embryo explant, the best result (7.90%) of embryogenic callus was
achieved onto Eeuwens media containing NAA (107.41µM) after 28 weeks. Highest
percentage (38.33%) of direct shoot development was also obtained from the same media
NAA (107.41µM). For female flower explant no callogenesis was observed during the study
period. After 4 subsequent subcultures onto the same medium with gradually reduction of
auxin concentration, the embryogenic callus and embryoid cells from young leaves and
mature zygotic embryo explants, failed to develop during the time require for this study.
Key words: Somatic embryogenesis, oil palm, young leaf, immature female flower, zygotic
embryo
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permission from IPB.
OIL PALM (Elaeis guineensis Jacq.) var. Tenera in Vitro
EMBRYOGENESIS EFFECTIVENESS FROM YOUNG LEAVES,
MATURE ZYGOTIC EMBRYO AND IMMATURE FEMALE FLOWER
EXPLANTS
MONDJELI CONSTANTIN
Thesis
Submitted to the Graduate School in partial fulfilment of the requirements for the degree
Master of Science
In Plant Breeding and Biotechnology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
External Examiner at final Exam: Prof.Dr.Ir. Sudirman Yahya, MSc.
Title
: Oil Palm (Elaeis guineensis Jacq.) var. Tenera In Vitro
Embryogenesis Effectiveness from Young Leaves, Mature
Zygotic Embryo and Immature Female Flower Explants
Name
: MONDJELI CONSTANTIN
Registration Number
: A253098021
Program
: Plant Breeding and Biotechnology
Approved by
Supervising Committee
Head
Member
Dr. Ir. Ni Made Armini Wiendi, MS
Dr. Ir. Ade Wachjar, MS
Endorsed by
Head of Study Program
Dr.Ir. Trikoesoemaningtyas, MSc.
Date of Examination: 30 December 2011
Dean of Graduate School
Dr. Ir. Dahrul Syah, MSc. Agr.
Date Graduated.................................
ACKNOWLEDGEMENTS
Glory to God in the highest and peace to his people on earth. Almighty God and Father,
I worship you and give you thanks for all your permanent presence, light, guidance, peace and
protection that I have always received during my studies and specially my research activities
in Bogor Agricultural University, without which all my efforts would not have borne any
fruits.
I would like to convey my sincere and special thanks to my supervising committee
Dr. Ir. Ni Made Armini Wiendi, MS and Dr. Ir. Ade Wachjar, MS for their advice, corrections
resourceful instructions and comments during the preparation, execution and presentation of
this research work.
Grateful thanks to Bapak Jo Daud Dharsono and Dr. Zok Simon, respectively the
Project Leader of Indonesian Palm Oil Board‟s (IPOB) and the former General Manager of
the Institute of Agricultural Research for Development (IRAD) Cameroon for their wisdom
and full goodwill, without which this project would never have surfaced.
I wish to convey my special thanks to Bapak Lalang Buana of PT SMART‟s V Team
Operations and all the members of IPOB mainly Ibu Tyas and Bapak Wasis, to Dr. Koona
Paul the Chief of Centre of the Specialised Centre on Oil Palm Research of La Dibamba
Cameroon (CEREPAH) and all the members for their constant support and encouragements
during my stay in Indonesia.
I want to take this opportunity to thank my family especially the Late Mouadiba
Stanislas who initiated the perfection cult that is expressing in me today; my mother Ango
Imelda, my wife Ngassa Nelly, my children Biba Mondjeli Audrey-stephane, Lihan Mondjeli
Cecile Santana, Mondjeli Mondjeli Constantin II, my brothers, sisters and cousins for their
constant prayers, moral support, sacrifices and encouragement.
I would like to thank the generosity of my classmates of Plant Breeding and
Biotechnology 2009 batch especially Yogo Adhi Nugroho, Nur Arifin, Vina Novita, Vitria
Pupistasari and Jose Maria Alves who were adopted me rapidly in this new environment of
Indonesia. My sincere acknowledgements to all the laboratory staff and technicians of Tissue
Culture Laboratory number 2 of IPB, especially Okti Hanayati and Yogo Adhi Nugroho.
Bogor, December 2011
Mondjeli Constantin
AUTOBIOGRAPHY
The author was born in Ebodié, Cameroon on the 10th of March 1972 from father
Mouadiba Stanislas and mother Ango Emilda. The author is eighth from a total of twenty five
children. The author is a father of two sons and one daughter with one wife.
In year 1995, the author passed the " Baccalauréat D " and was admitted into the
Faculty of Agronomy and Agricultural Sciences of University of Dschang through a
competitive entrance examination. In May 2001, the author was graduated with a Post
Graduate Certificate " Ingenieur Agronome " and was later recruited into the Ministry of
Scientific Research and Innovations of Cameroon in 2002.
The author was posted to Barong-bikang Multipurpose Agricultural Research Station
as assistant researcher on Perennial Crops (Cocoa and Coffee) from 2002 to 2004. Since
2004, the author has serving in Agronomy and Breeding sections of the Specialised Centre for
Oil Palm Research La-Dibamba as researcher.
In August 2009, the author obtained from the Bogor Agricultural University, the
opportunity to further his studies on Plant Breeding and Biotechnology at the Graduate School
with funding from the Indonesia Palm Oil Board and the Government of Cameroon.
During his studies in the Graduate School, the author joined the Community of Post
Graduate Students of Agronomy and Horticulture Department (FORSCA) and the
International Students Forum (ISF) of Bogor Agricultural University. The author attended the
National Exhibition on Perennial Crops to Overcome the Energy Crisis and Global Warming
in Jakarta in November 2010 where a scientific poster was presented. In December 2011, the
author attended the Regional Seminar- workshop on Advances in Tropical Genomics:
Conservation and Sustainable Utilization of Tropical Biodiversity in Bogor where another
scientific poster was submitted.
i
TABLE OF CONTENTS
Pages
LIST OF TABLES…………………………………………………………………………
iv
LIST OF FIGURES……………………………………………………………………….
v
LIST OF APPENDICES…………………………………………………………………..
vi
INTRODUCTION…………………………………………….……………………………
1
Background…………………………………………………………………………
1
Aim of Research……………………………………………………………………
3
Hypothesis………………………………………………………………………….
4
LITERATURE REVIEW…………………………………………………………………
5
Origin and History of Oil Palm (Elaeis guineensis Jacq)…………………………..
5
Taxonomy of Oil Palm (Elaeis guineensis Jacq)……………………………………
7
Botany of Oil Palm (Elaeis guineensis Jacq)………………………………………
7
Plant Tissue Culture…………………………..…………………………………………...
12
History and Definition………………………………………………………………
12
Medium of Tissue Culture…………………………………………………………..
13
Plant Growth Regulators…………………………………………………………….
14
Zygotic Embryo Culture……………………………………………………………….….
15
Ovary Culture…………………………………………………..………………………….
15
Tissue Culture problems and Preventions………………………………………………....
15
Genetic Changes in Culture…………………………………………………………
15
Blackening or Browning……………………………………………………………
16
Embryogenesis…………………………………………………………………………....... 16
Tissue Culture Field in Plant Breeding…………………………………………………..… 17
Oil Palm Genetic improvement………………………………………………………….…. 18
ii
Tissue Culture and Oil Palm improvement……………………………..………………….. 19
MATERIALS AND METHODS…………………………………………………………..
22
Location and Time…………………………………………………………………
22
Plant Materials and Equipments…………………………………………………....
22
Media Preparation…………………………………………………………………
22
Sterilization Compounds…………………………………………………………..
25
Laboratory Equipments and Tools…………...……………………………………
25
Methods……………………………………………………………………………………
25
Experiment 1. Callus and Somatic Embryos Induction from Young Leaf Explant... 25
Palm Tree Sampling and Explant Processing…………………………………
25
Callogenesis Mixing Medium (Combination).....……………………………………
26
Environmental Culture Conditions……………………………………………
27
Embryogenesis Mixing Medium………………………………………………
27
Culture conditions of Embryos……………………………………………….... 27
Subculture‟s Flow Chart…………………..…………………………….…………. 28
Experiment 2. Induction of Somatic Embryo from Mature Zygotic Embryo Explant
………………………………………………………………...………
28
Explant Sampling and Sterilization Process…………………………………
28
Callogenesis Mixing Medium (Combination)..………………….…………….
29
Environmental Culture Conditions………………………………….……….
29
Embryogenesis Mixing Medium…………………………………………….
30
Experiment 3.Induction of Somatic Embryo from Immature Female Flower Explant
.....……………………………………………………………………
30
Explant Sampling and Sterilization Process…………………………………
30
Callogenesis Mixing Medium (Combination)..………………………………..
31
Environmental Culture Conditions………………………..………………… 31
Data Analysis……………………………………………………………………..
31
RESULTS AND DISCUSSIONS…………………………………………………………
32
iii
CONCLUSIONS …………………………………………………..………………………
45
REFERENCES………………………………………………………………………….…
46
APPENDICES…………………………………………………………………………….
53
iv
LIST OF TABLES
Number
Pages
1.
Agroecological factors affecting growth and production of Oil Palm…………
12
2.
Murashige and Skoog (1962) Medium composition…………………………..
23
3.
Eeuwens and Blake (1976) Medium Composition…………………………….
24
4.
Percentage of contamination of oil palm leaves explant per medium composition
and rank of leaf after 36 weeks of culture (%) ………………………….…
32
5.
6.
7.
8
9.
10.
Influence of various medium composition (NAA and 2,4-D) and young leaves
rank of oil palm (E. guineensis) cv. Tenera spear on percentage of oxidized
calli after 4 subcultures.………………………………………………….……
33
Influence of various medium composition (NAA and 2,4-D) and young leaf
rank of oil palm (E. guineensis) cv. Tenera spear on the percentage of
explant bearing callus after 39 weeks ……………………….……………..
34
Mean number of young leaf explants of Tenera oil palm bearing callus per
media ……...................................................................................................
35
Effect of the spear leaf rank of oil palm var. Tenera on callogenesis after
39 weeks in medium ……………………………………………………….
36
Percentage of contamination per medium and type of zygotic embryo explant of
oil palm after 28 weeks of culture (%)………………………………………..
38
Effect of medium composition and type of zygotic embryo explants of oil palm
(E. guineensis) cv. Tenera on percentage of explant bearing callus after 32
weeks..............................................................................................................
39
11.
Mean number of zygotic embryo explants of oil palm var. Tenera bearing callus
per media composition ……………………………………………………
40
12.
Mean number of callogenesis per type of zygotic embryo explant of oil palm
var. Tenera …………………………………………………………………
41
13.
Effect of medium composition and type of zygotic embryo explants of oil
palm (E. guineensis) cv. Tenera on the percentage of explant producing
germinated shoot after 25 weeks in medium ……………………..………
41
Mean number of shoot germination production by zygotic embryo explant per
media composition ……………………………………………………….
42
14.
v
LIST OF FIGURES
Number
Pages
1.
Map showing the Extent of Oil Palm Cultivation ………………………………..
6
2.
Adult Oil Palm (Elaeis guineensis Jacq.) Tree……………………………………
8
3.
Male and Female Inflorescence of Oil Palm………………………………………
10
4.
Mature and Immature Fresh Fruit Bunches of Oil Palm…………………………..
10
5.
Cross section of the different varieties of oil palm (E. guineensis Jacq.)…………
11
6.
Different Type of Oil Palm Explants used………………………………………..
22
7.
Young Leaf Sample Started Material Preparation Process………………………..
26
8.
Mature Zygotic Embryo Explant Preparation Process……………………………..
29
9.
Immature Female Flower Explant Preparation Process……………………………
30
10. Friable nodular callus (NC) proliferation from young leaves explant …………….
33
Root-like callus initiation from young leaf explant of oil palm …………………..
34
12. Embryogenic callus (EC) obtained from young leaf of oil palm…………………..
36
13.
Direct embryoid cells obtained from young leaf of oil palm……………………..
37
14. Germinated shoots from mature zygotic embryo explant of oil palm………….....
38
15.
Root-like callus (RC) initiation from zygotic embryo explant …………………
39
16.
Embryogenic callus (EC) initiation of zygotic embryo………………………….
43
17.
Root initiation cells from immature female flower of oil palm………………….
44
11.
vi
LIST OF APPENDICES
Number
Pages
1.
ANOVA of callus induction from young leaves explant of oil palm…………….
53
2.
Student-Newman-Keuls test of different medium concentrations for callus
induction of young leaves explant…………………………………………….
53
3.
Student-Newman-Keuls test of different leaf ranks of oil palm for callus induction
of young leaves explant………………………..……………………………..
54
4.
Student-Newman-Keuls test of different basal medium for callus induction of
young leaf explant……………………………………………………..…….
54
5.
ANOVA of callus induction from zygotic embryo explant of oil palm…………
54
6.
Student-Newman-Keuls test of different medium for callus induction of mature
zygotic embryo explant…………………………………………..…………
55
7.
Student-Newman-Keuls test of different type of zygotic embryo explant for
callus induction……………………………………………………………..
55
8.
ANOVA of callus induction from zygotic embryo explant of oil palm for basal
medium and type of zygotic embryo……………………………….………
56
9.
Student-Newman-Keuls test of different basal medium for callus induction of
mature zygotic embryo explant……………………………………………..
56
10.
ANOVA of direct shoot formation from zygotic embryo explant of oil palm…..
56
11.
Student-Newman-Keuls test of different medium for direct shoot formation of
mature zygotic embryo explant……………………………………………..
57
12.
Student-Newman-Keuls test of different type of zygotic embryo explant for
shoots germination…………………………..………………………………
57
13.
ANOVA of shoot formation from zygotic embryo explant of oil palm for basal
medium and type of zygotic embryo……………………………………….
58
14.
Student-Newman-Keuls test of different basal medium for shoots germination
of mature zygotic embryo explant……………………………………………
58
1
INTRODUCTION
Background
Oil palm (Elaeis guineensis Jacq.) is an important commercial crop from all tropic producer
countries. Cultivated for palm oil and palm kernel oil, it is the highest yielding oil-bearing
crop, by producing more than five times the yield of oil per year per hectare of any annual oil
crop. The best oil palm is met in Indonesia, regularly producing 8 tons of oil/hectare/year
(Suprianto et al. 2008). The worldwide palm oil production since 2005 was 39.8 million
metric tons, of which 4.3 million tons was in the form of palm kernel oil. As compare to the
other oleaginous crops, Elaeis guineensis oil is the most widely-produced tropical oil, and
constitutes 30 % of total edible oil production worldwide. Since 2007, Indonesia emerged the
world's largest producer of palm oil, by producing approximately 50 % of world palm oil
volume, giving a total of 23 million ton/year (htt://Google Wikipedia oil palm, U.S
Department of Agriculture 2010). Palm oil production has contributed to economic benefits
such as government revenues, profits for companies, employment, and raised incomes for
smallholders. In Indonesia, the world‟s largest producer, the industry generated US$12.4
billion in foreign exchange from palm oil exports in 2009, and supports millions of jobs and
opportunities for rural farmers (Gingold 2011). Therefore, this economic sector employs
about 1 544 641 people in Indonesia and the planted area is increasing every year by 372 000
hectares. Oil palm was planted on a total land area of 8 036 431 hectares in the year 2010 in
Indonesia (D.T.E 2011).
Cameroon is the 13th world producer of palm oil with 190 000 metric tons and the 3rd
African country producer after Nigeria and Ivory Coast (U.S Department of Agriculture,
2010). Palm oil is mainly required for food purposes for about 80% as cooking oil. In
Europe, 10% of consumption high quality products sold contains palm oil. In addition, palm
oil is present into several industrial products and public restaurant food (www.econoecolo.org). Besides producing oils and fats, at present there is a continuous increasing interest
concerning oil palm renewable energy. One of the major attentions is bio-diesel from palm
oil. Bio-diesel implementation is important because of environmental protection and energy
supply security reasons. This palm oil bio-diesel is biodegradable, non-toxic, and has
significantly fewer emissions than petroleum-based diesel (petro-diesel) when burned. In
addition, oil palm is also a well known plant for its other sources of renewable energy, for
example huge quantities of biomass by-products are developed to produce value added
2
products such as methane gas, bio-plastic, organic acids, bio-compost, ply-wood, activated
carbon, and animal feedstock. Even waste effluent; palm oil mill effluent (POME) has been
converted to produce energy. Oil palm has created many opportunities and social benefits for
the locals. In the above perspective, the objective of the present work is to give a concise and
up-to-date picture of the present status of oil palm industry enhancing sustainable and
renewable energy (Sumathi et al. 2007). But the demand for palm oil has mainly increased in
recent years due to its use as a biofuel,
Oil palm (Elaeis guineensis Jacq.) is a monocot plant, originating from West and
Central Africa. Since the establishment of the first plantations in the 1920s and 1930s, five
generations of selection and breeding have been completed in oil palm. The oil palm breeding
program is limited by the long generation, selection cycles with typically 12 years and the
required resources (Hartley 1988).
The success of many in vitro techniques in plants depends on the success of plant
regeneration. Indeed, the application of some techniques such as in vitro mutant and
protoplast fusion are suitable to cell culture but may be limited because of the inability to
regenerate plants. A vast reservoir of genetic variability is available from in vitro plant
regeneration (Evans et al. 1983). The totipotency was first demonstrated with Solanaceous
species by using Nicotiana tabacum and they have been used as model systems of in vitro
studies (Vasil and Hildebrandt, 1965). The first production of haploid plants by the in vitro
culture of excised anthers was achieved by using Datura innoxia (Guha and Maheshwari,
1964). The first somatic hybrid was obtained from two Nicotiana species, N. glauca and N.
langsdorfii (Carlson et al. 1972). The initiation and development of embryos from somatic
tissues in plant culture was first recognized by Steward et al. (1958) and Reinert (1958, 1959)
in culture of Daucus carota tissue. According to oil palm, propagation through tissue culture
is also widely used. Initially tissue culture techniques were used to propagate elite oil palm
clones (Jones, 1974). Otherwise several explants sources have been used to establish tissue
culture. These include mature embryos (Rabechault et al. 1970), inflorescences (Smith and
Thomas 1973), roots (Jones 1974), seedlings (Ong 1977), and young leaves (Staritsky 1970;
Schwendiman et al. 1988). Unfortunately some early aberration were observed from plant
produced (Corley et al. 1986). However, oil palm tissue culture techniques have undergone
continuous improvement in these recent years. This resulted in the production of clonal palms
with minimal abnormality less than 5% (Jones, 1995, Rival et al. 1998). Moreover, Texeira et
al. (1994) reported that, complete plants have been successfully regenerated from various
3
explants of oil palm. Plant propagation using explants from flower organs either anther or
ovary through embryogenesis techniques have been successfully carried out although their
regeneration is more difficult than that using young leaves (Perera et al. 2007, 2008, Texeira
et al., 1994; Verdeil et al. 1994). Although these skills are applied, the frequencies for
complete plant regeneration from some explants are still inefficient (Rival et al. 1999). The
low response of embryogenesis is influenced mainly by genotype and donor plant age
(Ruslan, 1993), in addition to the influence of the appropriate media and explants source.
Explants of young tissues still undergoing cell division and generally form callus more readily
than those from older parts of plant, where the meristematic cell activities are reduced or lost.
In general, the more juvenile the explant material, the greater the likelihood of success (Edwin
and Sherrington, 1984; Lydiane and Kleyn, 1983). Embryogenesis rates of oil palm,
averaging usually less than 5% per ortet (Soh et al., 2006). Embryoids are multiplied until the
total number reaches the required 350. Of these 350 embryoids only 250 will produce shoots
(shooting percentage of 75%). Nevertheless, about 18% of the ortets will provide the bulk cultures
for mass propagation (Wong et al., 1997).
Thus some new protocols contributions are still needed for oil palm plant regeneration
based on embryogenesis. It is in this light that the study was carried out.
Aim of Research
The long term objective is to formulate the protocol of in vitro oil palm regeneration
using young leaves, mature zygotic embryo and immature female flower explants.
Research Activities
In short term, it was aimed to:
Find out the optimum medium for callus induction and somatic embryo formation from
young leaf explant of oil palm of Tenera variety.
Find out the optimum medium for callus induction and somatic embryo formation from
oil palm mature zygotic embryo explants of Tenera variety.
Find out the optimum medium for callus induction and somatic embryo formation from
oil palm immature female flower explant source of Tenera variety.
4
Importance of Study
This work will contribute to obtain protocols on elite oil palm plants propagation for
seedlings production centre. Protocols will be served as tool of clonal oil palm production to
ensure the best use of oil palm resources in germplasm management. The skills can be used
for the plant breeding program purpose and also during the oil palm in vitro selection study.
Hypothesis
1. There is an optimal type of medium composition for each explant source of Tenera
variety oil palm.
2. There is at least one explant for embryogenesis of Tenera variety of oil palm.
3. There is suitable young leaf rank explants for embryogenesis of Tenera variety oil
palm.
5
LITERATURE REVIEW
Origin and History of oil Palm
It is generally agreed that the Oil Palm (Elaeis guineensis Jacq.) is originated from the
equatorial tropical rain forest region of Africa, precisely along the gulf of guinea. It exists in
the wild type and cultivated state. The American oil palm, Elaeis oleifera is native to tropical
Central America and South America. The main belt runs through the southern latitudes of
Cameroon, Côte d‟Ivoire, Ghana, Liberia, Nigeria, Sierra Leone, Togo and into the equatorial
region of Angola and the Congo. Oil palm was first illustrated by Nicholaas Jacquin in 1763,
hence its name, Elaeis guineensis Jacq (en.wikipedia.org/wiki/Oil_palm#mw-head).
During the 14th to 17th centuries some palm fruits were taken to the Americas and
from there to the Far East. The plant appears to have thrived better in the Far East, thus
providing the largest commercial production of an economic crop far removed from its centre
of origin. Oil palms were introduced to Java (Indonesia) by the Dutch in 1848 (Lötschert and
Beese 1983) and to Malaysia (then British colony of Malaya) in 1910 by Scotsman William
Sime and English banker Henry Darby. The first plantations were mostly established and
operated by British plantation owners, such as Sime Darby and Boustead. The large plantation
companies remained listed in London until the Malaysian government engineered their
"Malaysianisation" throughout the 1960s and 1970s (Stevenson 2006).
The cameroon‟s wild oil palm material contains some interesting genotypes which are
present into many germplasm in the world. According to Hartley (1988), in Cameroon
Germans had identified thin shelled oil palm fruit with high oil content as early as 1902. Later
it was known as Tenera type. The material collected by Blaak and Sterling (1996) from some
Cameroon regions such as Ekona and Widikum in 1967 have been widely used to develop the
majority of oil palm planting seed found today in all the oil palm agroecological area of the
world (South East Asia, Africa, South and Central America). According to Chapman et al.
(2003), twenty years of breeding using Dami Deli material crossed with Cameroon and
Tanzanian selections of African oil palm have led to the development of precocious bearing
and cold tolerant oil palms. The oil palm was cultivated in Cameroon since 1907 in the early
days of the German colonial era. The first industrial plantations were created in 1910 by
Germans in Edea region where the current SPFS Company (Swaziland oil palm Farms
Company of Cameroon) is located. In 1929 the Unilever Group created the Pamol Plantations
Limited in the British area of the country. Later the British created the Commonwealth
Corporation in 1947 which is now known as the Cameroon Development Corporation (CDC).
6
In the French area of the country another oil palm plantations were created in 1959 by Rivaud
Group (Red Land) in Dizangue region and it is currently known as SAFACAM (Forestry and
Agricultural African Company of Cameroon). In 1963, the government of Cameroon decided
to create the Cameroon Oil Palm Company or SOCAPALM which is currently the largest oil
palm company in Cameroon. In 2008, Cameroon covered about 101 500 ha of land plantation
of commercial oil palm.
Figure 1. Map showing the extent of oil palm cultivation in 43 oil palm-producing
countries in 2006 (FAO 2007). Cited by Koh and Wilcove (2008)
7
Taxonomy of Oil Palm
The scientific classification of oil palm is as follows:
Kingdom
:
Plantae
Phylum/Division
:
Phanerogam/Spermatophyta
Subdivision
:
Angiospermae
Class
:
Monocotyledonae
Order
:
Arecales
Family
:
Arecaceae
Subfamily
:
Arecoideae
Tribe
:
Cocoseae
Subtribe
:
Elaeidinae
Genus
:
Elaeis
Species
:
guineensis and oleifera
Botany of Oil Palm
Oil palm is a diploid (2n=32) oleaginous tropical perennial crop (Dransfield et al.
2005). Elaeis guineensis is a single-stemmed palm which bears a single vegetative shoot
apical meristem, which is continuously active producing a new leaf every two weeks in
mature palms (Figure 2), under favourable climatic conditions (Adam et al. 2005).
Inflorescences are formed throughout the year in acropetal sequence in the axils of subtending
leaves of the plant. Separated male and female inflorescences are produced in same palm
8
plant in alternating cycle of variable duration depending on genetic factors, age and
environmental conditions (Corley, 1976).
Figure 2. Adult oil palm (Elaeis guineensis Jacq.) tree
The Foliage System of Oil Palm
At the mature stage, E. guineensis Jacq. is constituted with a large crown of 30 to 45
palms measuring 5 to 9 meters long topping the single cylindrical pseudo-trunk. Every year,
about 20 to 26 new pinnate leaves are produced and each mature leaf bears 250 to 300
leaflets. The leaf bases are persistent for years, and prominent leaf scars are arranged spirally
on the trunk of mature palms where bases have fallen. The leaflets cover the distal 2/3 of the
leaf, and the lower 1/3 is spined with spines increasing in length acropetally. The stem or
pseudo-trunk can extend a rate of 30 to 60 cm/year between the ages of 6 to 15 depend on
hereditary and environmental factors. It functions as a supporting, vascular and storage organ.
Generally, the height of trees determines the exploitation times of oil palm plantations. Thus,
palm plantations are exploited up to 25 to 30 years when the trees height is between 12 to 15
meters. Above of this, the harvest of fresh fruit bunches becomes difficult and replanting
should be required.
The Roots System of Oil Palm
The root architecture is a fundamental aspect of plant productivity through its
functional importance in the efficient acquisition of soil resources (water and nutrients
9
uptake). The different morphological types of oil palm roots have been distinguished
according to their development pattern and state of differentiation. As all the monocot, the
root system of E. guineensis is fasciculata. The oil palm has an adventitious root system; with
primary roots generally about 6-10 mm in diameter, originating from the base of the trunk and
either spreading horizontally or descending at varying angles into the soil. The primary roots
bear secondary roots, of about 2-4 mm in diameter. Tertiary roots, about 0.7-1.2 mm in
diameter, branch out from the secondary roots, which in turn bear the quaternary roots.
Quaternary roots are unlignified, about 0.1-0.3 mm in diameter and 1-4 mm long and they are
often assumed to be the main absorbing roots (Corley et al., 1976). The total length of tertiary
and quaternary roots in the soil is the most important root characteristic as they are the
absorbing roots that affect fertilizer use efficiency. Most of the root biomass is found within
1m of the soil surface, but tertiary and quaternary roots are found mostly in the upper 30 cm
from the soil surface. Primary roots can grow up to 20 m away from the base of the palm and
some primary roots could penetrate below the water table at 90 cm from the surface (Ng et al.,
2003). The distribution of roots depends largely on the nature of the soil. Oil palm being a
monocot needs a friable soil for root branching (Zuraidah et al. 2010).
The Reproductive System of Oil Palm
As monoecious plant, male and female flowers of oil palm occur separately on the
same tree. The male and female inflorescences are produced distinctly and alternatively on the
same plant. An inflorescence is initiated in the axil of every leaf. An inflorescence may bear
about 250 spikelets. Each female inflorescence (Figure 3B) can have about 12 to 30 flowers.
Sometimes hermaphrodites flowers can also occur but in rare cases. The female flower is
receptive for pollination between 36 to 48 hours. The male inflorescence (Figure 3A) contains
about 100 000 flowers. Most of the pollen released is shed between 2 to 4 days after opening.
The pollen remains viable for 6 days after release. The insufficient supply of water will
stimulate the plant to produce mostly male flowers. Irrigation and fertilization taken to
increase the flower ratio from male to female will have positive effect only between 22 to 40
months later.
10
A
B
Figure 3. A. Male inflorescence, and B. Female inflorescence of oil palm.
In the nature, the pollination is mainly entomophily (by insects) but can also be done
by the wind and the rain. The main pollinator of the oil palm is the insect from the genus
Elaeidobius and 14 species were found to be associated to the male and female inflorescences.
The most common one is Elaeidobius kamerunicus, which was introduced in Asia, South and
Central America. Its introduction led to an increase in yields of more than 35%. Upon
pollination at the anthesis stage, the female inflorescence may develop and give rise to a fruit
bunch 22 to 26 weeks later. In the field, fruit bunches (Figure 4) which are as compact and
ovoid mass spiked bearing spines can be found on the oil palm 2 to 3 years after planting. The
fruit bunches become heavier as the palms get older. On 10 years old palms, the bunch
weights can be ranged from 10 to 50 kg for a total of 500 to 4 000 fruits per bunch. The fruit
is a sessile drupe generally of ovoid shape measuring 2 to 5 cm length and weighing from 3 to
30g. The oil palm fruit consists of the pericarp which includes the outer and smooth exocarp,
the fibrous and fleshy mesocarp or pulp rich in palm oil, the hard endocarp or shell protecting
the seed and finally the kernel or seed. Inside the shell is the endosperm or kernel. The
endosperm contains large amount of fats and carbohydrates on which the seedling will be
entirely dependent after germination. The oil palm produces two main vegetable oils, namely
palm oil extracted from the mesocarp of the fruit and palm kernel oil extracted from seed.
Immature
Mature
Figure 4. Mature and Immature Fresh fruit bunches of oil palm
11
Commonly three fruit forms or “varieties” of oil palm can be identified based on the
thickness of the shell criterion (Figure 5). The Dura has a shell thickness between 2 to 8 mm,
while the Pisifera has no shell. The shell thickness of the hybrid Tenera is between 0.2 to 2
mm, the fleshy mesocarp of Dura yields between 15 to 17% oil while in Tenera yields
between 21 to 23% oil and Pisifera more than 23% oil. According to commercial purpose,
Pisifera is not cultivated on large scale because of its ability to fruits abortion and thereby
virtual empty bunches production. Tenera which is a hybrid of Dura and Pisifera varieties
remains the commercial variety with 60 to 96% of mesocarp. The hybrid produces more fruit
bunches than Dura.
B
A
C
Figure 5. Cross section of the different varieties of oil palm (E. guineensis Jacq.). A. Dura
palm fruit with thick shell, B. Tenera palm fruit with less thick shell, C. Pisifera
palm fruit with no shell
Climatic and Soil Requirements of Oil Palm
Oil palm culture is done well in low altitude (less than 500 m above sea level), 15º
from the equator in the humid tropics. Evenly distributed rainfall of 1,800 to 2,000 mm/year,
but will tolerate rainfall up to 5,000 mm/year, provided the soil is properly drained. Oil palm
is sensitive to poor drainage and drought. Potential yield is reduced where there are more than
three consecutive months with less than 100 mm rainfall per month. Irrigation may increase
economic returns in areas with pronounced dry periods. More than 2,000 sunshine hours (i.e.,
low cloud cover during daytime) are required.
It is adapted to a range of soil types. Tolerates low pH, but does not thrive at very high
pH (greater than 7.5). Soil must be free draining. The table below shows the suitable and
extreme values of climate and soil that are required by oil palm.
12
Table 1. Agro ecological factors affecting growth and production of oil palm
Characteristic
Rainfall (mm)
Temperature (oC)
Water deficit (mm)
Solar irradiation
Dry season (mths)
Wind (m/s)
pH
Slope (%)
Flooding
Drainage class
Good
2500
25-30
0-150
13-15
Suitability class
Moderate
1450-1700
20-22
150-250
9-11
Severe
1250-1450
10-20
250-400
7-9
Very severe
20
7
>30
Severe flooding
Very poor
PLANT TISSUE CULTURE
History of Plant Tissue Culture
Many authors have put forward various arguments on the origin of plant tissue culture.
Rehwald (1927) is thought to have been the first person who has obtained undifferentiated
callus tissue in sterile culture. But according to most scientists, the real scientific work on
tissue culture was done in 1934 by P.R. White in the USA (United States) and in the same
year, by Gautheret in France, using respectively tomato plant and the cambium of several tree
species (Acer pseudoplatanus, Salix caprea, sambucus) as sources of explant. Before that
period, in 1924, callus culture of carrots (Daucus carota) was reported by two physicians, R.
Blumenthal and P. Meyer in the pathological implication studies.
In 1934, the plant hormone or auxin named indole-3-acetic acid (IAA) was identified
the first time by F. Kögl, A.J. Haagen-Smit, and H. Erxleben. In 1955 C.O Miller has
discovered kinetin, which is a plant growth regulator known as cytokinin.
Plant tissue culture is a technique or a process by which small organs or pieces of tissue
of plants (explants) are grown in aseptic conditions within glass or clear plastic vessels. These
are usually termed in vitro techniques or propagation. It is also called micropropagation
because miniature shoots and plantlets are initially derived. The natural capability of plants to
multiply by asexual means is the basis for multiplication in vitro. Tissue culture was first used
13
on a large scale by the orchid industry in the 1950s. Later, it became clear that any plant
would respond to tissue culture as long as the right formula and the right processes were
developed for its culture. The process of tissue culturing plant the explant stage to the final
stage of plantlet requires four basic stages namely: establishment or initiation of explant
(Stage I), multiplication of cells (Stage II), rooting of shoots (Stage III), and acclimatization
or hardening off (Stage IV).
Explants
The kind of explant chosen, its size, age and the manner in which it is cultured, can all affect
whether tissue culture can be successfully initiated and whether morphogenesis can be
induced. The juvenile form had pre-existing competent cells that were able to respond to
auxin and become determined to form organs. However, the adult form appeared to lack cells
with pre-existing competence to form organs, but competence was acquired by some callus
cells once they had been initiated. Explants taken from mature shoots are frequently more
liable than juvenile material to suffer necrosis, especially when surface disinfested and placed
in culture (Hanus and Rohr, 1987). For tissue culture, juvenile explants are usually more
readily established in vitro and grow and proliferate at a more rapid rate than adult material.
This is particularly true with tree species where micropropagation of adult material is often
difficult (Edwin et al., 2008).
Medium of Tissue Culture
The success of plant tissue culture technique is greatly influenced by the nature of the
culture medium. The medium is the substrate for the started explant and it refers to an aseptic
environment of the mixture of a correct concentration and balanced chemical diet compounds
to form a nutrient-rich gel or liquid for growing cultures, whether cells, organs, or plantlets.
Plant tissue culture media are therefore made up from macronutrients salts which provide the
six major elements are N, P, K, Ca, Mg, and S, contributing in the growth of higher plants;
micronutrient salts are Fe, Mn, Zn, B, Cu, Co and Mo which are components of plant cell
proteins of metabolic and physiological importance: chlorophyll synthesis and chloroplast
function (Sundqvist et al., 1980); vitamin; amino acids and other nitrogen supplements; sugar,
buffers; solidifying agent; and growth regulators. The medium pH must be such that it will
not disrupt the function of plant cell membranes or the buffer pH of cytoplasm, and the
14
gelling efficiency of agar. The initial pH can often be selected to ensure the integrity of the
medium and the most rapid rate of culture growth (Edwin and Sherrington, 1984).
However a supply of reduced nitrogen is generally necessary for embryogenesis to occur in
callus or suspension cultures.
Plant Growth Regulators
Endogenous compounds occur naturally within plant tissues. They have a regulatory
role rather than nutritional activity in growth and development. These compounds are always
active at very low concentrations and known as plant growth substances or plant hormones.
Otherwise synthetic chemicals with similar physiological activities can be found and
generally supplied in the medium for growth and morphogenesis in vitro. They are termed
plant growth regulators (exogenous). Some recognized plant growth substances classes
include such chemical compounds as auxins, cytokinins, gibberellins, ethylene and abscisins.
The first two compound classes are by far the most important for regulating growth and
morphogenesis in plant tissue and organ cultures.
Auxins are phytohormones very widely used in micropropagation medium to promote
the growth of callus, cell suspensions or organs and to regulate morphogenesis, mainly when
combined with cytokinins. They influence cell enlargement, root initiation, and adventitious
bud formation. The potential levels of endogenous auxin in the explant tissues are found to
depend on the mother plant (donor plant growth state, growth conditions and season of
sampling) from which they were collected. Therefore, the natural auxin will vary between cell
strains of t
EMBRYOGENESIS EFFECTIVENESS FROM YOUNG
LEAVES, MATURE ZYGOTIC EMBRYO AND IMMATURE
FEMALE FLOWER EXPLANTS
MONDJELI CONSTANTIN
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
2012
DECLARATION OF ORIGINALITY AND AUTHENTICITY
This is to declare that the thesis titled « Oil Palm (Elaeis guineensis Jacq.) var.
Tenera In Vitro Embryogenesis Effectiveness from Young Leaves, Mature Zygotic
Embryo and Immature Female Flower Explants » is the result of my personal work under
the direction of the supervising committee and has never been presented in any form where
ever. Any other source of information that has been mentioned in this thesis from published or
unpublished works of other authors has been acknowledged in the text and included in the
reference chapter.
Bogor, December 2011
Mondjeli Constantin
NRP: A253098021
ABSTRAK
MONDJELI CONSTANTIN. 2011. Keefektifan Embriogenesis in Vitro Kelapa Sawit
(Elaeis guineensis Jacq. var. Tenera) dari Eksplan Daun Muda, Embrio Zigotik Matang
dan Bunga Betina Immature. Dibawah bimbingan NI MADE ARMINI WIENDI dan ADE
WACHJAR.
Tiga percobaan terpisah terkait sumber eksplan kelapa sawit varietas Tenera meliputi
daun muda, embrio zigotik matang dan bunga betina immature, yang diambil dari pohon
induk berumur 9 dan 13 tahun, telah dilakukan untuk menguji responnya terhadap produksi
embrio somatik. Pada berbagai komposisi media, eksplan-eksplan ini ditanam pada media
dasar MS dan Euwens yang mengandung kombinasi ZPT NAA (107.41µM) dan 2,4-D (0, 22.62,
45.24 atau 67.86 µM) dan media dasar MS dengan konsentrasi ZPT tinggi picloram 450µM
atau 2,4-D 450µM mengunakan 0,03% (w/v) arang aktif untuk menginduksi kalus embriogenik.
Persentase oksidasi berkisar antara 24 – 100% untuk eksplan daun dan antara 0 – 46.67%
pada eksplan embrio zigotik, sementara pada eksplan bunga 100%. Tingkat kontaminasi
berkisar antara 1.11 sampai 100 % pada eksplan daun, 0 – 35.56% pada eksplan embrio
zigotik dan 8.33 - 41.67% pada eksplan bunga betina. Pada 28 minggu setelah penanaman,
didapatkan kalus globular yang kompak dan putih dari daun nomor 5 (L5). Media MS dengan
kombinasi ZPT NAA (107.41µM) dan 2,4-D (67.86 µM) merupakan media yang optimal
untuk induksi kalus embriogenik (3.63%), sementara media MS dengan NAA (107.41µM)
menghasilkan persentase pembentukan kalus tertinggi (30.56%). Pada 36 minggu setelah
penanaman, didapatkan embrioid langsung dari daun nomor 6 (L6) pada media MS yang
mengandung kombinasi ZPT NAA (107.41µM) dan 2,4-D (45.24 µM). Pada eksplan embrio
zigotik dewasa, hasil kalus embriogenik terbaik (7,90%) didapatkan pada media Euwens
yang mengandung NAA (107.41µM) setelah 28 minggu. Persentase pertumbuhan tunas
tertinggi (38.33%) juga didapatkan pada media yang sama (NAA 107.41µM). Pada eksplan
bunga betina immature, tidak dijumpai kalogenesis dalam kurun waktu penelitian ini. Setelah
4 kali subkultur berturut turut pada media yang sama dengan konsentrasi zat pengatur
tumbuh yang menurun bertahap, kalus embriogenik dan embrioid tidak dapat berkembang
dalam kurun waktu penelitian ini, dari eksplan daun muda dan embrio zygotic matang.
Kata kunci: Embriogenesis somatik, kelapa sawit, daun muda, bunga betina muda, embrio
zigotik
SUMMARY
MONDJELI CONSTANTIN. 2011. Oil Palm (Elaeis guineensis Jacq.) var. Tenera in
Vitro Embryogenesis Effectiveness from Young Leaves, Mature Zygotic Embryo and
Immature Female Flower Explants. Under the supervision of NI MADE ARMINI
WIENDI and ADE WACHJAR.
Oil palm (Elaeis guineensis Jacq.) is an important commercial crop for all the
producer countries. It is the world‟s leading vegetable oil crop. In the last decade, the interest
in palm oil as biofuel was eventually caused constraints on worldwide supply for edible palm
oil. In addition, low production and lack of conventional seeds and seedlings are always
observed from the potential producer countries. More than one hundred million of tissue
culture plantlets are needed per year. Indonesia is the largest world producer country with
about 23 million tons of palm oil per year. The study was carried out to find in short term the
optimum medium for callus induction and somatic embryo formation from young leaves,
mature zygotic embryo and immature female flower explants of oil palm (E.guineensis Jacq.)
var.Tenera. Three different independent experiments related to explant sources of oil palm
(Elaeis guineensis Jacq.) var. Tenera include young leaf, mature zygotic embryo and
immature female flower explants isolated from 9 and 13 years old trees were investigated on
somatic embryos production. These explants were inoculated onto solid modified MS and
Eeuwens basal medium containing 107.41µM NAA and associated with 0, 22.62, 45.24 or
67.86 µM 2,4-D and onto another MS basal medium with high concentration 450µM picloram
or 450 µM 2,4-D in the presence of 0,03% (w/v) activated charcoal to induce embryogenic
calli. The percentage of oxidation was ranged from 25 to 100% for leaf explant and from 0 to
46.67% for zygotic embryo explant, while it was 100% for female flower explant. The level
of contamination was varied from 1.11 to 100 % for leaf explant, from 0 to 35.56% for
zygotic embryo explant, and 8.33 to 41.67% for female flower explant. After 28 weeks of
culture, compact and pearly-white, globular callus was obtained from the leaf number five
(L5). The media 2,4-D and NAA concentration (67.86 and 107.41µM) respectively
combination was the optimal media for embryogenic callus induction, while the media
containing only NAA (107.41µM) induced the highest percentage of calli formation
(30.56%). After 36 weeks of culture, direct embryoids was obtained from leaf number six
(L6) onto the media containing the combination of NAA (107.41µM) and 2,4-D (45.24 µM).
According to zygotic embryo explant, the best result (7.90%) of embryogenic callus was
achieved onto Eeuwens media containing NAA (107.41µM) after 28 weeks. Highest
percentage (38.33%) of direct shoot development was also obtained from the same media
NAA (107.41µM). For female flower explant no callogenesis was observed during the study
period. After 4 subsequent subcultures onto the same medium with gradually reduction of
auxin concentration, the embryogenic callus and embryoid cells from young leaves and
mature zygotic embryo explants, failed to develop during the time require for this study.
Key words: Somatic embryogenesis, oil palm, young leaf, immature female flower, zygotic
embryo
© Copy right IPB, 2012
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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 reviews; and
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No part or all of this work may be reproduced in any form or by any means without
permission from IPB.
OIL PALM (Elaeis guineensis Jacq.) var. Tenera in Vitro
EMBRYOGENESIS EFFECTIVENESS FROM YOUNG LEAVES,
MATURE ZYGOTIC EMBRYO AND IMMATURE FEMALE FLOWER
EXPLANTS
MONDJELI CONSTANTIN
Thesis
Submitted to the Graduate School in partial fulfilment of the requirements for the degree
Master of Science
In Plant Breeding and Biotechnology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
External Examiner at final Exam: Prof.Dr.Ir. Sudirman Yahya, MSc.
Title
: Oil Palm (Elaeis guineensis Jacq.) var. Tenera In Vitro
Embryogenesis Effectiveness from Young Leaves, Mature
Zygotic Embryo and Immature Female Flower Explants
Name
: MONDJELI CONSTANTIN
Registration Number
: A253098021
Program
: Plant Breeding and Biotechnology
Approved by
Supervising Committee
Head
Member
Dr. Ir. Ni Made Armini Wiendi, MS
Dr. Ir. Ade Wachjar, MS
Endorsed by
Head of Study Program
Dr.Ir. Trikoesoemaningtyas, MSc.
Date of Examination: 30 December 2011
Dean of Graduate School
Dr. Ir. Dahrul Syah, MSc. Agr.
Date Graduated.................................
ACKNOWLEDGEMENTS
Glory to God in the highest and peace to his people on earth. Almighty God and Father,
I worship you and give you thanks for all your permanent presence, light, guidance, peace and
protection that I have always received during my studies and specially my research activities
in Bogor Agricultural University, without which all my efforts would not have borne any
fruits.
I would like to convey my sincere and special thanks to my supervising committee
Dr. Ir. Ni Made Armini Wiendi, MS and Dr. Ir. Ade Wachjar, MS for their advice, corrections
resourceful instructions and comments during the preparation, execution and presentation of
this research work.
Grateful thanks to Bapak Jo Daud Dharsono and Dr. Zok Simon, respectively the
Project Leader of Indonesian Palm Oil Board‟s (IPOB) and the former General Manager of
the Institute of Agricultural Research for Development (IRAD) Cameroon for their wisdom
and full goodwill, without which this project would never have surfaced.
I wish to convey my special thanks to Bapak Lalang Buana of PT SMART‟s V Team
Operations and all the members of IPOB mainly Ibu Tyas and Bapak Wasis, to Dr. Koona
Paul the Chief of Centre of the Specialised Centre on Oil Palm Research of La Dibamba
Cameroon (CEREPAH) and all the members for their constant support and encouragements
during my stay in Indonesia.
I want to take this opportunity to thank my family especially the Late Mouadiba
Stanislas who initiated the perfection cult that is expressing in me today; my mother Ango
Imelda, my wife Ngassa Nelly, my children Biba Mondjeli Audrey-stephane, Lihan Mondjeli
Cecile Santana, Mondjeli Mondjeli Constantin II, my brothers, sisters and cousins for their
constant prayers, moral support, sacrifices and encouragement.
I would like to thank the generosity of my classmates of Plant Breeding and
Biotechnology 2009 batch especially Yogo Adhi Nugroho, Nur Arifin, Vina Novita, Vitria
Pupistasari and Jose Maria Alves who were adopted me rapidly in this new environment of
Indonesia. My sincere acknowledgements to all the laboratory staff and technicians of Tissue
Culture Laboratory number 2 of IPB, especially Okti Hanayati and Yogo Adhi Nugroho.
Bogor, December 2011
Mondjeli Constantin
AUTOBIOGRAPHY
The author was born in Ebodié, Cameroon on the 10th of March 1972 from father
Mouadiba Stanislas and mother Ango Emilda. The author is eighth from a total of twenty five
children. The author is a father of two sons and one daughter with one wife.
In year 1995, the author passed the " Baccalauréat D " and was admitted into the
Faculty of Agronomy and Agricultural Sciences of University of Dschang through a
competitive entrance examination. In May 2001, the author was graduated with a Post
Graduate Certificate " Ingenieur Agronome " and was later recruited into the Ministry of
Scientific Research and Innovations of Cameroon in 2002.
The author was posted to Barong-bikang Multipurpose Agricultural Research Station
as assistant researcher on Perennial Crops (Cocoa and Coffee) from 2002 to 2004. Since
2004, the author has serving in Agronomy and Breeding sections of the Specialised Centre for
Oil Palm Research La-Dibamba as researcher.
In August 2009, the author obtained from the Bogor Agricultural University, the
opportunity to further his studies on Plant Breeding and Biotechnology at the Graduate School
with funding from the Indonesia Palm Oil Board and the Government of Cameroon.
During his studies in the Graduate School, the author joined the Community of Post
Graduate Students of Agronomy and Horticulture Department (FORSCA) and the
International Students Forum (ISF) of Bogor Agricultural University. The author attended the
National Exhibition on Perennial Crops to Overcome the Energy Crisis and Global Warming
in Jakarta in November 2010 where a scientific poster was presented. In December 2011, the
author attended the Regional Seminar- workshop on Advances in Tropical Genomics:
Conservation and Sustainable Utilization of Tropical Biodiversity in Bogor where another
scientific poster was submitted.
i
TABLE OF CONTENTS
Pages
LIST OF TABLES…………………………………………………………………………
iv
LIST OF FIGURES……………………………………………………………………….
v
LIST OF APPENDICES…………………………………………………………………..
vi
INTRODUCTION…………………………………………….……………………………
1
Background…………………………………………………………………………
1
Aim of Research……………………………………………………………………
3
Hypothesis………………………………………………………………………….
4
LITERATURE REVIEW…………………………………………………………………
5
Origin and History of Oil Palm (Elaeis guineensis Jacq)…………………………..
5
Taxonomy of Oil Palm (Elaeis guineensis Jacq)……………………………………
7
Botany of Oil Palm (Elaeis guineensis Jacq)………………………………………
7
Plant Tissue Culture…………………………..…………………………………………...
12
History and Definition………………………………………………………………
12
Medium of Tissue Culture…………………………………………………………..
13
Plant Growth Regulators…………………………………………………………….
14
Zygotic Embryo Culture……………………………………………………………….….
15
Ovary Culture…………………………………………………..………………………….
15
Tissue Culture problems and Preventions………………………………………………....
15
Genetic Changes in Culture…………………………………………………………
15
Blackening or Browning……………………………………………………………
16
Embryogenesis…………………………………………………………………………....... 16
Tissue Culture Field in Plant Breeding…………………………………………………..… 17
Oil Palm Genetic improvement………………………………………………………….…. 18
ii
Tissue Culture and Oil Palm improvement……………………………..………………….. 19
MATERIALS AND METHODS…………………………………………………………..
22
Location and Time…………………………………………………………………
22
Plant Materials and Equipments…………………………………………………....
22
Media Preparation…………………………………………………………………
22
Sterilization Compounds…………………………………………………………..
25
Laboratory Equipments and Tools…………...……………………………………
25
Methods……………………………………………………………………………………
25
Experiment 1. Callus and Somatic Embryos Induction from Young Leaf Explant... 25
Palm Tree Sampling and Explant Processing…………………………………
25
Callogenesis Mixing Medium (Combination).....……………………………………
26
Environmental Culture Conditions……………………………………………
27
Embryogenesis Mixing Medium………………………………………………
27
Culture conditions of Embryos……………………………………………….... 27
Subculture‟s Flow Chart…………………..…………………………….…………. 28
Experiment 2. Induction of Somatic Embryo from Mature Zygotic Embryo Explant
………………………………………………………………...………
28
Explant Sampling and Sterilization Process…………………………………
28
Callogenesis Mixing Medium (Combination)..………………….…………….
29
Environmental Culture Conditions………………………………….……….
29
Embryogenesis Mixing Medium…………………………………………….
30
Experiment 3.Induction of Somatic Embryo from Immature Female Flower Explant
.....……………………………………………………………………
30
Explant Sampling and Sterilization Process…………………………………
30
Callogenesis Mixing Medium (Combination)..………………………………..
31
Environmental Culture Conditions………………………..………………… 31
Data Analysis……………………………………………………………………..
31
RESULTS AND DISCUSSIONS…………………………………………………………
32
iii
CONCLUSIONS …………………………………………………..………………………
45
REFERENCES………………………………………………………………………….…
46
APPENDICES…………………………………………………………………………….
53
iv
LIST OF TABLES
Number
Pages
1.
Agroecological factors affecting growth and production of Oil Palm…………
12
2.
Murashige and Skoog (1962) Medium composition…………………………..
23
3.
Eeuwens and Blake (1976) Medium Composition…………………………….
24
4.
Percentage of contamination of oil palm leaves explant per medium composition
and rank of leaf after 36 weeks of culture (%) ………………………….…
32
5.
6.
7.
8
9.
10.
Influence of various medium composition (NAA and 2,4-D) and young leaves
rank of oil palm (E. guineensis) cv. Tenera spear on percentage of oxidized
calli after 4 subcultures.………………………………………………….……
33
Influence of various medium composition (NAA and 2,4-D) and young leaf
rank of oil palm (E. guineensis) cv. Tenera spear on the percentage of
explant bearing callus after 39 weeks ……………………….……………..
34
Mean number of young leaf explants of Tenera oil palm bearing callus per
media ……...................................................................................................
35
Effect of the spear leaf rank of oil palm var. Tenera on callogenesis after
39 weeks in medium ……………………………………………………….
36
Percentage of contamination per medium and type of zygotic embryo explant of
oil palm after 28 weeks of culture (%)………………………………………..
38
Effect of medium composition and type of zygotic embryo explants of oil palm
(E. guineensis) cv. Tenera on percentage of explant bearing callus after 32
weeks..............................................................................................................
39
11.
Mean number of zygotic embryo explants of oil palm var. Tenera bearing callus
per media composition ……………………………………………………
40
12.
Mean number of callogenesis per type of zygotic embryo explant of oil palm
var. Tenera …………………………………………………………………
41
13.
Effect of medium composition and type of zygotic embryo explants of oil
palm (E. guineensis) cv. Tenera on the percentage of explant producing
germinated shoot after 25 weeks in medium ……………………..………
41
Mean number of shoot germination production by zygotic embryo explant per
media composition ……………………………………………………….
42
14.
v
LIST OF FIGURES
Number
Pages
1.
Map showing the Extent of Oil Palm Cultivation ………………………………..
6
2.
Adult Oil Palm (Elaeis guineensis Jacq.) Tree……………………………………
8
3.
Male and Female Inflorescence of Oil Palm………………………………………
10
4.
Mature and Immature Fresh Fruit Bunches of Oil Palm…………………………..
10
5.
Cross section of the different varieties of oil palm (E. guineensis Jacq.)…………
11
6.
Different Type of Oil Palm Explants used………………………………………..
22
7.
Young Leaf Sample Started Material Preparation Process………………………..
26
8.
Mature Zygotic Embryo Explant Preparation Process……………………………..
29
9.
Immature Female Flower Explant Preparation Process……………………………
30
10. Friable nodular callus (NC) proliferation from young leaves explant …………….
33
Root-like callus initiation from young leaf explant of oil palm …………………..
34
12. Embryogenic callus (EC) obtained from young leaf of oil palm…………………..
36
13.
Direct embryoid cells obtained from young leaf of oil palm……………………..
37
14. Germinated shoots from mature zygotic embryo explant of oil palm………….....
38
15.
Root-like callus (RC) initiation from zygotic embryo explant …………………
39
16.
Embryogenic callus (EC) initiation of zygotic embryo………………………….
43
17.
Root initiation cells from immature female flower of oil palm………………….
44
11.
vi
LIST OF APPENDICES
Number
Pages
1.
ANOVA of callus induction from young leaves explant of oil palm…………….
53
2.
Student-Newman-Keuls test of different medium concentrations for callus
induction of young leaves explant…………………………………………….
53
3.
Student-Newman-Keuls test of different leaf ranks of oil palm for callus induction
of young leaves explant………………………..……………………………..
54
4.
Student-Newman-Keuls test of different basal medium for callus induction of
young leaf explant……………………………………………………..…….
54
5.
ANOVA of callus induction from zygotic embryo explant of oil palm…………
54
6.
Student-Newman-Keuls test of different medium for callus induction of mature
zygotic embryo explant…………………………………………..…………
55
7.
Student-Newman-Keuls test of different type of zygotic embryo explant for
callus induction……………………………………………………………..
55
8.
ANOVA of callus induction from zygotic embryo explant of oil palm for basal
medium and type of zygotic embryo……………………………….………
56
9.
Student-Newman-Keuls test of different basal medium for callus induction of
mature zygotic embryo explant……………………………………………..
56
10.
ANOVA of direct shoot formation from zygotic embryo explant of oil palm…..
56
11.
Student-Newman-Keuls test of different medium for direct shoot formation of
mature zygotic embryo explant……………………………………………..
57
12.
Student-Newman-Keuls test of different type of zygotic embryo explant for
shoots germination…………………………..………………………………
57
13.
ANOVA of shoot formation from zygotic embryo explant of oil palm for basal
medium and type of zygotic embryo……………………………………….
58
14.
Student-Newman-Keuls test of different basal medium for shoots germination
of mature zygotic embryo explant……………………………………………
58
1
INTRODUCTION
Background
Oil palm (Elaeis guineensis Jacq.) is an important commercial crop from all tropic producer
countries. Cultivated for palm oil and palm kernel oil, it is the highest yielding oil-bearing
crop, by producing more than five times the yield of oil per year per hectare of any annual oil
crop. The best oil palm is met in Indonesia, regularly producing 8 tons of oil/hectare/year
(Suprianto et al. 2008). The worldwide palm oil production since 2005 was 39.8 million
metric tons, of which 4.3 million tons was in the form of palm kernel oil. As compare to the
other oleaginous crops, Elaeis guineensis oil is the most widely-produced tropical oil, and
constitutes 30 % of total edible oil production worldwide. Since 2007, Indonesia emerged the
world's largest producer of palm oil, by producing approximately 50 % of world palm oil
volume, giving a total of 23 million ton/year (htt://Google Wikipedia oil palm, U.S
Department of Agriculture 2010). Palm oil production has contributed to economic benefits
such as government revenues, profits for companies, employment, and raised incomes for
smallholders. In Indonesia, the world‟s largest producer, the industry generated US$12.4
billion in foreign exchange from palm oil exports in 2009, and supports millions of jobs and
opportunities for rural farmers (Gingold 2011). Therefore, this economic sector employs
about 1 544 641 people in Indonesia and the planted area is increasing every year by 372 000
hectares. Oil palm was planted on a total land area of 8 036 431 hectares in the year 2010 in
Indonesia (D.T.E 2011).
Cameroon is the 13th world producer of palm oil with 190 000 metric tons and the 3rd
African country producer after Nigeria and Ivory Coast (U.S Department of Agriculture,
2010). Palm oil is mainly required for food purposes for about 80% as cooking oil. In
Europe, 10% of consumption high quality products sold contains palm oil. In addition, palm
oil is present into several industrial products and public restaurant food (www.econoecolo.org). Besides producing oils and fats, at present there is a continuous increasing interest
concerning oil palm renewable energy. One of the major attentions is bio-diesel from palm
oil. Bio-diesel implementation is important because of environmental protection and energy
supply security reasons. This palm oil bio-diesel is biodegradable, non-toxic, and has
significantly fewer emissions than petroleum-based diesel (petro-diesel) when burned. In
addition, oil palm is also a well known plant for its other sources of renewable energy, for
example huge quantities of biomass by-products are developed to produce value added
2
products such as methane gas, bio-plastic, organic acids, bio-compost, ply-wood, activated
carbon, and animal feedstock. Even waste effluent; palm oil mill effluent (POME) has been
converted to produce energy. Oil palm has created many opportunities and social benefits for
the locals. In the above perspective, the objective of the present work is to give a concise and
up-to-date picture of the present status of oil palm industry enhancing sustainable and
renewable energy (Sumathi et al. 2007). But the demand for palm oil has mainly increased in
recent years due to its use as a biofuel,
Oil palm (Elaeis guineensis Jacq.) is a monocot plant, originating from West and
Central Africa. Since the establishment of the first plantations in the 1920s and 1930s, five
generations of selection and breeding have been completed in oil palm. The oil palm breeding
program is limited by the long generation, selection cycles with typically 12 years and the
required resources (Hartley 1988).
The success of many in vitro techniques in plants depends on the success of plant
regeneration. Indeed, the application of some techniques such as in vitro mutant and
protoplast fusion are suitable to cell culture but may be limited because of the inability to
regenerate plants. A vast reservoir of genetic variability is available from in vitro plant
regeneration (Evans et al. 1983). The totipotency was first demonstrated with Solanaceous
species by using Nicotiana tabacum and they have been used as model systems of in vitro
studies (Vasil and Hildebrandt, 1965). The first production of haploid plants by the in vitro
culture of excised anthers was achieved by using Datura innoxia (Guha and Maheshwari,
1964). The first somatic hybrid was obtained from two Nicotiana species, N. glauca and N.
langsdorfii (Carlson et al. 1972). The initiation and development of embryos from somatic
tissues in plant culture was first recognized by Steward et al. (1958) and Reinert (1958, 1959)
in culture of Daucus carota tissue. According to oil palm, propagation through tissue culture
is also widely used. Initially tissue culture techniques were used to propagate elite oil palm
clones (Jones, 1974). Otherwise several explants sources have been used to establish tissue
culture. These include mature embryos (Rabechault et al. 1970), inflorescences (Smith and
Thomas 1973), roots (Jones 1974), seedlings (Ong 1977), and young leaves (Staritsky 1970;
Schwendiman et al. 1988). Unfortunately some early aberration were observed from plant
produced (Corley et al. 1986). However, oil palm tissue culture techniques have undergone
continuous improvement in these recent years. This resulted in the production of clonal palms
with minimal abnormality less than 5% (Jones, 1995, Rival et al. 1998). Moreover, Texeira et
al. (1994) reported that, complete plants have been successfully regenerated from various
3
explants of oil palm. Plant propagation using explants from flower organs either anther or
ovary through embryogenesis techniques have been successfully carried out although their
regeneration is more difficult than that using young leaves (Perera et al. 2007, 2008, Texeira
et al., 1994; Verdeil et al. 1994). Although these skills are applied, the frequencies for
complete plant regeneration from some explants are still inefficient (Rival et al. 1999). The
low response of embryogenesis is influenced mainly by genotype and donor plant age
(Ruslan, 1993), in addition to the influence of the appropriate media and explants source.
Explants of young tissues still undergoing cell division and generally form callus more readily
than those from older parts of plant, where the meristematic cell activities are reduced or lost.
In general, the more juvenile the explant material, the greater the likelihood of success (Edwin
and Sherrington, 1984; Lydiane and Kleyn, 1983). Embryogenesis rates of oil palm,
averaging usually less than 5% per ortet (Soh et al., 2006). Embryoids are multiplied until the
total number reaches the required 350. Of these 350 embryoids only 250 will produce shoots
(shooting percentage of 75%). Nevertheless, about 18% of the ortets will provide the bulk cultures
for mass propagation (Wong et al., 1997).
Thus some new protocols contributions are still needed for oil palm plant regeneration
based on embryogenesis. It is in this light that the study was carried out.
Aim of Research
The long term objective is to formulate the protocol of in vitro oil palm regeneration
using young leaves, mature zygotic embryo and immature female flower explants.
Research Activities
In short term, it was aimed to:
Find out the optimum medium for callus induction and somatic embryo formation from
young leaf explant of oil palm of Tenera variety.
Find out the optimum medium for callus induction and somatic embryo formation from
oil palm mature zygotic embryo explants of Tenera variety.
Find out the optimum medium for callus induction and somatic embryo formation from
oil palm immature female flower explant source of Tenera variety.
4
Importance of Study
This work will contribute to obtain protocols on elite oil palm plants propagation for
seedlings production centre. Protocols will be served as tool of clonal oil palm production to
ensure the best use of oil palm resources in germplasm management. The skills can be used
for the plant breeding program purpose and also during the oil palm in vitro selection study.
Hypothesis
1. There is an optimal type of medium composition for each explant source of Tenera
variety oil palm.
2. There is at least one explant for embryogenesis of Tenera variety of oil palm.
3. There is suitable young leaf rank explants for embryogenesis of Tenera variety oil
palm.
5
LITERATURE REVIEW
Origin and History of oil Palm
It is generally agreed that the Oil Palm (Elaeis guineensis Jacq.) is originated from the
equatorial tropical rain forest region of Africa, precisely along the gulf of guinea. It exists in
the wild type and cultivated state. The American oil palm, Elaeis oleifera is native to tropical
Central America and South America. The main belt runs through the southern latitudes of
Cameroon, Côte d‟Ivoire, Ghana, Liberia, Nigeria, Sierra Leone, Togo and into the equatorial
region of Angola and the Congo. Oil palm was first illustrated by Nicholaas Jacquin in 1763,
hence its name, Elaeis guineensis Jacq (en.wikipedia.org/wiki/Oil_palm#mw-head).
During the 14th to 17th centuries some palm fruits were taken to the Americas and
from there to the Far East. The plant appears to have thrived better in the Far East, thus
providing the largest commercial production of an economic crop far removed from its centre
of origin. Oil palms were introduced to Java (Indonesia) by the Dutch in 1848 (Lötschert and
Beese 1983) and to Malaysia (then British colony of Malaya) in 1910 by Scotsman William
Sime and English banker Henry Darby. The first plantations were mostly established and
operated by British plantation owners, such as Sime Darby and Boustead. The large plantation
companies remained listed in London until the Malaysian government engineered their
"Malaysianisation" throughout the 1960s and 1970s (Stevenson 2006).
The cameroon‟s wild oil palm material contains some interesting genotypes which are
present into many germplasm in the world. According to Hartley (1988), in Cameroon
Germans had identified thin shelled oil palm fruit with high oil content as early as 1902. Later
it was known as Tenera type. The material collected by Blaak and Sterling (1996) from some
Cameroon regions such as Ekona and Widikum in 1967 have been widely used to develop the
majority of oil palm planting seed found today in all the oil palm agroecological area of the
world (South East Asia, Africa, South and Central America). According to Chapman et al.
(2003), twenty years of breeding using Dami Deli material crossed with Cameroon and
Tanzanian selections of African oil palm have led to the development of precocious bearing
and cold tolerant oil palms. The oil palm was cultivated in Cameroon since 1907 in the early
days of the German colonial era. The first industrial plantations were created in 1910 by
Germans in Edea region where the current SPFS Company (Swaziland oil palm Farms
Company of Cameroon) is located. In 1929 the Unilever Group created the Pamol Plantations
Limited in the British area of the country. Later the British created the Commonwealth
Corporation in 1947 which is now known as the Cameroon Development Corporation (CDC).
6
In the French area of the country another oil palm plantations were created in 1959 by Rivaud
Group (Red Land) in Dizangue region and it is currently known as SAFACAM (Forestry and
Agricultural African Company of Cameroon). In 1963, the government of Cameroon decided
to create the Cameroon Oil Palm Company or SOCAPALM which is currently the largest oil
palm company in Cameroon. In 2008, Cameroon covered about 101 500 ha of land plantation
of commercial oil palm.
Figure 1. Map showing the extent of oil palm cultivation in 43 oil palm-producing
countries in 2006 (FAO 2007). Cited by Koh and Wilcove (2008)
7
Taxonomy of Oil Palm
The scientific classification of oil palm is as follows:
Kingdom
:
Plantae
Phylum/Division
:
Phanerogam/Spermatophyta
Subdivision
:
Angiospermae
Class
:
Monocotyledonae
Order
:
Arecales
Family
:
Arecaceae
Subfamily
:
Arecoideae
Tribe
:
Cocoseae
Subtribe
:
Elaeidinae
Genus
:
Elaeis
Species
:
guineensis and oleifera
Botany of Oil Palm
Oil palm is a diploid (2n=32) oleaginous tropical perennial crop (Dransfield et al.
2005). Elaeis guineensis is a single-stemmed palm which bears a single vegetative shoot
apical meristem, which is continuously active producing a new leaf every two weeks in
mature palms (Figure 2), under favourable climatic conditions (Adam et al. 2005).
Inflorescences are formed throughout the year in acropetal sequence in the axils of subtending
leaves of the plant. Separated male and female inflorescences are produced in same palm
8
plant in alternating cycle of variable duration depending on genetic factors, age and
environmental conditions (Corley, 1976).
Figure 2. Adult oil palm (Elaeis guineensis Jacq.) tree
The Foliage System of Oil Palm
At the mature stage, E. guineensis Jacq. is constituted with a large crown of 30 to 45
palms measuring 5 to 9 meters long topping the single cylindrical pseudo-trunk. Every year,
about 20 to 26 new pinnate leaves are produced and each mature leaf bears 250 to 300
leaflets. The leaf bases are persistent for years, and prominent leaf scars are arranged spirally
on the trunk of mature palms where bases have fallen. The leaflets cover the distal 2/3 of the
leaf, and the lower 1/3 is spined with spines increasing in length acropetally. The stem or
pseudo-trunk can extend a rate of 30 to 60 cm/year between the ages of 6 to 15 depend on
hereditary and environmental factors. It functions as a supporting, vascular and storage organ.
Generally, the height of trees determines the exploitation times of oil palm plantations. Thus,
palm plantations are exploited up to 25 to 30 years when the trees height is between 12 to 15
meters. Above of this, the harvest of fresh fruit bunches becomes difficult and replanting
should be required.
The Roots System of Oil Palm
The root architecture is a fundamental aspect of plant productivity through its
functional importance in the efficient acquisition of soil resources (water and nutrients
9
uptake). The different morphological types of oil palm roots have been distinguished
according to their development pattern and state of differentiation. As all the monocot, the
root system of E. guineensis is fasciculata. The oil palm has an adventitious root system; with
primary roots generally about 6-10 mm in diameter, originating from the base of the trunk and
either spreading horizontally or descending at varying angles into the soil. The primary roots
bear secondary roots, of about 2-4 mm in diameter. Tertiary roots, about 0.7-1.2 mm in
diameter, branch out from the secondary roots, which in turn bear the quaternary roots.
Quaternary roots are unlignified, about 0.1-0.3 mm in diameter and 1-4 mm long and they are
often assumed to be the main absorbing roots (Corley et al., 1976). The total length of tertiary
and quaternary roots in the soil is the most important root characteristic as they are the
absorbing roots that affect fertilizer use efficiency. Most of the root biomass is found within
1m of the soil surface, but tertiary and quaternary roots are found mostly in the upper 30 cm
from the soil surface. Primary roots can grow up to 20 m away from the base of the palm and
some primary roots could penetrate below the water table at 90 cm from the surface (Ng et al.,
2003). The distribution of roots depends largely on the nature of the soil. Oil palm being a
monocot needs a friable soil for root branching (Zuraidah et al. 2010).
The Reproductive System of Oil Palm
As monoecious plant, male and female flowers of oil palm occur separately on the
same tree. The male and female inflorescences are produced distinctly and alternatively on the
same plant. An inflorescence is initiated in the axil of every leaf. An inflorescence may bear
about 250 spikelets. Each female inflorescence (Figure 3B) can have about 12 to 30 flowers.
Sometimes hermaphrodites flowers can also occur but in rare cases. The female flower is
receptive for pollination between 36 to 48 hours. The male inflorescence (Figure 3A) contains
about 100 000 flowers. Most of the pollen released is shed between 2 to 4 days after opening.
The pollen remains viable for 6 days after release. The insufficient supply of water will
stimulate the plant to produce mostly male flowers. Irrigation and fertilization taken to
increase the flower ratio from male to female will have positive effect only between 22 to 40
months later.
10
A
B
Figure 3. A. Male inflorescence, and B. Female inflorescence of oil palm.
In the nature, the pollination is mainly entomophily (by insects) but can also be done
by the wind and the rain. The main pollinator of the oil palm is the insect from the genus
Elaeidobius and 14 species were found to be associated to the male and female inflorescences.
The most common one is Elaeidobius kamerunicus, which was introduced in Asia, South and
Central America. Its introduction led to an increase in yields of more than 35%. Upon
pollination at the anthesis stage, the female inflorescence may develop and give rise to a fruit
bunch 22 to 26 weeks later. In the field, fruit bunches (Figure 4) which are as compact and
ovoid mass spiked bearing spines can be found on the oil palm 2 to 3 years after planting. The
fruit bunches become heavier as the palms get older. On 10 years old palms, the bunch
weights can be ranged from 10 to 50 kg for a total of 500 to 4 000 fruits per bunch. The fruit
is a sessile drupe generally of ovoid shape measuring 2 to 5 cm length and weighing from 3 to
30g. The oil palm fruit consists of the pericarp which includes the outer and smooth exocarp,
the fibrous and fleshy mesocarp or pulp rich in palm oil, the hard endocarp or shell protecting
the seed and finally the kernel or seed. Inside the shell is the endosperm or kernel. The
endosperm contains large amount of fats and carbohydrates on which the seedling will be
entirely dependent after germination. The oil palm produces two main vegetable oils, namely
palm oil extracted from the mesocarp of the fruit and palm kernel oil extracted from seed.
Immature
Mature
Figure 4. Mature and Immature Fresh fruit bunches of oil palm
11
Commonly three fruit forms or “varieties” of oil palm can be identified based on the
thickness of the shell criterion (Figure 5). The Dura has a shell thickness between 2 to 8 mm,
while the Pisifera has no shell. The shell thickness of the hybrid Tenera is between 0.2 to 2
mm, the fleshy mesocarp of Dura yields between 15 to 17% oil while in Tenera yields
between 21 to 23% oil and Pisifera more than 23% oil. According to commercial purpose,
Pisifera is not cultivated on large scale because of its ability to fruits abortion and thereby
virtual empty bunches production. Tenera which is a hybrid of Dura and Pisifera varieties
remains the commercial variety with 60 to 96% of mesocarp. The hybrid produces more fruit
bunches than Dura.
B
A
C
Figure 5. Cross section of the different varieties of oil palm (E. guineensis Jacq.). A. Dura
palm fruit with thick shell, B. Tenera palm fruit with less thick shell, C. Pisifera
palm fruit with no shell
Climatic and Soil Requirements of Oil Palm
Oil palm culture is done well in low altitude (less than 500 m above sea level), 15º
from the equator in the humid tropics. Evenly distributed rainfall of 1,800 to 2,000 mm/year,
but will tolerate rainfall up to 5,000 mm/year, provided the soil is properly drained. Oil palm
is sensitive to poor drainage and drought. Potential yield is reduced where there are more than
three consecutive months with less than 100 mm rainfall per month. Irrigation may increase
economic returns in areas with pronounced dry periods. More than 2,000 sunshine hours (i.e.,
low cloud cover during daytime) are required.
It is adapted to a range of soil types. Tolerates low pH, but does not thrive at very high
pH (greater than 7.5). Soil must be free draining. The table below shows the suitable and
extreme values of climate and soil that are required by oil palm.
12
Table 1. Agro ecological factors affecting growth and production of oil palm
Characteristic
Rainfall (mm)
Temperature (oC)
Water deficit (mm)
Solar irradiation
Dry season (mths)
Wind (m/s)
pH
Slope (%)
Flooding
Drainage class
Good
2500
25-30
0-150
13-15
Suitability class
Moderate
1450-1700
20-22
150-250
9-11
Severe
1250-1450
10-20
250-400
7-9
Very severe
20
7
>30
Severe flooding
Very poor
PLANT TISSUE CULTURE
History of Plant Tissue Culture
Many authors have put forward various arguments on the origin of plant tissue culture.
Rehwald (1927) is thought to have been the first person who has obtained undifferentiated
callus tissue in sterile culture. But according to most scientists, the real scientific work on
tissue culture was done in 1934 by P.R. White in the USA (United States) and in the same
year, by Gautheret in France, using respectively tomato plant and the cambium of several tree
species (Acer pseudoplatanus, Salix caprea, sambucus) as sources of explant. Before that
period, in 1924, callus culture of carrots (Daucus carota) was reported by two physicians, R.
Blumenthal and P. Meyer in the pathological implication studies.
In 1934, the plant hormone or auxin named indole-3-acetic acid (IAA) was identified
the first time by F. Kögl, A.J. Haagen-Smit, and H. Erxleben. In 1955 C.O Miller has
discovered kinetin, which is a plant growth regulator known as cytokinin.
Plant tissue culture is a technique or a process by which small organs or pieces of tissue
of plants (explants) are grown in aseptic conditions within glass or clear plastic vessels. These
are usually termed in vitro techniques or propagation. It is also called micropropagation
because miniature shoots and plantlets are initially derived. The natural capability of plants to
multiply by asexual means is the basis for multiplication in vitro. Tissue culture was first used
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on a large scale by the orchid industry in the 1950s. Later, it became clear that any plant
would respond to tissue culture as long as the right formula and the right processes were
developed for its culture. The process of tissue culturing plant the explant stage to the final
stage of plantlet requires four basic stages namely: establishment or initiation of explant
(Stage I), multiplication of cells (Stage II), rooting of shoots (Stage III), and acclimatization
or hardening off (Stage IV).
Explants
The kind of explant chosen, its size, age and the manner in which it is cultured, can all affect
whether tissue culture can be successfully initiated and whether morphogenesis can be
induced. The juvenile form had pre-existing competent cells that were able to respond to
auxin and become determined to form organs. However, the adult form appeared to lack cells
with pre-existing competence to form organs, but competence was acquired by some callus
cells once they had been initiated. Explants taken from mature shoots are frequently more
liable than juvenile material to suffer necrosis, especially when surface disinfested and placed
in culture (Hanus and Rohr, 1987). For tissue culture, juvenile explants are usually more
readily established in vitro and grow and proliferate at a more rapid rate than adult material.
This is particularly true with tree species where micropropagation of adult material is often
difficult (Edwin et al., 2008).
Medium of Tissue Culture
The success of plant tissue culture technique is greatly influenced by the nature of the
culture medium. The medium is the substrate for the started explant and it refers to an aseptic
environment of the mixture of a correct concentration and balanced chemical diet compounds
to form a nutrient-rich gel or liquid for growing cultures, whether cells, organs, or plantlets.
Plant tissue culture media are therefore made up from macronutrients salts which provide the
six major elements are N, P, K, Ca, Mg, and S, contributing in the growth of higher plants;
micronutrient salts are Fe, Mn, Zn, B, Cu, Co and Mo which are components of plant cell
proteins of metabolic and physiological importance: chlorophyll synthesis and chloroplast
function (Sundqvist et al., 1980); vitamin; amino acids and other nitrogen supplements; sugar,
buffers; solidifying agent; and growth regulators. The medium pH must be such that it will
not disrupt the function of plant cell membranes or the buffer pH of cytoplasm, and the
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gelling efficiency of agar. The initial pH can often be selected to ensure the integrity of the
medium and the most rapid rate of culture growth (Edwin and Sherrington, 1984).
However a supply of reduced nitrogen is generally necessary for embryogenesis to occur in
callus or suspension cultures.
Plant Growth Regulators
Endogenous compounds occur naturally within plant tissues. They have a regulatory
role rather than nutritional activity in growth and development. These compounds are always
active at very low concentrations and known as plant growth substances or plant hormones.
Otherwise synthetic chemicals with similar physiological activities can be found and
generally supplied in the medium for growth and morphogenesis in vitro. They are termed
plant growth regulators (exogenous). Some recognized plant growth substances classes
include such chemical compounds as auxins, cytokinins, gibberellins, ethylene and abscisins.
The first two compound classes are by far the most important for regulating growth and
morphogenesis in plant tissue and organ cultures.
Auxins are phytohormones very widely used in micropropagation medium to promote
the growth of callus, cell suspensions or organs and to regulate morphogenesis, mainly when
combined with cytokinins. They influence cell enlargement, root initiation, and adventitious
bud formation. The potential levels of endogenous auxin in the explant tissues are found to
depend on the mother plant (donor plant growth state, growth conditions and season of
sampling) from which they were collected. Therefore, the natural auxin will vary between cell
strains of t