UNIVERSITY OF CAPE COAST

DEVELOPMENT OF OIL PALM (ELAEIS GUINEENSIS JACQ) TISSUE CULTURE PROTOCOL

SOLOMON KUUKU MENSAH

2010

UNIVERSITY OF CAPE COAST

DEVELOPMENT OF OIL PALM (ELAEIS GUINEENSIS JACQ) TISSUE CULTURE PROTOCOL

BY SOLOMON KUUKU MENSAH

THESIS SUBMITTED TO THE DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY OF THE SCHOOL OF BIOLOGICAL SCIENCES OF THE UNIVERSITY OF CAPE COAST IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR AWARD OF A MASTER OF PHILOSOPHY DEGREE IN BOTANY

SEPTEMBER, 2010

DECLARATION Candidate’s Declaration I hereby declare that this thesis is the result of my own original work and that no part of it has been presented for another degree in this University or elsewhere.

.................................................... SOLOMON KUUKU MENSAH

DATE: ................................

(Candidate)

Supervisors’ Declaration We hereby declare that the preparation and presentation of the thesis were supervised in accordance with the guidelines on supervision of thesis laid down by the University of Cape Coast. .................................................... DR. ELIZABETH ACHEAMPONG

DATE: ................................

(Principal Supervisor)

DATE: ................................

........................................................ DR. GEORGE OKYERE-BOATENG (Co-Supervisor)

................................................... DR. ISAAC GALYUON

DATE: ................................

(Co-Supervisor)

ii

ABSTRACT This research was carried out to find the most appropriate conditions and requirements needed to propagate Oil palm (Elaeis guineensis) through tissue culture. The explants used were young non-chlorophyllous leaves or ‘cabbage’, immature inflorescence and zygotic embryos from Dura and Tenera seeds. Decontamination of Dura and Tenera seeds as well as cabbage explants was achieved by stirring the explants in a 10% Sodium hypochlorite solution for 5 minutes. A modified Murashige and Skoog (MS) tissue culture medium containing 84mg / l 2,4-Dichlorophenoxyacetic acid (2,4-D) was much more successful in inducing callus from all the explants. Tissue culture media usually contain amino acids like Arginine, Asparagine, and Glutamine. In this experiment, complex organic compounds were used in place of standard amino acids. These were Casein hydrolosate, malt extract and coconut milk. The results obtained showed that, although the media containing complex organic compounds were able to induce callus, the best results were obtained from media containing standard amino acids. The media containing malt extract induced more callus than those containing Casein hydrolysate and coconut milk. With the successful decontamination of Oil palm explants and the initiation of callus from various explants on MS medium containing 84 mg/l 2,4-D, it is recommended that further work should be carried out to produce somatic embryos from callus produced from Oil palm explants.

iii

ACKNOWLEDGEMENTS My special thanks and appreciation go to my supervisors, Dr. Elizabeth Acheampong of the Department of Botany, University of Ghana, Legon, Dr. G. Okyere-Boateng of the Council for Scientific and Industrial Research - Oil Palm Research Institution (CSIR – OPRI) and Dr. Isaac Galyuon of the Department of Molecular Biology and Biotechnology of University of Cape Coast (UCC), for their insightful comments and invaluable suggestions and tremendous assistance which has made this project possible. My special thanks go to the management of CSIR – OPRI for providing me with all the explants material. I further wish to acknowledge the sponsorship of the Council for Scientific and Industrial Research, under the Agriculture, Forestry and Fisheries Sector Programme (AgSSIP). My profound gratitude goes to Mrs. Bernice Asante of the Plant Tissue Culture

Laboratory of the University of Ghana, Legon who shared her

expertise in plant tissue culture with me. I further wish to express my heartfelt gratitude to Prof. E C. Quaye, Mr. Aaron T. Asare, Dr. E. Plas Otwe, Dr. D.H.A.K. Ameworwor and Mr. Daniel Sakyi Agyirifo all of the School of Biological Sciences, UCC for their support, encouragement and concern. My final thanks go to Mrs. Theresa Maison and Miss Olivia Acquah of the Department of Molecular Biology and Biotechnology, UCC, as well as Beyebenwo Kabenla. I would like to acknowledge all the lecturers and staff of the Department of Molecular Biology and Biotechnology for their support.

iv

DEDICATION This work is dedicated to the memory of my deceased parents Mr. and Mrs. Moses Kwesi Mensah and my wife Mrs. Emma Dakwaa Mensah.

v

TABLE OF CONTENTS CONTENT

PAGE

DECLARATION

II

ABSTRACT

III

ACKNOWLEDGEMENTS

IV

DEDICATION

V

TABLE OF CONTENTS

VI

LIST OF TABLES

IX

LIST OF FIGURES

X

LIST OF PLATES

XI

CHAPTER ONE GENERAL INTRODUCTION

1

Background to the study .................................................................................... 1 Propagation of oil palm...................................................................................... 6 The purpose of the study ................................................................................... 9

CHAPTER TWO LITERATURE REVIEW

10

The origin of oil palm ...................................................................................... 10 The distribution of the oil palm plant .............................................................. 11 Oil palm cultivation

12 vi

CONTENT

PAGE

Classification.................................................................................................... 14 Cultivars of Oil palm ....................................................................................... 14 Botanical description ...................................................................................... 22 Leaves

...................................................................................................... 23

Flowers.......................................................................................................... ..24 Fruits

...................................................................................................... 25

Stem

...................................................................................................... 25

Roots

...................................................................................................... 26

Challenges in oil palm breeding ...................................................................... 27 Tissue culture ................................................................................................... 29 Tissue culture media ........................................................................................ 30 Macronutrients ................................................................................................. 30 Micronutrients .................................................................................................. 31 Vitamins

...................................................................................................... 31

Amino acids ..................................................................................................... 32 Carbohydrate source ........................................................................................ 32 Solidifying agents ............................................................................................ 33 Undefined organic additives ............................................................................ 34 Activated charcoal ........................................................................................... 35 pH of plant tissue culture media ...................................................................... 35 Plant growth substances ................................................................................... 36 Oil palm tissue culture ..................................................................................... 39 Problems of oil palm tissue culture.................................................................. 45

CHAPTER THREE MATERIALS AND METHODS

49

Types and parentages of explants used. ........................................................... 49 General methods .............................................................................................. 50 Sterilization of oil palm kernels ....................................................................... 55 vii

CONTENT

PAGE

Sterilization of leaves....................................................................................... 56 Sterilization of inflorescence ........................................................................... 57 Callus initiation experiments ........................................................................... 58 Callus initiation experiments using non-chlorophyllous young leaves or ‘cabbage’

...................................................................................................... 58

Callus initiation experiments using immature inflorescence ........................... 59 Callus initiation experiments using embryos from oil palm kernels ............... 59 Data analyses ................................................................................................... 60

CHAPTER FOUR RESULTS

61

Results of decontamination experiments ......................................................... 61 Leaf decontamination experiments .................................................................. 64 Callus initiation experiments ........................................................................... 66 Callus initiation on MPOB containing different concentrations of naphthalene acetic acid and dichlorophynoxy acetic acid ......................... 67 Callus initiation experiments using a combination of amino acids and various additives ........................................................................................ 69 Callus initiation experiments with inflorescence ............................................. 71 Callus initiation experiments using seed embryo explant................................ 74 Callus initiation experiments using tenera embryo explant ............................. 75 Comparison of dura and tenera callus initiation experiment results ................ 78

CHAPTER FIVE DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

80

REFERENCES . ............................................................................................. 89 APPENDICES .............................................................................................. 112

viii

LIST OF TABLES TABLE

PAGE

1: Types and Parentage of Explants Used (Seeds).......................................... 49  2: Types and Parentage of explants used ('Cabbage’ and Immature inflorescence) .................................................................. 50  3: Modified MS Medium for Oil Palm Tissue Culture (MPOB, 1989) ................................................................................ 51 4: Modified MS Medium for Oil palm Tissue Culture (Texiera et al., 1993) ...................................................... 53  5: Effectiveness of Different Concentrations of NaOCl on the Sterilization of Oil palm seeds (Dura) .............................. 62  6: Effectiveness of Different Concentrations of NaOCl on the Sterilization of Oil palm seeds (Tenera) ......................... 62  7: Effectiveness of Different Concentrations of NaOCl on the Sterilization of Oil palm leaves..................................... 64  8: Results of Callus Initiation Experiment Using Various Media Formulations. (Tenera ‘cabbage’ Explants) ............. 66  9: Callus Initiation from ‘cabbage’ on MPOB Media Containing Different Concentrations of 2,4-D and NAA (K28:B50) ................................. 68 10: Callus Initiation of ‘Cabbage’ MPOB Media Containing Various Amino Acids and Various additives (ME, CH, and CM). (K28:B37) ......................................................................................... 70  11: Callus Initiation of Inflorescence explants on MPOB Media Containing Amino Acids and various additives. (K28:B37) ............................... 72 ix

LIST OF FIGURES FIGURE

PAGE

1: Comparison of Yield of Major Oil seeds. ..................................................... 4  2: Global Palm Oil Production.......................................................................... 5  3: Oil Palm Distribution in Africa .................................................................. 12  4: Oil Palm Production Worldwide (2006) ..................................................... 13  5: Propagation of Oil Palm by Tissue Culture ................................................ 45  6: Callus Initiation of Dura Embryos on MPOB Medium Containing 2,4-D, Amino Acids and Various Additives. .................................. 74 7: Callus Initiation of Tenera Embryos on MPOB Medium Containing 2,4-D, Amino Acids and various Additives. .................................. 76 8: Comparison of Callus Initiation of Dura and Tenera Embryos at 48 Weeks on MPOB Medium Containing Amino Acids and Various Additives. ........................................................................ 78 

x

LIST OF PLATES PLATE

PAGE

1: Pisifera Fruit (Shell-less)

................................................... 16 

2: Tenera Fruit (Thin Shell)

................................................... 17 

3: Dura Fruit (Thick Shell)

................................................. 18 

4: Types of Oil Palm Kernels.......................................................................... 19  5: Fruit Types of Oil Palm .............................................................................. 21  6: Oil Palm Tree (Elaeis guineensis Jacq) ...................................................... 23  7: Young Sterile Non-chlorophyllous Leaves ‘Cabbage’

...................... 65 

8: Callus Initiated from Tenera Embryo after 24 Weeks of Incubation

76 

9: Callus Initiated from Tenera Embryo after 48 Weeks of Incubation

78 

xi

CHAPTER ONE GENERAL INTRODUCTION

Background to the Study The Oil palm is one of the most important oil producing crops in the world. The Oil palm produces two different types of oil, palm oil which is obtained from the fleshy mesocarp and palm kernel oil which is produced from the kernel of the seed. Palm kernel oil is made up of short chain saturated fatty acids similar to coconut oil (PORIM, 1991). Palm oil is one of the most important edible oils accounting for 30% of global edible oil consumption (FAO, 2003). Both palm oil and palm kernel oil are used for culinary purposes like cooking and frying. They are also used industrially in the manufacture of soaps, candles, lubricants, grease, toothpaste, non-dairy creamers, baking fats, mayonnaise, toilet soaps and ice cream (PORIM, 1991; Purseglove, 1972). Refined bleached deodorized palm oil is now an important raw material in the manufacture of several products (Choo, Ma, Yap, Ooi & Basiron, 1993). Palm oil can be used to create biodiesel either as a simply processed palm oil mixed with petrol diesel or it can be processed through a transesterification process to create a palm oil methyl blend (Faessler, Folmetz, Seang & Lee, 2007). The use of palm oil and Oil palm residues in the production of biodiesel and biogas is gaining much prominence in the face of declining global crude oil 1

production (Choo et al., 1993). The cake which is left behind after the extraction of palm kernel oil from the kernel of the seed is also used in the formulation of animal feed (Hartley, 1988). The Oil palm also provides building materials, fibre and brooms. Another product of the Oil palm is palm wine which is obtained from the sap by tapping. Palm wine is sweet when it is freshly tapped and contains 4.5g/100ml of sucrose and 3.4g/100ml of glucose, it can be drunk fresh or fermented and distilled to produce gin or ethanol. Palm wine can also be fermented to produce vinegar (Okigbo, 1980). The Oil palm plant has so many uses that it has been described as nature’s gift to humankind and has been consumed worldwide for more than 5,000 years (MacFarlane, Swerman & Cousey, 1984). Palm Oil is a natural source of antioxidants and tocopherol and tocotreinols which are constituents of Vitamin E. It is also rich in Vitamin A (Kato, Yamoka, Komiyama & Umezewa, 1985; Temple & Basu, 1988). Palm oil contains several saturated and unsaturated fats like laurate (0.1% saturated), myrisate (0.1% saturated), palmitate (44% saturated), stearate (5% saturated), oleate (39% monosaturated), linolenate (10% polysaturated) and linolenate (0.3% polysaturated), it does not contain cholesterol(Cottrell,1990). Palm kernel Oil however is more saturated than Palm Oil (USFDA, 1990). Linoleate is an unsaturated omega-6-fatty acid and oleate is an omega9-fatty acid. These two are essential fatty acids required by humans. They help lower blood cholesterol levels and are necessary for growth of bones and the proper functioning of joints and healthy skin (Mukherjee & Mitra, 2009). Red palm oil contains an assortment of vitamins and phytochemicals. It is the 2

richest dietary source of Pro-Vitamin A carotene which is essential to protect the body from heart attacks, cataracts and arthritis (Kamen, 1999). Palm Oil is the second most widely produced edible oil after soybean oil. World production of palm oil in the 2006-2007 growing season was 38.0 million metric tonnes and palm kernel oil production for the same period was 3.8 million metric tonnes (Gustoni, 2009).

Palm oil is the most widely

produced tropical oil and makes up 31% of total edible oil produced Worldwide (USDA, 2004). Soybean accounts for 50% of global oil seed production and about 160 million metric tonnes of soybeans was produced worldwide in 2000 (FAO, 2003). The Oil palm is the highest yielding of all oil crops (Mayes, Jack, Marshall & Corley, 1997). It is the leading tropical vegetable oil crop but uses the least acreage, saving large tracks of land (Datuk, Basiron & Yaw, 2009). It produces more oil per unit area than any other oil seed crop (Fig 1). Oil palm produces 5-6 tonnes of oil per hectare per year, soybean produces 1 tonne of oil per hectare per year, coconut produces 2.6 tonnes of oil per hectare per year and groundnut produce 400kg of oil per hectare per year (Paranjothy, 1984). Potential yields of 17 metric tonnes of oil per hectare per year have been estimated by Corley (1985) but the average yield from commercial plantations in Malaysia is 6-7 tonnes of oil per hectare per year. Oil palm and soybean produced similar metric tonnes of oil in 2003 despite the approximately 8-fold greater acreage for soybean (FAO, 2003). Global production of oil and fats stood at 160 million tonnes in 2008. Palm oil and palm kernel oil were jointly the largest contributors accounting

3

for 48 million tonnes or 30% of the total global output. Soybean was second with 37 million tonnes or 23% of total global output of oil (MPOID,2008).

Figure 1: Comparison of Yield of Major Oil Seeds. Source: USDA-FAS, 2009

The ten largest palm oil producing countries are Indonesia (44%), Malaysia (43%), Thailand (2%), Nigeria (2%), Columbia (2%), La Cote d’Ivoire (1%), Ecuador (1%), Cameroun (1%) Congo (1%) and Ghana (1%) and others (2%), Fig 2 (BERNAMA, 2005). The production of Palm Oil in Malaysia has been an important part in the economic and industrial development of that country. Malaysia has been the world leader in Oil palm cultivation and palm oil production until it was over-taken by Indonesia in 2006/2007. During the 2006/2007 session, Indonesia produced 16.8 million metric tonnes of Palm Oil, while Malaysia produced 15.8 million metric tonnes of palm oil during the same period 4

(Gustoni, 2009). Palm oil production in Indonesia rose to 20.9 million metric tonnes in 2009 and will out space that of Malaysia in the foreseeable future. Indonesia and Malaysia account for 87% of global Oil palm production and 89% of global palm oil exports (USDA, 2009).

Figure 2: Global Palm Oil Production Source: USDA- FAS, 2009.

In Ghana, most of the Oil Palm cultivation is done by small-holder producers with a few large-scale plantations like Twifo Oil Palm Plantations (TOPP), Benso Oil Palm Plantations (BOPP) and the Ghana Oil Palm Development Company (GOPDC). About 1 million metric tonnes of Oil palm fruits were produced in Ghana in 2002 from an estimated area of 282,000 hectares (FASDEP, 2002). The expansion of Oil palm cultivation was one of the Presidential Special Initiatives (P.S.I.) of the New Patriotic Party government and it was envisaged that 100,000 hectares of land will be put 5

under oil palm cultivation annually from 2004 to 2009. This was supposed to increase Oil palm cultivation from 282,000 hectares to 882,000 hectares by 2010. Malaysia the second largest producer of Oil Palm has a total area of 4.5 million hectares of land under oil palm production employing more than 570,000 people (MPOID, 2008; FAO, 2004). The economic and industrial development of Malaysia has been fuelled in part by the Oil palm industry (BERNAMA, 2005) and the four fold anticipated increase in Oil palm production will make Ghana a major producer and exporter of Palm Oil. This will enable Ghana to produce enough raw materials for industries that depend on palm oil and other products produced by the Oil palm plant. Oil palm has the potential to meet the increased demand for domestic and industrial edible oils, it is however a “land hungry” plant and has been blamed for increasing the rate of deforestation in Indonesia, Malaysia, Thailand and the Philippians (FAO, 2003).

It is therefore

imperative that planting materials with the highest genetic potential are produced for growers to maximize yield.

Propagation of Oil Palm Oil palm is propagated by seed using hybrid seeds. These hybrids are developed from crosses between Dura (thick shell) as female parents and Pisifera (Shell-less) as pollen source. The thickness character is controlled by a monofactorial gene (Beirnaert & Vanderweyen, 1941). The Tenera hybrid gives the best yield of palm oil and a reasonable yield of palm kernel oil, but because it is a hybrid it does not breed true to type. When it is selfed it segregates in a ratio of 1:2:1, producing all three fruit forms. This is an 6

indication of incomplete dominance of Dura over Pisifera. Because of this the cross of Dura and Pisifera is repeated each time to produce Tenera hybrids (Krikorian, 1994). There is also a very high degree of heterogeneity among hybrids with some plants yielding as much as 60% more oil than the average of a given cross (Noiret, 1981). Oil palm also has a long breeding cycle of 1012 years (Okyere-Boateng, 2001), the production of improved seeds depends on the setting up of seed orchards which in the case of oil palm has 143 plants per hectare planting at 9 meters triangular spacing. The low planting density of oil palm means large tracts of land are needed for the establishment of seed orchards. Oil palm breeding programmes are labour intensive, time consuming and expensive, as such, these problems have made genetic improvement of Oil palm to be very slow (Jacquemard, Baudouin & Noiret, 1997). The constraints indicated earlier and the need to produce materials from elite high yielding Tenera hybrids have necessitated the need to employ vegetative propagation techniques in the breeding of Oil palm. Oil palm however does not produce suckers or off shoots (Tisserat, 1979 (a)) and so the only method which can be used to produce disease free clonal true-to type planting material is tissue culture. Tissue culture research on Oil palm started in 1970 (Durand-Gasselin, Le Guen, Konan & Duval, 1990). Work on Oil palm tissue culture has mostly employed non chlorophyllous tissue (Okyere-Boateng, 2001). The source of explants used in Oil palm tissue culture is usually apical meristem, young roots, young leaves and immature inflorescence (Corley, Wooi & Wong, 1979). Cloning of Oil palm has been achieved by somatic embryogenesis of callus derived from various explants. The embryogenesis of callus according 7

to Jones (1974) is a function of the source of explants. Callus which can be used to induce embryogenesis have been produced from segments of Oil palm leaf laminae (Rabechault & Martin, 1976). Roots have also been induced to produce

callus

when

inoculated

on

media

containing

2,4-

Dichlorophenoxyacetic acid (2,4-D) or 1-Naphathaleneacetic acid (NAA) (Smith & Thomas, 1970). Ong (1975) was able to induce callus from the roots of Oil palm which was cultured on modified MS media containing 2,4D. Callus has also been induced from immature inflorescence (Lioret, 1981). Callogenesis can also be induced from mature embryos cultured on a medium containing 2,4-D and IBA (Kanchanapoom & Domyoas, 1999). Tissue culture research on Oil palm has been going on over the last three decades in several laboratories such as the ORSTOM – CIRAD laboratories in France, the Unilever laboratories in England, the PORIM laboratories in Malaysia, and the Marihat Research Centre in Indonesia. The work done so far at the various laboratories has led to refinements of existing protocols but due to commercial and other considerations, much of the research findings remain unpublished and most of the published protocols do not work as they are expected to. For Ghana to develop a clonal propagation programme, there is the need to use tissue culture in the production of Oil palm seedlings. Tissue culture is being used to produce Oil palm planting material by Indonesia, Malaysia and Thailand and it is no wonder that these countries are the leading producers of palm oil.

8

The Purpose of the Study The increase in production of Oil palm in Ghana has been hampered by the problem of producing adequate quantities of good quality seeds. The Institution which has the mandate and resources to produce good quality planting material is the Oil Palm Research Institute of the Council for Scientific and Industrial Research. Presently the production of seeds depends on the use of Dura and Pisifera (D x P) crosses to produce Tenera hybrids. This process is laborious, time consuming and expensive and places limitations on the number of planting materials which can be produced. It is therefore justified that a study be conducted into the use of tissue culture to produce clonal planting materials in Ghana, besides the advantages of producing true to type, clean and high yielding planting materials. The main purpose of this study was to find out the most appropriate method of initiating callus from various explants of the Oil palm plant.

The specific objectives of the study were to: •

Determine the best method for decontaminating explants



Optimize levels of auxins for Callus initiation

9

CHAPTER TWO

LITERATURE REVIEW

The Origin of Oil Palm Oil palm was first reported by the Portuguese sailor Eannes in 1434 (Hartley, 1988). The Oil palm is believed to have its origin from West Africa (Zeven, 1964; Chevalier, 1943). Cook (1942) and Corner (1966) however suggested an American origin because the plant grows spontaneously in the coastal regions of Brazil and most of the related taxa like the American oil palm, Elaeis melanococca originates from South America. Zeven (1964) and Rees (1964) made a strong case for the African origin of Oil palm by basing their facts on the work of earlier botanists like Clusus and Sloan (Hartley, 1988) and the discovery of fossil pollen in West Africa which is similar to the pollen of Oil palm plants found in Nigeria. Historical accounts and writings from the earliest explorers to Africa indicate that Oil palm groves were a common occurrence in tropical Africa (Purseglove, 1972).

Hartley (1988) has suggested that Oil palm was

introduced to South America during the transatlantic slave trade. It is now generally accepted that Oil palm originated from the tropical rain forest of West Africa.

10

The Distribution of the Oil Palm Plant Oil palm is native to West and Central Africa and natural semi-wild grooves of the plant are found along the African coastline from the Cape Verde Islands to Angola (Chevalier, 1943). Oil palm flourishes on the island of Madagascar and around Lake Mweru and along the basins of the Manawibolo and Tsiribihina rivers (Hartley, 1988).

It is a plant of the

lowlands and flourishes from Sea level to 300m (Opeke, 1992).

The

establishment of the Oil palm plant is however restricted within 20oN and South of the equator (Zeven, 1967). The main Oil palm belt of West Africa stretches from Sierra Leone to Cameroon and then into the equatorial region of Africa (Fig. 3). Oil palm does well in areas with altitudes lower than 400 m with 3000 mm to 5000 mm of rainfall distributed throughout the year (Surre & Ziller, 1963). Oil palm was introduced to Java, Singapore and Sumatra from West Africa and at present the development of industrial Oil palm plantations is centred in South East Asia. Oil palm plantations are also found in Central and South America and West Africa. About 60% of the total global acreage of 6 million hectares of Oil palm is situated in Indonesia and Malaysia (FAO, 2005).

11

Figure 3: Oil Palm Distribution in Africa LEGEND:

The yellow shaded areas represent the oil palm belt. The areas of heaviest population and production are shaded red. The green dots represent the positions or boundaries of isolated areas of colonisation. (Adopted from Zeven (1967) and earlier authors)

Oil Palm Cultivation Oil palm is a plant of the lowlands and requires frequent and adequate rainfall. It needs 3000 mm or more of rainfall distributed evenly throughout the year. The temperature range suitable for Oil palm is 22-33oC and the ideal relative humidity (R.H.) is 70%. The Oil palm also needs at least five hours of solar radiation daily (Hartley, 1988). It thrives best on deep well drained soils which must be free of iron concentration (Opeke, 1992). 12

The plant can

tolerate a wide range of soil pH but neutral soils are the best. Large scale plantations of Oil palm are situated 7oN and 7oS of the equator at altitudes lower than 400m and high rainfall of (4000-5000mm) (Surre & Ziller, 1963). The industrial plantations of Oil palm began in South East Asia at the beginning of the 20th Century, plantations were developed in Africa along the Gulf of Guinea and in Brazil in the 1920’s (Duval, Engelmann & DurrandGasselin, 1995). The development of Oil palm plantations has since been extended to South American and Central American countries like Ecuador, Colombia, Peru and Costa Rica (Fig. 4) (Rival, 2000).

Figure 4: Oil Palm Production Worldwide (2006) Source:Saadat,2008

13

3Classification Oil palm belongs to the Kingdom Plantae, Order Aracales, Family Arecaceae, Subfamily Arecoideae, Tribe Cocoeae, Genus Elaeis and species guineensis, (USDA, 2004).

The genus was founded on Oil palm plants

introduced to Martinique in the Caribbean and it received its name from Nicolas Jacquin (Bailey, 1933). The generic name Elaeis is derived from the Greek word ELAION which is the word for oil in Greek. The species name refers to the place of origin of the plant which is the Guinea coast of Africa. The genus has three species Elaeis odora, Elaeis oleifera and Elaeis guineensis. At present only Elaeis guineensis is being exploited for oil production. The Oil palm (2n=32) has 16 pairs of chromosomes consisting of repetitive sequences of DNA (Castilho, Vershinin & Heslop-Harrison, 2000). The genome size is estimated to be 1.95 billion base pairs. It is about four times the size of the rice genome and two thirds the 33size of the maize genome (Snaidy, Beckers, Herram, Ritter & Rohde, 2003).

Cultivars of Oil Palm Cultivars do not occur in oil palm in the strict sense of the word because the palm is monoecious and cross pollinated individual plants are usually heterozygous.

They can however be classified in terms of the

structure of the shell of the fruit as follows:

14

Dura Type: Dura fruits are characterized by a thin mesocarp (Plate 3) and a thick endocarp or shell (Plate 4). The kernels are large. The Dura type is genetically denoted by DD as the plant is a diploid.

The Pisifera Type: It has a thick mesocarp and lacks an endocarp (Plate 1). It is thus shell-less and has small kernels. The Pisifera is genetically homozygous and recessive for shell. This variety is denoted as dd.

The Tenera Type: It also has a thick mesocarp (Plate 3), a thin endocarp (Plate 4) and a reasonably sized seed or kernel. The Tenera is the preferred type for the establishment of plantations since it produces more palm oil than the other fruit types and a reasonable quantity of kernel oil. genetically heterozygous and is denoted by Dd.

15

The Tenera form is

FRUIT FORMS

Plate 1: Pisifera Fruit (Shell-less) Source: NIFOR,2008

16

x4

Plate 2: Tenera Fruit (Thin Shell) Source: NIFOR,2008

17

x4

Plate 3: Dura Fruit (Thick Shell) Source: NIFOR,2008

18

x4

(a) Dura Oil Palm Kernels

x2

(b) Tenera Oil Palm Kernels

x2

Plate 4: Types of Oil Palm Kernels

19

The Oil palm can also be classified based on the pigmentation of the exocarp as follows: Albescens: This type of fruit lacks the typical reddish colour at maturity and contains little carotene. When it ripens it is pale yellow or waxy in colour and has a black or green upper half (Plate 5). The Albescens is rare and the colour condition is recessive.

Virescens: It has green unripe fruits which ripen to assume a light reddish orange coloration with a small green tip (Plate 5). This fruit type is more common than the Albescens.

Nigrescens: It is the most common fruit variety. The unripe fruit is deep violet to black at the apex and Ivory towards the base and when it ripens (Plate 5), it has a deep red coloration (Pursglove, 1972).

20

Nigrescens

Unripe Fruit

Ripe Fruit

x1

Albescens

Unripe Fruit

Ripe Fruit

x1

Ripe Fruit

x1

Virescens

Unripe Fruit

Plate 5: Fruit Types of Oil Palm (Source: Hartley, 1988)

21

Botanical Description The Oil palm plant is a large feather palm with a solitary columnar stem which has short internodes (Hartley, 1988). The stately stem can reach up to 80 feet in height when it grows in the wild but when cultivated it attains a height of 20 to 30 feet. The trunk of the Oil palm is straight and stout and has a diameter of 1 to 2 feet (Plate 6). The crown of the Oil palm consists of 30 to 50 leaves and about 20 to 40 leaves are produced each year. The Oil palm tree has a single growing point or terminal meristem which produces about two leaves per month (Rival, 2000).

22

Plate 6: Oil Palm Tree (Elaeis guineensis Jacq) Leaves The leaves are pinnate and the leaflets are borne on each side of the leaf stalk or rachis (Broekmans, 1957). The leaflets are produced when an entire leaf splits up during the elongation of the leaf axis. The leaves are spread out between 10 to 25 feet in length with leaflets numbering 200-300 leaflets per leaf. The leaflets are 3-4 feet long and 1.5 to 2 inches wide (Corley, 2001). The leaves are almost parallel to the ground. Stomata are 23

found only on the lower surface of the leaflets (Okyere-Boateng, 2001). Each leaf has short thorns at its base. The leaves are produced in a regular sequence with older leaves progressively displaced centrifugally as younger leaves are produced. The leaflets are not continuous and the irregular appearance of the frond is a characteristic of the species (Hartley, 1988). A well grown mature Oil palm has 41 to 50 leaves each having a dry weight of 4.5 to 5.5 kg and a leaf area of 10m2. (Corley & Tinker, 2000).

Flowers Oil palm is a monoecious plant and it produces male and female inflorescences in leaf axils.

It is a temporal dioecious species and thus

alternates male and female flowering cycles (Cruden, 1988).

The

inflorescences of both male and female flowers are a compound spadix enclosing two spathes or bracts. The male inflorescence has finger-like spikes which contain 400 to 1500 flowers. Each individual flower is less than ⅛ of an inch in length and contains 6 anthers that protrude beyond the petals. The female inflorescence has shorter spikes which contain hundreds of flowers. The female flowers are larger than male flowers and are borne in triads with two abortive males flanking one female flower and all enclosed in spiny bracts (Beirnaert, 1935). Female flowers have off-white tri-lobed stigma visible among the bracts. Some inflorescences are hermaphrodite and both male and female spiklets occurs on the same bunch (Hartley, 1988). The inflorescences arise from the leaf axils and the female inflorescence matures into a bunch after it has been pollinated and fertilized. Oil palm is pollinated by wind and insects. In Africa, weevils (Elaeidobius 24

spp) are the pollinators. These weevils have been introduced to the far East since the native pollinator Thrips hawaiienis is not as efficient as the African pollinator. In Latin America a native beetle Mystrops costaricensis along with the introduced Elaeidobius spp are the pollinators (Corley & Tinker, 2000).

Fruits The bunch of the Oil palm which is made up of pollinated and fertilized female flowers is made up of spiklets which are borne on the peduncles. The fruits are sessile drupes 2-5 cm long and weigh 20 to 30g. The mesocarp and endocarp vary in thickness. In the Dura variety the endocarp is thick and the mesocarp not so thick, the Tenera variety has a thicker mesocarp and a thinner endocarp. The colour of the exocarp is green and changes to orange in the virescens variety. In the nigrescens variety the colour changes from green to orange with brown or black check colours. The fruit is obovoid in shape (Hartley, 1988), the mesocarp is fibrous and oily and the seed which is a nut is opaque white in colour and encased in a brown endocarp or shell. Botanically the kernel of the oil palm is the seed (Purgslove, 1972). Each female inflorescence which develops into a bunch of fruits contains 5000-8000 fruits. Fruits ripen about 5-6 months after pollination (Corley, 2001).

Stem The stem or trunk of the Oil palm which is also called a stiple has a wide central core or cylinder which is separated from a narrow cortex. The cylinder is made up of a zone of vascular bundles containing fibrous phloem sheaths embedded in parenchyma tissue. There is no cambium present in the 25

trunk of the oil palm tree and because of this no secondary thickening takes place, and the plant reaching its full width just below the apex. The apex of the Oil palm is a conical structure buried in the crown and the apical meristem can be found in a concave-like depression at the apex of the stem (Rees, 1964).

The stem of the Oil palm tree supports the leaves and vascular

structures which transport mineral salts and water upwards and the products of photosynthesis downwards. The stem also acts as a storage organ and it stores some of the products of photosynthesis. The sieve tubes (Phloem) of oil palm remain functional throughout the lifespan of the plant (Okyere-Boateng, 2001).

Roots The Oil palm has an adventitious roots system. The primary roots which are 6 to 10mm in diameter arise from the bole and spread into the soil at various angles or horizontally. New primary roots are produced continuously to replace dead ones (Yampolsky, 1922; Okyere-Boateng, 2001). The primary roots carry secondary roots which are 2 to 4mm in diameter. The secondary roots in turn give rise to tertiary roots, which are 0.7 to 1.2mm in diameter. The tertiary roots which bear quaternary roots are unlignified and are 0.1 to 0.3mm in diameter. The quaternary roots are the main absorbing roots and are thus responsible for the uptake of water and mineral salts (Okyere-Boateng, 2001). The penetration of the adventitious roots system depends on the depth of the water table. If the water table is high most of the primary roots will be found close to the surface of the soil (Lambourne, 1935). According to Purvis

26

(1956) the root system of the Oil palm in free draining sandy soils can reach great depths of up to 45 metres. The adventitious root system of the Oil palm plant ensures that it is firmly anchored in the soil and it enables the Oil palm to obtain water and nutrients from the soil. The total length of roots of a mature Oil palm tree may exceed 60 km. (Corley & Tinker, 2000).

Challenges in Oil Palm Breeding Oil palm is usually propagated by seed.

The seeds planted in

plantations are Tenera hybrids obtained by crossing Pisifera (shell-less) and Dura (thick shelled) varieties. The thickness character is controlled by a monofactorial gene (Beirnaert & Vanderweyen, 1941). Breeding schemes utilize pollen from Pisifera to fertilize female Dura flowers, the fertilized ovules develop into Dura × Pisifera Crosses which are then germinated and planted. The first filial generation then gives Tenera seeds. The process of producing good parent stock for producing quality planting materials involves the selection of high yielding Dura plants which are further improved using the recurrent reciprocal selection method in order to concentrate the favourable genes that would otherwise be dispersed within the large population (Okyere-Boateng, 2001). The development of good Pisifera male parents for commercial Dura x Pisifera seed production (D x P) also involves the use of the recurrent reciprocal selection method.

27

In Oil palm, the breeding cycle takes a minimum of 12 years and the steps involved are outlined below: •

Pollen collection = 2 years (The selected plant may be in the female cycle when pollen is required).



Crossing programme = 2-3 years (The required plant may be in the male cycle when required for pollination).



Seed germination = 6 months.



Pre-nursery and nursery = 1 ½ years.



Field planting, growth and maturation = 3-4 years.



Fruit and Bunch analysis for selection = 2 years.

(Okyere–Boateng, Personal Communication, 2nd August 2010. Plant Biotechnologist/Breeder, C.S.I.R. – Oil Palm Research Institute, Kusi, Ghana.) Because of this, genetic improvement is very slow and not much progress has been achieved over the last 50 years (Jacquemard et al., 1997). Secondly since Tenera is a hybrid, it produces all three fruit forms in a ratio of 1:2:1 indicating the incomplete dominance of Dura over Pisifera. Because segregation occurs when the Tenera hybrid is selfed, the Dura x Pisifera cross is repeated each time to produce Tenera hybrids for commercial oil palm plantations (Krikorian, 1994). There is a very high degree of heterogeneity among hybrids with some plants producing as much as 60% more oil than the average of a given cross (Noiret, 1981). When these bottlenecks are considered in addition to the low planting density of 143 palms per hectare coupled with the need to establish seed orchards for producing commercial planting seeds, Oil palm improvement is 28

evidently labour intensive, time consuming and expensive (Rival, 2000). These constraints existing in Oil palm breeding programmes have made it imperative that vegetative propagation techniques be exploited in Oil palm breeding. The Oil palm plant however does not produce suckers or off shoots like the Date palm (Tisserat, 1979(b)). The biological characteristics of the Oil palm do not make it possible for it to be propagated by normal horticultural techniques and the only way to clonally propagate Oil palm is through tissue culture techniques (Rival, Barre, Bule, Harmon, Duval, & Noiret, 1981).V

Tissue Culture Tissue culture is the technique of growing differentiated plant parts like cells, tissues or organs taken from a mother plant on an artificial medium in vitro under aseptic conditions. Cultures can be initiated from explants made up of small pieces of meristematic or embryonic tissue which are surface sterilized and then cultured on sterile media under asceptic conditions (Coombs, 1994). Plants cells are totipotent and so they are capable of developing into whole plants under suitable conditions. In higher plants, various structures are organised to perform specific functions (Okyere-Boateng, 2001). In issue culture, the explants undergo far reaching changes and reach a ‘crisis’ situation (Meins, 1983). The explants loose their normal supplies of water, minerals, carbohydrates and plant growth substances. The restoration of the several supplies to the explants is the first requirement to induce growth and

29

so the culture medium must contain minerals, water, sugars, plant growth substances and sometimes vitamins (Schwabe, 1984).

Tissue culture media The growth and morphogenesis of plant tissue in culture is greatly affected by the composition of the culture medium used (Constable & Shyluk, 1994). The culture media usually used in plant tissue culture are made up of some or all of the following constituents, Macronutrients, Micronutrients, Vitamins, Amino Acids, Energy Source, Solidifying Agents, Plant Growth Substances, Undefined Organic Additives or Supplements and Activated Charcoal (Dixon, 1987).

Macronutrients Macronutrients are needed in large amounts. There are six major elements that plant cell and tissue culture require. These are Nitrogen (N), Potassium (K), Magnesium (Mg), Phosphorus (P), Calcium (Ca), and Sulphur (S) (Razdan, 2003). Plant Cell and tissue culture medium should contain at least 30-60mM of inorganic nitrogen for adequate growth (George, 1993). Plant cells can grow fairly well when the medium contains nitrates, but for optimum growth the medium must contain both a nitrate and an ammonium nitrate source (Pierik, 1989). Plants need potassium for the growth of cells and most media used in plant cell and tissue culture contain potassium in the nitrate or chloride form at a concentration of 20-40mM (George, 1993). According to Pierik (1989), the optimum concentrations of Magnesium, Calcium, Sulphur and Phosphorus range from 2 -5mM. 30

Micronutrients The essential micronutrients for plant cell and tissue culture are Iron (Fe), Manganese (Mn), Zinc (Zn), Boron (B), Copper (Cu) and Molybdenum (Mo), Nickel, (Ni), and Chlorine (Cl). Iron is the most critical of the micro nutrients and since iron easily precipitates out of solution, the use of ethylene diaminotetraacetic acid (EDTA) – Iron Chelate is employed. Iron and Molybdenum are added to plant cell and tissue culture media at a concentration of 1μM/l, Copper and Chlorine at a concentration of 0.1μM/l, Zinc at a concentration of 5 – 301μM/l, Manganese at a concentration of 20-901μM/l and Boron at a concentration of 25-1001μM/l, (George, 1993). According to Razdan (2003), an element can be regarded as essential for plant growth if i.

The plant is not able to complete its life cycle without it.

ii.

The action of the element is specific and that it cannot be replaced by any other element.

iii.

It is a constituent of a molecule which is known to be essential.

Vitamins Plants normally synthesize the vitamins they require for their proper growth and development. Vitamins are required by plants as catalysis in various metabolic and anabolic processes (Butcher & Ingram, 1976). In tissue cultured plants the lack of some vitamins may become limiting factors which can affect cell growth. Because of this, vitamins may be included in plant cell and tissue culture media. Vitamins normally used in plant cell and tissue

31

culture include Thiamine or vitamin (B1), Pyridoxine or vitamin (B6), Nicotinic acid, Myo Inositol (Roberts & Shuler, 1997) Thiamine is required by all cells for growth and is normally added to culture media at a concentration of 0.1 to 10.0mg/l. Nicotinic acid is added to culture media at a concentration of 0.1-5.0mg/l, pyridoxine and Myo Inositol are added to culture media at concentrations of 0.1 to 10mg/l and 50-5000mg/l respectively (Dodds & Roberts, 1985).

Amino acids Tissue cultured plants can synthesize the amino acids they need, but the addition of certain amino acids can stimulate cell growth. Amino acids added to tissue culture media provide plants cells with an immediate available source of nitrogen which generally can be taken up more rapidly by plant cells than inorganic nitrogen (George, 1993). Amino acids used in plant cell and tissue culture include the following: Glycerine, Asparagine, Arginine, Cysteine and Tyrosine. (Dubuis, Kurt & Prenosil, 1995)

Carbohydrate source Tissue cultured plants require a source of carbohydrate because only a few plant cell lines cultured in vitro are capable of supplying all their carbohydrate needs (Bahmani, Karami & Gholami, 2009). According to Zhang,Zhong and Yu (1996), carbohydrate source is a significant factor in plant cell metabolism. The most commonly used carbohydrate is sucrose. It is easily assimilated and relatively stable. Other carbohydrates like glucose, maltose, 32

galactose, fructose, maltose, rafinose and lactose can also be used (Rappaport, 1954).

Solidifying agents Plant tissue culture media can be in ‘liquid’ or ‘solid’ forms. Agar which is produced from seaweed is the most common gelling agent used for most routine operations. Agar is a high molecular weight polysaccharide obtained from some species of red algae mostly from the genera Gracilaria and Gelidium( Whyte, Englar & Hosford, 1984). When agar is mixed with water, it forms a gel that melts at between 60-100 0C and solidifies at around 40o C (Cameron, 2006). Agar gels are therefore stable at incubation temperatures. Agar gels do not react with the components of the media and are not affected by plant enzymes (Klimasewska, Berner-Cardou & Sutton, 2000; Whyte et al., 1984). Agar concentrations at 0.5 to 1.0% can produce a firm gel (Mao, Tang & Swanson, 2001). Agarose which is a purified extract of agar can also be used as a gelling agent. It is however required in lesser quantities because it has a higher gelling strength, Agarose is used in culturing protoplast (Dodds & Roberts., 1985). Gellum gum which is known commercially as gelite or phytogel, an artificial gelling agent, is also used in plant tissue culture. It is able to withstand up to 120oC and can be used to detect contamination and also to culture thermophilic organisms (Krieg & Gerhardt, 1981)

33

Undefined organic additives Tissue culture media are often supplemented with a variety of organic extracts which have constituents of an undefined nature. These include casein hydrolysate, malt extract, peptone, endosperm fluids, fruit juice and animal by-products (Thorpe, 1994). Casein hydrolysate can be used as a cheap source of a mix of amino acids and can be helpful in inducing callus formation (Krikorian, 1994 ; Aktar, Nasiruddin & Hossain, 2008). Casein hydrolysate concentrations added to media at 0.5 - 2.0g/l can induce callus in Date Palm (Abdel-Rahim, Abdel-Fatal, Kobasse, El-Shemy & Abd-El-Samei, 1998). Casein hydrolysate has been successfully used to induce callus formation in Tobacco (Heimer & Filner, 1970), beans (Crocomo, Sharp & Peter, 1976) carrot (Weatherell & Dougall, 1976) and fenugreek (Singh, Kokate & Tipnis, 1981). Cardi and Monti (1993) found out that addition of casein hydrolysate at 2.0g/l is important for callus production in pea and kidney bean. In tissue culture, the success achieved with casein hydrolysate (0.05 – 1.0g/l) for some species has been significant (Razdan, 2003). Coconut Milk commonly used at 5-20% (v/v) can be used to produce callus (Razdan,2003). Coconut milk is also known to as a supplier of growth regulators, it contains sugars, minerals, vitamins, amino acids and phytormones and can be used in plant tissue culture (Yong, Ge, Ng & Tan, 2009). Malt extract contains Kinetin, Myo Inositol, Urea and Arginine and can be used to induce callus. (Duchooa & Ramburn, 2004).

34

Activated charcoal Activate charcoal was initially added to tissue culture media in order to stimulate soil conditions (Van Winkle & Pulman, 1995). It is now used in plant tissue culture to improve cell growth and development. Activated charcoal plays a critical role in micropropagation, somatic embryogenesis, anther culture, protoplast culture and stem elongation (Wang & Huang, 1976). Activated charcoal irreversibly absorbs inhibitory compounds in culture medium and substantially decreases toxic metabolites, phenolic exudation, brown exudates accumulation and absorbs toxic pigments (Thomas, 2008). Activated charcoal is also involved in several stimulatory and inhibitory activities including the release of substances naturally present in it which promote growth. Activated charcoal darkens the medium, absorbs vitamins, metal ions and growth regulatory substances which are gradually released and become available for use by plants (Butcher & Ingram, 1976). Activated charcoal added to media promotes greater plant survival, improved plant growth, callus initiation, production of somatic embryos and improved plant vigour (Van Winkle & Pulman, 1995).

pH of plant tissue culture media Plant cell and tissues require optimum pH for proper growth and development. The pH of culture media must be such that it does not disrupt the plant tissue (George, 1993). The pH of the media also determines whether salt remain in a soluble form. pH influences the uptake of medium ingredient, affect chemical reactions especially those catalyzed by enzymes and affect the gelling efficiency of agar (Razdan, 2003). 35

The effective pH of culture media is therefore restricted and as a rule of thumb, it is usually set at 5.0 to 6.0 before sterilization (George, 1993). If the pH is high, the medium becomes too hard and if it is low, the agar does to solidify satisfactorily (Skirvim, Chu, Mann, Young, Sullivan & Fermariam, 1986)

Plant growth substances Plant growth substances are substances which at low concentration influence plant growth and differentiation. The major classes of plant growth substances are Abscisic Acid, Auxins, Cytokinins, Ethylene and Gibberellic Acid. Other plant growth substances are Steroids and Phenol derivatives (Mader, 1997). Auxins influence cell elongation, cell division, adventitious root formation, induction of primary vascular tissue, induction of callus and growth of cells in suspensions (de Klerk, 1993; Coombs, 1994). Auxins used in plant tissue culture include Indole-3-Acetic Acid (IAA) and Indol-3-Butyric Acid (IBA). Both these Auxins induce adventitious root formation. Other Auxins are Phenylacetic Acid (PAA), 1 Naphatalenacetic Acid

(NAA),

2,4-Dichlorophenoxyacetic

Trichlorophenoxyacetic

Acid

(2,4,5-T),

Acid

Picloram

(2,4-D),

2,4,5-

(4-amino-3,5,6

tri-

chloropicolinic acid) and Dicamba (3,6-dichloro-2-methoxybenzoic acid). 2,4D, NAA, 2,4,5-T and Picloram can be used to induce callus formation and growth (Davis, 1995).

36

Cytokinins are used in tissue culture to promote cell division, adventitious shoot formation, prevention of senescence, cell growth and differentiation (Postlethewaith, Hopson & Hadson, 1989). When Cytokinins are used in conjunction with Auxins, cell division and morphogenesis takes place (Okyere-Boateng, 2001). Cytokinins used in plant

tissue

culture

isopentenyladenine

include

(iP),

Zeatin

(Z),

isopentenyladenosine

Zeatinriboside (iPA)

(ZR),

and

6-

Benzylaminopurine (BAP). Abscisic Acid (ABA) aids the maturation of somatic embryos; it also inhibits the growth of callus. ABA facilitates the acclimatization of plantlets before they are taken to the field (de Klerk, 1993). Absisic Acid can also be used to hasten the rate of morphogenesis (Li, Rice, Rohobaugh & Wender, 1970 ; Shepard, 1980). Gibberellic Acids (GA) are plant growth substances which can be used in plant tissue culture to induce the growth of undifffentiated cells (Schroeder & Spector, 1957). Gibberellic Acids are also used in plant tissue culture in combination with Auxins and Cytokinins to induce callogenesis (Engelke, Hamzi & Skoog, 1973). Gibberellic Acids can induce cell enlongation and release dormancy in somatic embryos (de Klerk, 1993). Gibberellic Acids are isolated from the fungus Gibberella fujikuroi and the first Gibberellic Acids isolated were GA, GA2, and GA3. More than 30 Gibberellic Acids have now been isolated (Solomon, Berg, Martin & Villee, 1993) Tissue Culture can be grouped into the following (a) Organ culture (b) Callus culture (c) Protoplast culture and (d) Anther and Microspore culture.

37

Organ culture In organ culture shoot explants which are essentially equivalent to sterile cuttings are grown in vitro. The reduction in apical dominance leads to the formation of auxiliary shoots in every leaf axil leading to the production of several shoots which can be separated and either subcultured to yield more shoots or rooted and nursed into plantlets (Schwabe, 1984). Organ cultures include meristem cultures as described earlier and embryo cultures which involve fertilized or unfertilized zygotic seed embryos which are dissected out and cultured in vitro until they mature into seedlings. Other organ cultures are root cultures and flower bud cultures (Alemanno, Berthouly & MichanxFerrieve, 1996).

Callus cultures In unorganized callus cultures, callus which are unorganized masses of cells are induced to form embryoids by the introduction of relatively high levels of auxins into the growth medium. This makes the explants to lose its organization and proliferate in a rather random manner.

Callus tissue can be

cultured on solid media containing agar or other gelling agents like gelite. Callus tissue can also be dispersed in an agitated liquid medium to form suspension or cell cultures (Gupta, 1996). Callus tissue can be induced to undergo differentiation by drastically lowering the auxin content of the media and raising the cytokinin levels.

Callus can also be induced to form

‘embryoids’ which can be germinated to produce plantlets. (Schwabe, 1984)

38

Protoplast cultures Protoplast cultures can also be initiated using plant cells whose cell walls have been removed. The protoplasts can be used for somatic cell fusion as well as for the absorption of foreign DNA, cell organelles, bacteria and viral particles (Gupta, 1996). Another method of tissue culture is anther and microspore culture which is the culture of anthers or microspores on artificial media to produce haploid plants (Okyere-Boateng, 2001; Gupta, 1996). Tissue culture can be used to preserve endangered species and also as an aid to conventional breeding programmes. Tissue culture can also be used as a valuable tool in genetic engineering.

Oil palm tissue culture: Callus Initiation The first step in Oil palm tissue culture is the production or initiation of callus from various explants. Calli are actively growing undifferentiated tissue produced in higher plants in response to wounding and some infections. A callus can also be formed in vitro during tissue culture (Coombs, 1994). The source of explants may be embryos, young roots, young stem, stem apex tissue and young inflorescence. The media used for this phase of Oil palm tissue culture is Murashige and Skoog (MS) media and according to Paranjothy and Rohani (1982) this media is adequate for initiation of callus. It has also been found out that the presence of an auxin in the MS medium is essential for the initiation of callogenesis. (de Touchet, Duval & Pannetier, 1991)

39

The addition of 2-4 Dichlorophenoxyacetic acid (2,4-D) and Naphthalene Acetic Acid (NAA) to the MS medium speeds up the initiation of callogenesis (Smith & Thomas, 1973). Although 2,4-D and NAA when added to MS media induces callogenesis, 2,4-D has been found to be more effective than NAA in inducing callus formation (Paranjothy, 1984). Browning of explants in Oil palm tissue culture occurs frequently due to the action of agar and cytokinins (Martin & Rabechault, 1978). This problem is solved by adding activated charcoal to the medium. The addition of activated charcoal to the medium used in oil palm tissue culture apart from preventing browning of the explants also promoted callogenesis (Reynolds & Murashige, 1979). Callus formation originates from cells connected with the vascular system and the callus generated can be sub-cultured onto fresh MS media at regular intervals, the growth of callus is however very slow (Paranjothy, 1984).

Somatic embryogenesis Somatic embryogenesis is the formation of an embryo from a somatic cell instead of from sex cells or gamete. Somatic embryogenesis relies on the fact that an individual somatic cell contains the blue print of the genetic information of the plant. Somatic embryogenesis results when the existing gene expression programme of the somatic cells of an explant is terminated and replaced with an embryonic gene expression programme in the somatic cells leading to the formation of somatic embryos (Okyere-Boateng, 2001). Oil palm is an arborescent monocotyledon and cannot be multiplied by conventional methods of vegetative propagation. Somatic embryogenesis would be of great interest due to its high potential in plant breeding. Success 40

in plant regeneration by means of somatic embryogenesis has already been reported in many species of palms (Valvende, Avias & Thorpe, 1987). Because Oil palm does not produce auxiliary shoots, the process of producing new plants using tissue culture methods makes use of the process of somatic embryogenesis by using callus generated from various explants (Jones, 1974). Callus initiation is therefore a prerequisite for somatic embryogenesis. Although other factors like light, molality, culture media and the initial inoculum are factors in the promotion of somatic embryogenesis, (Rabechault, Ahee & Guenin, 1972), the induction of somatic embryogenesis is not very well understood (Okyere-Boateng, 2001).

The formation of callus and

somatic embryos remains one of the bottlenecks in Oil palm tissue culture. The rate of callogenesis of Oil palm explants is unpredictable. According to Corley and Tinker (2000), the rate of callogenesis is about 19%. Wooi (1995) stated an even lower rate of callogenesis from leaf explants of about 6%. It has however been observed that after successive sub-culturing of callus, the callus cells become capable of undergoing embryogenesis. This phenomenon was observed at both high and low auxin concentrations by Turnham and Northcote (1982).

Despite the economic importance of Oil palm tissue

culture, little is known about the chemical characteristics and molecular changes associated with callogenesis and embryogenesis in Oil palm (Eng-Ti, Halimah, Soo-Heong, Elyana, Chi-Yee, Lesli, . . . Rajinder, 2008). It has however been observed that the production of somatic embryos involves three pathways and this process depends on the type of callus from which the somatic embryos originate (Duval, Engelmann & Durrand-Gasselin, 1995). 41

Embryogenesis on nodular callus In this pathway the cambium-like zone undergoes modification and the meristematic cells of the nodular callus evolve into cells with high nucleocytoplasmic ratio, large central nucleus with single nucleolus dense cytoplasm and numerous droplets.

These characteristics are typical of

embryogenic cells (Duval et al., 1995). These cambium like cells which undergo the evolution then further undergo intense multiplication and then give rise to embryoids. Somatic embryogenesis however occurs at a very low frequency.

Embryogenesis of fast-growing callus Nodular callus produces a new type of callus called Fast-growing callus (FGC) (Smith & Jones, 1970), when it is cultured on a Medium containing a high concentration of auxin. Fast Growing Callus has a fast growth rate and is made up of clumps of meristematic cells, large cells and lacunae (Ahee, Arthuis, Cas, Duval, Guenin, Hanower, . . . Zukerman, 1981). These cells originate from the disorganization of the procambial zone. When fast growing callus is cultivated on media with reduced auxin concentration and or supplemented with cytokinin, embryogenesis is observed (Duval et al., 1995).

Embryogenesis on granular callus Granular callus can develop on the surface of nodular callus. Granular calli however are located at the periphery of the parenchymatous tissue unlike 42

nodular calli which have an internal origin.

When granular nodules are

transferred to liquid medium containing 2,4-D and activated charcoal they produce embryonic cell cluster suspensions, which can give rise to somatic embryos (de Touchet et al., 1991).

Germination of somatic embryos Nodular calli and fast growing calli can develop into polyembryonic tissue which is made up of somatic embryos at various stages of development and produce adventitious somatic embryos and simultaneously the most advanced somatic embryos can develop into shoots when cultured on hormone free media (Duval et al., 1995). Callus obtained from roots have been able to develop roots on media containing low concentrations of auxins (Martin, Cas & Rabechault, 1972).

Roots and shoots can be regenerated from callus

induced from apical meristem (Rabechault, Ahee & Guenin, 1972). When shoots developed from germinated somatic embryos reach a suitable size (5 to 7 cm) they are separated and rooted on media containing 2-5 x 10-6 to 10-6M NAA and sometimes GA3 (Nwankwo & Krikorian, 1983). Recent work on somatic embryogenesis have focused on the production of plantlets using embryogenic cell suspensions by developing systems based on the artificial seed concept (Redenbaugh, 1993) in which the somatic embryogenesis process is used to produce individual embryos with a relatively synchronous development (Rival, Bertrand, Beule, Combs, Trouslot & Lashermess, 1998). The artificial seed concept has made mass propagation of Oil palm using tissue culture possible and a further step is the use of bioreactors for the production of cell suspensions of Oil palm for somatic 43

embryogenesis (Goret, Rosli, Oppenheim, Willis, Lessard, Rha & Sinsky, 2004).

Plantlets developed from somatic embryos have to be gradually

hardened as the leaves produced in tissue cultured plantlets fail to develop a normal cuticle if introduced directly into the field (Wooi, 1984). A high humidity is also needed for the survival of newly transplanted plantlets (Wooi, Wong & Corley, 1981). After acclimatization, plantlets are transferred to the pre-nursery and treated like ordinary seedlings before they are transferred to the field. A few plant species are at present propagated on a large scale using plantlets obtained through embryogenesis. This method of morphogenesis has some advantages. It is a cost effective method of producing large quantities of clonal planting material (Dixon & Gonzales, 1995). There are about 20 Oil palm tissue culture laboratories in operation throughout the world with capacities ranging form 10,000 to 200,000 plantlets per year (Zamzuri, Mohd, Rajunaidu & Rohani, 1999). As compared to seed production, tissue culture of Oil palm offers several advantages. It allows the rapid multiplication of uniform planting material with desired characteristics. This enables the improvement of planting materials using existing individual plants with desirable qualities and characteristics (Wahid, Abdulla & Henson, 2004).

44

The diagram of propagation of Oil palm by tissue culture is illustrated in Figure 5.

Fast Growing Callus (FGC)

Stage 3

Explant

(Roots, young leaves, inflorescences, embryo)

Primary Callus

Stage 1 Callus Initiation

Embryogenesis of FGC Polyembryogenic material Multiplication of Polyembryogenic Material Plantlets without roots Complete Axenic Plantlets Plantlets in normal growing condition

Stage 2 Embryogenesis on Primary Callus

Stage 4 Shoot Development

Stage 5 Rhizogenesis

Stage 6 Hardening and Establishing

Adopted from Lioret, 1981 Figure 5: Propagation of Oil Palm by Tissue Culture

Problems of Oil palm tissue culture: Sterilization of explants Sterilization is a procedure which kills or removes microorganisms or their spores from explants. If this is not done, the culture medium will be contaminated. Roots are more difficult to sterilize than internal plant tissue and seeds which are generally free from contamination (Okyere-Boateng, 2001). Explants are normally washed under running water and then soaked in 45

water containing a wetting agent and suitable pesticide. The explants are then placed in a suitable container with a lid containing disinfectants. The explants are then washed in a sterilant solution and sterile distilled water before they are transferred under asceptic conditions onto sterile media. A variety of sterilants are used to obtain complete asepsis. hypochlorite,

Mercuric

chloride,

Hydrogen

permanganate (Okyere-Boateng, 2001).

These include Sodium

peroxide

and

Potassium

Surface sterilization can also be

obtained using 70% ethanol. Well concealed parts like the stem apex, young inflorescence and seed embryos are easier to sterilize than roots and leaves and all precautions must be taken to ensure that explants are sterilized properly to ensure contamination- free cultures are obtained. It must also be noted that the source of the explants is important since materials from vigorously growing palms are not heavily contaminated (Starisky, 1970) and produce less contaminated cultures than materials from palms which are not very healthy.

Somaclonal variation Somaclonal variation is a variation which occurs in plants propagated from cell or tissue cultures. It may arise as a result of the influence of culture media, the culturing process and the condition of the plant. Clonal propagation of Oil palm through somatic embryogenesis has led to the production of millions of plantlets. In vitro propagation of Oil palm provides an efficient way of multiplying selected clones of Oil palm. Plants regenerated through somatic embryogenesis are usually morphologically and cytologically normal but sometimes a proportion of 46

aberrant plants are produced (George, Hall & de Klerk, 1996). Genetically abnormal plants are more likely to occur when embryogenesis is initiated in callus or suspension cultures after a period of unorganised growth or when embryogenic cultures are maintained for several months (Orton, 1985). So despite its considerable potential, somatic embryogenesis has a serious drawback, because tissue culture-derived plants of Oil palm can develop abnormal flowers in which the stamen primordial are converted in carpel-like (Mantled Fruits) (Corley, Lee, Law & Wong,1996).

This abnormality which is

unpredictable can be heritable (Matthes, Singh, Cheah & Karp, 2001). The mantled somaclonal variation of oil palm affects the formation of floral organs. In Oil palm, male and female inflorescences are produced alternatively (temporal dioecy).

In the mantled variant, there is the

transformation of male floral organs (Stamens and Staminodes) into pseudocarpels which lead to fruit abortion in most severe cases (Hartley, 1988). Mantled plants occur in 5 to 10% of tissue cultured Oil palm plants (CIRAD, 2003). Studies of ploidy levels and RAPD polymorphism do not reveal any genomic chains that can be linked to the variant phenotype and so it is believed that mantling is of epigenetic origin (Morcillo, Gamgeur, Adam, Richaud, Singh, Cheah, . . . Trangear, 2006). The occurrence of the mantled variant hampers the scaling up of clonal plant micropropagation. To improve the reliability of the clonal micropropagation process developed for Oil palm it is necessary to identify molecular markers of the mantled flowering abnormality. The early detection of the ‘mantled’ variant is thus very critical in Oil palm clonal micropropagation. To find out whether callus cultures have or lack the mantled abnormality novel genes are being 47

used, for example (EgM39A and EgAAI) are being used as molecular makers (Morcillo et al., 2006). The identification of clonal conformity markers first at the mRNA level and secondly by studying sequence specific DNA methylation is also being pursued so that the fidelity of plantlets developed through tissue culture can be assured. It has also been noted that using safe tissue culture methods can reduce floral abnormalities (Corley & Law, 1997). Several works have been carried out on the problem of mantling in order to characterise gene expression in calli and embryoids producing normal and abnormal plants with the hope of identifying an early marker of the mantled phenotype (Tregear, Morcillo, Ricchaud, Berger, Singh, Cheah, . . . Duval, 2002). Molecular maps of Oil palm have been published (Mayes, James, Hormner, Jack & Corley, 1996) and the use of DNA markers can be used for fingerprinting to determine the identity of clones in order to increase the efficiency of breeding programmes (Mayes, Jack, Marshall & Corley, 1997)

48

CHAPTER THREE MATERIALS AND METHODS The explants used were Dura and Tenera seeds, young nonchlorophyllous leaves or ‘cabbage’ and young immature inflorescences. All the explant materials were obtained from the Council for Scientific and Industrial Research - Oil Palm Research Institute (CSIR-OPRI). Below are Tables 1 and 2 showing parentages of the seed, ‘cabbage’ and Inflorescence explants used.

TYPES AND PARENTAGES OF EXPLANTS USED A. Seeds Table 1: Types and Parentage of Explants Used (Seeds)

Code

Parentage

Shell Type

21

K4.904T x K4.904T

Tenera

22

K1.2793T x K1.2793T

Tenera

23

K6.815T x K14.218D

Tenera

24

K6.942D x K4.974T

Dura

25

K6.942D x K4.987T

Dura

26

K6.942D x K4.450T

Dura

49

B. Cabbage and Immature Inflorescence Table 2: Types and Parentage of explants used ‘Cabbage’ and Immature inflorescence

No.

Parentage

Shell Type

K28:32

K1.3747D x K3.880P

Tenera

K28B:50

K1.3747D x K3.880P

Tenera

K28:B37

K4.3747D x K3.880P

Tenera

General methods The media used for the research was modified Murashige and Skoog (MS) media. The method used for preparing the stock solutions used was that outlined by Dixon, (1987). In this procedure, stock solutions of macro nutrients (A), micro nutrients (B), iron source (C), were prepared and stored in a refrigerator at a temperature of +4oC. Stock solutions of vitamins (D) and amino acids (E) were prepared and stored in 5ml aliquots in a deep freezer at -20oC. The formulations for preparing plant tissue culture media used was based on the protocol outlined by Texeira, Sondahl and Kirby (1993) and that of the Malaysian Palm Oil Board (MPOB, 1989). These tissue culture cloning protocols are outlined in Table 3 and Table 4. 50

Table 3: Modified MS Medium for Oil Palm Tissue Culture (MPOB, 1989)

Constituents

Concentration of Stock Solution (x100)

A. Macro Nutrients

1000ml

NH4 NO3

165.0g

KNO3

190.0g

CaCl2H20

44.0g

MgSO4.7H20

37.0g

KH2 PO4

17.0g

NaH2 PO4.2H20

17.0g

B. Micro Nutrients

1000ml

H3BO3

6.20mg

KI

0.83mg

MuSO4.7H2O

22.3mg

ZNSO4.7H2O

8.6mg

Cu SO4. 5H2O

2. 5mg

CoCl2. 6H2O

2. 5mg

Na2 M002 . 2H2O

25. 0mg

C. Iron Source

1000ml

FeSO4 . 7H2O

55. 6mg

Na2EDTA

74. 6mg

or NaFeEDTA

37. 5mg

51

Volume of Stock Solution per Litre of Medium (ml)

Storage of Stock Solution (0C)

50ml

+40C

5ml

+40C

5ml

+40C

5ml

Constituents

Concentration of Stock Solution (x100)

D. Vitamins

100ml

Myo Inositol

100mg

Nitronic acid

100mg

Pyridoxines HCl

100mg

Thiamine HCl

100mg

E. Amino Acids

100ml

L – Arginine L –asparagine L – glutamine

Volume of Stock Solution per Litre of Medium (ml)

Storage of Stock Solution (0C)

5ml

-20OC (in 5ml aliquots)

5ml

-20OC (in 5ml aliquots)

100mg 100mg 100mg

Add as a solid (30g/l) Add as a solid (7g/l)

F. Sucrose G. Agar

Add as a solution of 3g in 10ml distilled H2O (pH 7)

H. Activated Charcoal I. 2,4 –D or

56mg/l

NAA

186mg/l

Used to replace Amino Acids Casein hydrolysate

1g

or Malt extract

1g 52

150ml

or Coconut milk

Table 4: Modified MS Medium for Oil Palm Tissue Culture (Texeira et al., 1993)

Constituents

Concentration of Volume of Stock Stock Solution Solution per Litre of (x100) Medium (ml)

A. Macro Nutrients

1000ml

NH4 NO3

165.0g

KNO3

190.0g

CaCl2H20

44.0g

MgSO4.7H20

37.0g

KH2 PO4

17.0g

B. Micro Nutrients

1000ml

H3BO3

6.20mg

KI

0.83mg

MuSO4.7H2O

22.3mg

ZnSO4.7H2O

8.6mg

Cu SO4. 5H2O

2. 5mg

CoCl2. 6H2O

2. 5mg

Na2 M002 . 2H2O

25. 0mg

C. Iron Source

1000ml

FeSO4 . 7H2O

55. 6mg

Na2EDTA

74. 6mg

or NaFeEDTA

37. 5mg

53

Storage of Stock Solution (0C)

25ml

+40C

5ml

+40C

5ml

+40C

5ml

Constituents

Concentration of Volume of Stock Stock Solution Solution per Litre of (x100) Medium (ml)

D. Vitamins Cysteine

100ml 500mg

Myo Inositol

100mg

Nicotinic acid

50mg

Thiamine HCl

25mg

Pyridoxine HCl

8mg

Biotin

20mg

Pantothenic acid

20mg

5ml

Storage of Stock Solution (0C)

-20OC (in 5ml aliquots)

Add as a solid (30g/l)

F. Sucrose

Add as a solid (7g/l)

G. Agar

Add as a solution of 3g in 10ml distilled H2O (pH 7)

H. Activated Charcoal

4.65mg/l

I. 2,4 –D

6.65mg/l

Picloram

Casein hydrolysate, coconut milk and yeast extract were also used. These organic additives are source of extra nutrients and growth regulators. The malt extract and casein hydrolysate were obtained from the Biochemistry Department of the University of Ghana, Legon. The coconut milk was obtained by draining the endosperm from several fresh coconut fruits into a 54

beaker and then filtered through Whatman No1 filter paper before autoclaving and storing in a freezer at a temperature of -10OC until required. The actual formulation of the tissue culture media was prepared using the steps outlined below: •

To make up 1 litre of medium, required volumes of stock solutions were pipetted into a 2 litre glass beaker on a magnetic stirrer.



The required amount of sucrose was added and allowed to fully dissolve by adding more water.



The auxin used was dissolved in ethanol and added to the mixture.



The volume of the mixture was topped up to 950ml with distilled water.



The pH of the mixture was adjusted to 5.8 - 5.9 with dilute HCl or NaOH solution.



A 0.3% activated charcoal solution which had been prepared separately and adjusted to a pH of 7 was added to the mixture.



Agar (7g) was added and the mixture topped up to 1 liter with distilled water.



The mixture was heated in a microwave oven to melt the agar and stirred to allow thorough mixing.



Batches of medium (15ml) were transferred into Tissue culture tubes, capped and autoclaved for 15minutes at 121oC at a pressure of 1. 06kg/cm3.

55

Sterilization of Oil Palm Kernels Embryos of Oil palm seeds were used as source of explant to initiate callus cultures. The right treatment to be used to ensure that the embryos isolated from the seeds were sterile is a very important step. The procedure used for the decontamination of oil palm kernels and seeds are outlined below: •

The kernels were washed in running water for 10 minutes and briefly immersed in 70% ethanol for 15 seconds.



The kernels were then placed in various concentrations of Sodium hypochlorite (NaOCl) i.e. 0%, 5%, and 10%. Two drops of Tween 20, a non-phytotoxic wetting agent was added to the Sodium hyprochlorite solution.



The kernels in the Sodium hypochlorite solution were stirred for 20 minutes to ensure that all contaminants were destroyed.



The kernels were then removed from the Sodium hypochlorite solution and cracked to facilitate the removal of the embryos. The seeds were rinsed briefly in 70% ethanol for 15 seconds.



The seeds were then washed in various concentrations of Sodium hypochlorite i.e. 0%, 5%, or 10% containing 2 drops of Tween 20 for 5 or 10 minutes.



The seeds were rinsed 3 times with sterile distilled water.



The operculum of the seeds was cut off and the embryo removed and placed on sterile media.

56

Sterilization of Leaves Non-chlorophyllous leaves of Oil palm were used as explants materials for the production of callus. The leaves were sterilized using the procedure below: •

The Oil palm leaves were cut into small strips of 1cm2.



The strips of Oil palm leaves were washed in sterile distilled water for 10 minutes.



The explants were then removed from sterile distilled water and briefly immersed in 70% ethanol for 10 seconds and then washed in 0%, 5% and 10% Sodium hypochlorite solutions containing drops of Tween 20 for 5 or 10 minutes.



The samples were rinsed three times in sterile distilled water.



Finally, the explants were dipped in a mixture of 0.1g/l ascorbic and 0.15g/l citric acid for 15 minutes to prevent oxidation and then placed on sterile media.

Sterilization of Inflorescence The immature inflorescence was sterilized using the procedure below: •

The immature inflorescence was cut into small pieces of about 1cm.



The pieces of the immature inflorescence were washed in sterile distilled water for 10 minutes.



The explants were then removed from sterile distilled water and briefly immersed in 70% ethanol for 10 seconds and then washed in 10%

57

Sodium hypochlorite solutions containing drops of Tween 20 for 5 minutes. •

The samples were rinsed three times in sterile distilled water.



Finally, the immature inflorescence was dipped in a mixture of 0.1g/l ascorbic and 0.15g/l citric acid for 15 minutes to prevent oxidation and then placed on sterile media.

Callus Initiation Experiments Callus initiation experiments were conducted using explant materials from non-chlorophyllous young leaves also referred to as ‘cabbage’, immature inflorescence and mature embryo from oil palm kernels.

Callus Initiation Experiments using Non-chlorophyllous Young Leaves or ‘Cabbage’ Non-chlorophyllous young leaves or cabbage was used to initiate the production of callus. The ‘cabbage’ was obtained from the Oil palm plant using the non-destructive method. The outer branches were cut away with a sharp cutlass until the innermost branches were exposed. These branches were cut off to expose the young immature non-chlorophylus tissue. This portion of the crown was then cut off and the exposed surface of the crown of the mother plant was sprayed with a fungicide and pesticide to prevent the cut surface from being attacked by pathogens. This was done so that the mother plant could be protected from infection as it recovered. The ‘cabbage’ was immediately placed in a sterile flask as soon as it was removed from the crown of the mother plant. It was then taken from the Oil Palm Research Institute to 58

the Tissue Culture Laboratory at the Department of Botany of the University of Ghana. The ‘cabbage’ was removed from the sterile flask and placed in the laminar flow hood as soon as it arrived at the tissue culture laboratory. The ‘cabbage’ was sterilized using the procedure for sterilizing leaves outlined earlier and incubated at a temperature of 250C in the dark and observed weekly.

Callus Initiation Experiments using Immature Inflorescence The immature inflorescence used for callus initiation experiments were obtained from mature Oil palm trees at the plantations of the Oil Palm Research Institute using the non-destructive method. The spathe containing the inflorescence was carefully cut and placed in a sterile flask. The flask containing the inflorescence was then taken to the Tissue Culture Laboratory of the Department of Botany, University of Ghana, Legon. The spathe was removed from the flask under aseptic conditions in a Laminar flow hood. The outer layers of the spathe were removed until the immature inflorescence was exposed. The inflorescence was then cut into 1cm long pieces and then washed in sterile distilled water for 10 minutes. The inflorescence explants were rinsed three-times with sterile distilled water and then dipped into a mixture of 0.1g/l Ascorbic acid and 0.15g/l Citric acid for 15 minutes. The sterile inflorescence explants were then placed on callus induction media in culture tubes and sealed with parafilm and placed in a dark growth chamber at 25oC and observed weekly.

59

Callus Initiation Experiments Using Embryos from Oil Palm Kernels. The embryos used were obtained from mature Oil palm kernels obtained from Council for Scientific and Industrial Research – Oil Palm Research Institute (CSRI-OPRI). Both Dura and Tenera kernels were used for these experiments. The seeds were washed in running water for 10 minutes and briefly immersed in 70% ethanol for 15 seconds. The seeds were placed in 10% NaOCl solution containing 2 drops of Tween 20. The seeds were then removed and cracked to facilitate the removal of embryos. The kernels obtained were then placed in 70% ethanol for 15 seconds and then placed in 10% NaOCl containing Tween 20 for 10 seconds. The kernels were rinsed thrice with sterile distilled water. The operculums of the kernels were cut off and the embryo removed and placed on sterile callus induction media in Baby food jars and sealed with parafilm and placed in a dark growth chamber at 250C and observed weekly.

Data Analyses The data obtained were analysed using the Analysis of Variance (ANOVA) in Minitab Statistical Package Software.

60

CHAPTER FOUR RESULTS Results of Decontamination Experiments Sterilization of explants to ensure complete decontamination is the first step in plant tissue culture.

This procedure is a pre-requisite for the

establishment of sterile cultures. Plant materials taken from the field and even those grown on sterilized soil may harbour insects, microorganisms or spores which can contaminate the explants. It is therefore very important that any explants used in plant tissue culture must be thoroughly decontaminated if sterile cultures are to be obtained. Experiments were carried out to find the best way of decontaminating the explants used. The explants used were young non-chlorophyllous leaves referred to as cabbage, oil palm seeds and inflorescences. These explants have been used successfully in Oil palm tissue culture (Corley, 2001). Various methods of decontaminating them have been described by several researchers. Decontamination experiments were carried out on Dura and Tenera seeds using 0%, 5% and 10% (v/v) concentration of sodium hypochlorite. The results of these experiments are indicated in Tables 5 and 6.

61

Table 5: Effectiveness of Different Concentrations of NaOCl on the Sterilization of Oil Palm Seeds (Dura) Concentration of Sodium Hypochlorite Solution (NaOCl) 0%

Time

5%

10%

Clean

Contaminated

Clean

Contaminated

Clean

Contaminated

0

10

4

6

7

3

1

9

4

6

7

3

1

9

4

6

7

3

0

10

4

6

7

3

5 min

10 min

Table 6: Effectiveness of Different Concentrations of NaOCl on the Sterilization of Oil Palm Seeds (Tenera) Concentration of Sodium Hypochlorite Solution (NaOCl) 0%

Time

5%

10%

Clean

Contaminated

Clean

Contaminated

Clean

Contaminated

0

10

4

6

7

3

1

9

4

6

7

3

1

9

4

6

7

3

0

10

4

6

7

3

5 min

10 min

62

Analysis of the results of the Dura seeds sterilisation in Table 5 shows that, there was a significant difference in the number of clean cultures in relation to the concentration of sodium hypochlorite (p0.05) [Appendix 1]. The results of the Tenera sterilization experiment when analysed show that the various concentrations of the sterilant that is 0%, 5% and 10% exhibited varying degrees of the sterilizing ability of the sterilant. There was a significant difference in the number of clean cultures for the various concentrations of Sodium hypochlorite solutions used (P New.

113

APPENDIX 3 LEAF MTB > AOVOneway

'0' '5' '10'.

One-way ANOVA: 0, 5, 10 Source Factor Error Total

DF 2 9 11

S = 0.6236

Level 0 5 10

N 4 4 4

SS 92.167 3.500 95.667

MS 46.083 0.389

R-Sq = 96.34%

Mean 0.2500 4.2500 7.0000

StDev 0.5000 0.5000 0.8165

F 118.50

P 0.000

R-Sq(adj) = 95.53%

Individual 95% CIs For Mean Based on Pooled StDev --+---------+---------+---------+------(--*--) (--*--) (--*--) --+---------+---------+---------+------0.0 2.5 5.0 7.5

Pooled StDev = 0.6236 MTB > AOVOneway

'5MIN' '5MIN'.

One-way ANOVA: 5MIN, 10MIN Source Factor Error Total

DF 1 10 11

S = 3.011

Level 5MIN 10MIN

SS 0.00 90.67 90.67

MS 0.00 9.07

R-Sq = 0.00%

F 0.00

P 1.000

R-Sq(adj) = 0.00%

Individual 95% CIs For Mean Based on Pooled StDev N Mean StDev ----+---------+---------+---------+----6 3.667 3.011 (-----------------*------------------) 6 3.667 3.011 (-----------------*------------------) ----+---------+---------+---------+----1.5 3.0 4.5 6.0

Pooled StDev = 3.011

114

APPENDIX 4

One-way ANOVA: 56mg/l, 84mg/l, 112mg/l Source Factor Error Total

DF 2 6 8

SS 14.000 4.000 18.000

S = 0.8165

Level 56mg/l 84mg/l 112mg/l

N 3 3 3

MS 7.000 0.667

F 10.50

R-Sq = 77.78%

Mean 4.0000 3.0000 1.0000

StDev 1.0000 1.0000 0.0000

2, 4 - D

P 0.011

R-Sq(adj) = 70.37%

Individual 95% CIs For Mean Based on Pooled StDev -+---------+---------+---------+-------(-------*------) (-------*-------) (-------*------) -+---------+---------+---------+-------0.0 1.5 3.0 4.5

Pooled StDev = 0.8165

APPENDIX 5

One-way ANOVA: 186mg/l, 279mg/l, 372mg/l N A A Source Factor Error Total

DF 2 6 8

S = 0.3333

Level 186mg/l 279mg/l 372mg/l

N 3 3 3

SS 8.222 0.667 8.889

MS 4.111 0.111

F 37.00

R-Sq = 92.50%

Mean 0.0000 2.3333 1.0000

StDev 0.0000 0.5774 0.0000

P 0.000

R-Sq(adj) = 90.00%

Individual 95% CIs For Mean Based on Pooled StDev -----+---------+---------+---------+---(----*----) (---*----) (----*----) -----+---------+---------+---------+---0.0 1.0 2.0 3.0

Pooled StDev = 0.3333

115

APPENDIX 6 ANOVA: response versus conc, complx org Factor conc complx org

Type fixed fixed

Levels 3 4

Values 1, 2, 3 1, 2, 3, 4

Analysis of Variance for response Source conc complx org conc*complx org Error Total

S = 0.799305

DF 2 3 6 24 35

SS 13.3889 2.8889 1.2778 15.3333 32.8889

R-Sq = 53.38%

MS 6.6944 0.9630 0.2130 0.6389

N 12 12 12

complx org 1 2 3 4

N 9 9 9 9

P 0.001 0.238 0.913

R-Sq(adj) = 32.01%

Means conc 1 2 3

F 10.48 1.51 0.33

response 1.1667 2.4167 1.0833

response 2.0000 1.2222 1.5556 1.4444

116

APPENDIX 7

ANOVA: res infl versus conc infl, complx org infl Factor conc infl complx org infl

Type fixed fixed

Levels 4 3

Values 1, 2, 3, 4 1, 2, 3

Analysis of Variance for res infl Source conc infl complx org infl conc infl*complx org infl Error Total

S = 0.816497 Means conc infl 1 2 3 4

N 9 9 9 9

complx org infl 1 2 3

DF 3 2 6 24 35

R-Sq = 52.94%

SS 6.0000 6.5000 5.5000 16.0000 34.0000

MS 2.0000 3.2500 0.9167 0.6667

F 3.00 4.88 1.38

R-Sq(adj) = 31.37%

res infl 3.0000 2.3333 2.0000 2.0000

N 12 12 12

res infl 1.7500 2.7500 2.5000

ANOVA: resp versus variety, concen Factor variety concen

Type fixed fixed

Levels 2 4

Values 1, 2 1, 2, 3, 4

Analysis of Variance for resp Source variety concen variety*concen Error Total

S = 0.75

DF 1 3 3 8 15

SS 7.5625 28.6875 1.6875 4.5000 42.4375

R-Sq = 89.40%

MS 7.5625 9.5625 0.5625 0.5625

F 13.44 17.00 1.00

P 0.006 0.001 0.441

R-Sq(adj) = 80.12%

117

P 0.050 0.017 0.265

Means variety 1 2

concen 1 2 3 4

N 8 8

N 4 4 4 4

resp 7.1250 8.5000

resp 9.5000 8.7500 6.5000 6.5000

APPENDIX 8 DURA

One-way ANOVA: 84AA, 112AA, 84ME, 112ME, 84CH, 112CH, 84CM, 112CM Source Factor Error Total

DF 7 24 31

S = 1.997

Level 84AA 112AA 84ME 112ME 84CH 112CH 84CM 112CM

N 4 4 4 4 4 4 4 4

SS 62.97 95.75 158.72

MS 9.00 3.99

R-Sq = 39.67%

Mean 6.250 7.250 5.750 5.250 2.750 4.500 3.500 4.000

StDev 1.708 1.708 2.217 2.986 1.708 1.291 2.082 1.826

F 2.25

P 0.065

R-Sq(adj) = 22.08%

Individual 95% CIs For Mean Based on Pooled StDev -------+---------+---------+---------+-(-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) -------+---------+---------+---------+-2.5 5.0 7.5 10.0

Pooled StDev = 1.997

118

APPENDIX 9 TENERA

One-way ANOVA: 84AA_1, 112AA_1, 84ME_1, 112ME_1, 84CH_1, 112CH_1, 84CM_1, ... Source Factor Error Total

DF 7 24 31

S = 1.885

SS 56.72 85.25 141.97

MS 8.10 3.55

F 2.28

R-Sq = 39.95%

P 0.062

R-Sq(adj) = 22.44%

Individual 95% CIs For Mean Based on Pooled StDev Level 84AA_1 112AA_1 84ME_1 112ME_1 84CH_1 112CH_1 84CM_1 112CM_1

N 4 4 4 4 4 4 4 4

Mean 7.750 8.750 5.500 7.500 5.750 6.500 4.500 5.500

StDev 2.217 2.217 2.887 1.291 1.258 1.291 1.291 1.915

+---------+---------+---------+--------(-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) (-------*-------) +---------+---------+---------+--------2.5 5.0 7.5 10.0

Pooled StDev = 1.885

Dura and Tenera ANOVA: resp versus variety, conc, additive Factor variety conc additive

Type fixed fixed fixed

Levels 2 2 4

Values 1, 2 1, 2 1, 2, 3, 4

Analysis of Variance for resp Source variety conc additive Error Total

DF 1 1 3 26 31

S = 0.735436

SS 11.281 9.031 41.594 14.063 75.969

MS 11.281 9.031 13.865 0.541

R-Sq = 81.49%

F 20.86 16.70 25.63

P 0.000 0.000 0.000

R-Sq(adj) = 77.93%

Means

119

variety 1 2

conc 1 2

N 16 16

additive 1 2 3 4

N 16 16

resp 6.8750 8.0625

resp 6.9375 8.0000

N 8 8 8 8

resp 9.0000 8.1250 6.2500 6.5000

120