DEVELOPMENT AND POLYMORPHISM OF MICROSATELLITE MARKERS IN Neolamarckia cadamba (Roxb.) BOSSER (KELAMPAYAN)

USING ISSR SUPPRESSION PCR METHOD

Phui Seal Lol

Master or Science 2012

DEVELOPMENT AND POLYMORPHISM OF MICROSATELLITE MARKERS IN Neolamarckia cadamba (ROXB.) BOSSER (KELAMPAYAN) USING ISSR SUPPRESSION PCR METHOD

PHUI SENG LOI

A thesis submitted in fulfillment of the requirement for the Degree of

Master of Science (Plant Biotechnology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARA W AK

2012

I

I

Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SAKAWA)(

DEVELOPMENT AND POLYMORPHISM OF MICROSATELLITE MARKERS IN Neolamarckia cadamba (Roxb.) Bosser

(KELAMPA YAN) USING ISSR SUPPRESSION PCR METHOD

PHUI SENG LOI

Thesis submitted for the degree of Master of Science

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2012

DECLARATION

I hereby declare that the work in this dissertation is my own except for quotations which

have been duly acknowledged. No portion of the work referred to in this dissertation has

been submitted in support of an application for another degree or qualification.

PHUI SENG LOI Matric no: 07021297

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my supervisor, Dr. Ho Wei Seng for his

patience, guidance, advice and dedication throughout the project. My sincere appreciation

also goes to Dr. Pang Shek Ling for her guidance and helps throughout this work.

Besides, I also would like to thank Mr. lulaihi Abdullah and staff of Seed Bank, Sarawak

Forestry Corporation (SFC), Kuching for supplying the plant materials and Sarawak

Timber Association (STA) for entitling the scholarship to me. Last but not least, my

special thanks to my family, colleagues and lab assistants for their endless support and

encouragement throughout the project.

11

ABSTRACT

Reliable information on the distribution of genetic variation is a crucial point for

applicability and efficiency of any breeding, preservation and conservation programmes

for forest trees. The emergence of DNA marker technologies provides new tools for rapid

genetic analysis, fingerprinting and studying relatedness among cultivars of many forest

tree species. A vailability of good numbers of polymerase chain reaction (PCR)­

compatible DNA markers which is heritably co-dominant and multiallelic has become a

priority due to the genetic complexity of breeder's populations and high levels of

heterozygosity in individual genotypes. Simple sequence repeats (SSRs) or

microsatellites, are tandemly repeated motifs of 1-6 nuc1eotides found in all prokaryotic

and eukaryotic genomes. The uniqueness and value of micro satellites arises from their

multiallelic nature, codominant transmission, relative abundance and extensive genome

coverage. Because of these attributes, microsatellites are currently the excellent markers

of choice for comparative genetic and genomic analysis, high-throughput genotyping and

studies of gene flow in forest trees. Neolamarckia cadamba (Roxb.) Bosser or locally

known as Kelampayan, belongs to the Rubiaceae, was chosen in the present study due to

its high commercial value and fast growing ability. Although N cadamba becomes one

of the most frequently planted trees in the tropics, but genetic information about this

species is limited and none of the DNA markers has been developed from N cadamba

compared to other economically important tropical trees. In the present study, 15 SSR

markers specific for N cadamba were developed using inter-simple sequence repeat

(ISSR) suppression PCR method, a method which is relatively simple and rapid without

enrichment steps. Considerable allelic amplifications were obtained for all SSR markers

111

across the tested genotypes whereby 66 alleles were detected with an average of 4 per

locus. Most of the detected loci analyzed showed high polymorphism as indicated by

their PIC value which was above 0.5. The most polymorphic loci were: ACll

(PIC=0.849), ACl2 (PIC=0.722) and AGOl (PIC=0.712). Besides, cross-speCies

transferability of micro satellites was also confirmed in this study and the newly

developed SSR markers showed good cross species amplification efficiency. These SSR

markers represent a potent tool for genetic diversity study, estimate pollen contamination

in seed orchards, germplasm identification and to assist with the construction of genetic

linkage map for N cadamba. The derived SSR-based map later will be an important pre­

requisite for marker-assisted selection (MAS) to increase the efficiency of N cadamba

breeding.

Key Words: SSR markers, Neolamarckia cadamba (Roxb.) Bosser, ISSR suppression­PCRmethod

IV

ABSTRAK

Pengembangan dan Sifat Polimotfik Penanda Mikrosatelit Dalam Neolamarckia cadamba (Roxb.) Bosser (Kelampayan) Dengan Menggunakan Teknik Ulangan

Antara Jujukan Mudah (ISSR) Melalui Penindasan peR

Kepastian maklumat dalam pengagihan variasi genetik merupakan prasyarat penting

untuk kebolehgunaan dan kecekapan mana-mana program pembiakan, pengawetan dan

pemuliharaan bagi pokok-pokok hutan. Kemunculan teknologi-teknologi penanda DNA

menyediakan peralatan baru untuk analisa genetik pesat, fingerprinting dan

pembelajaran hubungan antara kultivar-kultivar pelbagai spesies pokok hutan. Penanda

DNA yang sepadan dengan PCR (Polymerase Chain Reaction) dan mampu diwarisi

secara ko-dominan daripada pelbagai aiel telah menjadi suatu keutamaan dalam

kerumitan genetik populasi pembiak baka, serta tahap variasi yang tinggi dalam setiap

genotip individu. Simple Sequence Repeats (SSRs) atau mikrosatelit merupakan ulangan

motif sejajar yang terdiri daripada 1-6 nukleotida yang terdapat dalam genom

prokaryotik dan eukaryotik. Keunikan mikrosatelit terhasil daripada sifat kepelbagaian

aiel yang semulajadi, transmisi secara ko-dominan, perkaitan dan liputan genom yang

meluas. Sifat-sifat ini membolehkan mikrosatelit kini menjadi penanda pilihan bagi

analisa perbandingan genetik dan genomik, daya pemprosesan genotip yang tinggi dan

kajian-kajian aliran gen dalam pokok-pokok hu~an. Neolamarckia cadamba (Roxb.)

Bosser atau dikenali dengan nama tempatannya, kelampayan berasal daripada famili

Rubiaceae, terpilih dalam kajian ini disebabkan nilai komersialnya yang tinggi dan

keupayaan pertumbuhan yang cepat. N. cadamba telah menjadi salah satu daripada

pokok yang paling banyak ditanam di kawasan tropika. Walau bagaimanapun, maklumat

v

genetik mengenai spesies ini adalah terhad dan belum ada penanda DNA yang

dikembangkan daripada N cadamba berbanding dengan spesies tropika lain yang

berkepentingan ekonomi. Dalam kajian ini, 15 penanda SSR yang spesijik untuk N

cadamba telah dikembangkan menggunakan kaedah yang mudah dan cepat iaitu teknik

ulangan antara jujukan mudah (ISSR) melalui penindasan PCR. Amplijikasi aiel yang

boleh dipertimbangkan telah diperoleh untuk semua penanda SSR yang diuji genotipnya,

di mana 66 aiel telah dikesan dengan purata 4.4 per lokus. Kebanyakan lokus yang

dianalisa menunjukkan sifat polimorjik yang tinggi seperti yang ditunjukkan oleh nilai

PIC, iaitu lebih tinggi daripada 0.5. Lokus yang paling polimorjik adalah: AC11 (PIC:

0.849), AC12 (PIC: 0.722) dan AG01 (PIC: 0.712). Selain itu, pemindahan mikrosatelit

secara lintas-spesies juga telah dikenalpasti dalam kajian ini, dan penanda SSR yang

dikembangkan ini menunjukkan keberkesanan amplijikasi yang baik antara lintasan

spesies. Kesemua penanda SSR ini menjadi satu alat yang berkesan dalam kajian

kepelbagaian genetik, anggaran pencemaran debunga, pengenalpastian germplasma

serta membantu dalam pembentukan rangkaian genetik untuk N cadamba. Rangkaian

berasaskan SSR ini kemudiannya akan menjadi satu pra-syarat penting bagi pemilihan

penanda yang dibantu (MAS) bagi meningkatkan keberkesanan pembiak bakaan N

cadamba.

Kala Kunci: penanda SSR, Neolamarckia cadamba ·(Roxb.) Bosser, teknik ulangan antara jujukan mudah (ISSR) melalui penindasan PCR

VI

Pusat Kbidmat Maldu..at Akadem~ UNlVERSm MALAYSIA SARAWAJ(

TABLE OF CONTENTS

DECLARATION ACKNOWLEDGEMENTS ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

11

III

V

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CHAPTER I INTRODUCTION 1

CHAPTER II

2.1 Selection of Species Studied 4

2.2 Molecular Marker Techniques in Plant Sciences 8

2.3 Simple Sequence Repeats (SSRs)

CHAPTER III

3.1 Sampling of Plant Materials 35

3.2 DNA Extraction 35

3.3 DNA Purification 37

3.4 DNA Quantification by UV Spectrophotometric Analysis 37

3.5 Cloning and Sequencing of Amplified Fragments 38

LITERA TURE REVIEW

2.3.1 Background of SSRs 13

2.3.2 SSR Polymorphism 19

2.3.3 Models of Microsatellite Mutation 22

2.3.4 Advantages of SSR Analysis 24

2.3.5 SSR Marker Development 27

2.3.6 SSR Applications 31

MATERIALS AND METHODS

of Inter-simple Sequence Repeats

V11

3.6 DNA Libraries Construction 40

3.7 Detennination of the Sequence beyond the Determined 41

ISSR Sequence

3.8 SSR Search 42

3.9 Primer Design 43

3.10 PCR Amplification with Developed SSR Primer Pairs 43

3.11 Data Analysis 44

3.12 Cross Species Amplification Ability of Developed SSR 45

Primer Pairs

CHAPTER IV RESULTS AND DISCUSSION

4.1 DNA Extraction and Purification 46

4.2 Re-optimization of DNA Extraction Protocol 47

4.3 Quantification of DNA by UV Spectrophotometric 48

Analysis

4.4 Cloning and Sequencing of Amplified Fragments of

Inter-simple Sequence Repeats

4.4.1 ISSR-PCR Optimization 49

4.4.2 Cloning of Amplified ISSR Fragments 50

4.5 Determination of the sequence beyond the detennined

ISSR sequence by Genome Walking Method

4.5.1 DNA Libraries Construction 56

4.5.2 Nested PCR 58

4.6 SSR Search and Primer Design 60

4.7 Pre-screening with the Newly Developed SSR Primer Pairs 67

4.8 SSR Marker Validation 68

4.9 SSR Data Analysis 71

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4.10 Cross Species Amplification ofSSR Markers 73

CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 77

REFERENCES 79

APPENDIX I ISSR FRAGMENT DNA SEQUENCES 93

IX

~~ ,..------------------------------------------------­

TABLE NO.

Table 2.1

Table 4.1

Table 4.2

Table 4.3

Table 4.4

Table 4.5

Table 4.6

LIST OF TABLES

PAGE

Comparison of various aspects of frequently used molecular marker techniques

12

DNA Concentration 49

Optimum PCR conditions for different primers 49

Total number of fragments that had been cloned from different SSR primers

54

Kelampayan microsatellite locus, repeat motif, complexity, type, primer pair nucleotide sequences and product size

61

SSR data analysis by Power Marker software 71

Potential cross-species amplification of N cadamba SSR loci in Coffea canephora (coffee), Duabanga moluccana (sawih), Shorea parvifolia subsp. parvifolia (meranti sarang punai) and Canarium odontophyllum (dabai)

76

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LIST OF FIGURES

FIGURE NO. PAGE

Figure 2.1 Seedling of Neolamarckia cadamba (Roxb.) Bosser 5

Figure 2.2 Neolamarckia cadamba (Roxb.) Bosser; 1) Habit of young tree; 2) Twig with inflorescence; 3) Flower; and 4) Infructescence

6

Figure 2.3 Mechanisms of unequal crossing-over and replication slippage for producing SSRPs

20

Figure 2.4 A general protocol for developing SSR markers with a SSR-enrichment step

28

Figure 4.1 Purified DNA samples obtained on 0.8% agarose gel. DNA was obtained via primary DNA extraction protocol

46

Figure 4.2 Purified DNA samples obtained on 0.8% agarose gel. DNA was obtained via re-optimized DNA extraction protocol

48

Figure 4.3 PCR amplification from three primers. (a): (AC)IO; (b): (AG)IO; (c): (GTG)6. The PCR products obtained on 1.5% agarose gel

50

Figure 4.4 Cloning of amplified ISSR fragments. (a) ISSR fragments amplified from (GTG)6primer. (b) Gel purification. (c) Colony PCR analysis. Lanes 1-2: Negative control; Lanes 3-4: Recombinant clones. (d) Plasmid isolation

51

Figure 4.5 Cloning of amplified ISSR fragments. (a) ISSR fragments amplified from (AG) 10 primer. (b) Gel purification. (c) Colony PCR analysis. (d) Plasmid isolation

51

Figure 4.6 Cloning of amplified ISSR fragments. (a) ISSR fragments amplified from (AC) 10 primer. (b) Gel purification. (c) Colony PCR analysis. (d) Plasmid isolation

52

Figure 4.7 Colour screening on LB agar plates. The recombinant clones (white colonies) were selected and cultured for subsequent analysis

52

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Figure 4.8 Isolated plasmid from recombinant clones. Lane 1: AC500bp; Lane 2: AG700bp; Lane 3: GTG500bp; Lane 4: GTG700bp; Lane 5: GTG750bp; Lane 6: GTG800bp; Lane 7: GTGI000bp; Lane M: Supercoiled DNA Ladder

53

Figure 4.9 Example of nested fragment

PCR primer design for amplified ISSR 54

Figure 4.10 Digested DNA samples obtained on 0.8% agarose HaellI; R: RsaI; S: SspI; A: AluI and E: EcoRV

gel. H: 57

Figure 4.11 Example of amplification products obtained using walking method to sequence the other flanking region of a microsatellite. Lanes 1-4: Primary PCR products amplified with the adaptor primer API and ISSR-specific primer IPI. Lanes 5­6: Second nested PCR products amplified with the adaptor primer AP2 and ISSR-specific primer IP2. Lane M is a 100 bp ladder of DNA size markers

58

Figure 4.12 Second nested PCR products amplified with different ISSR-Specific primers from different restricted DNA libraries. Lane A: Alul restricted library; Lane E: EcoRV restricted library; Lane H: HaelII restricted library; Lane R: Rsal restricted library; Lane S: Sspl restricted library; Lane M: 100 bp ladder of DNA size markers

59

Figure 4.13 SSR search by SSR Finder software 63

Figure 4.14 Example of primer pair designed for flanking the identified SSR core motif

64

Figure 4.15 Types of SSR markers 64

Figure 4.16 Example of primer pairs that failed to amplify poorly

or amplified 67

Figure 4.17 SSR marker validation for 15 loci 70

xu

LIST OF ABBREVIATIONS

API AP2 BAC CAPs CIA crAB ddHzO DNA dNTP EDTA ESTs EtBr GSM H HWE lAM IPI IP2 IP3 IPTG IRAP ISSR ISSR..PCR KAM LB MAS MgClz NaCI NaOAc PCR PIC PS PVP QTL RAPDs REMAP RFLPs RNase SPAs SFC SMM SSR

Adapter Primer 1 Adapter Primer 2 Bacterial Artificial Chromosome Cleaved Amplified Polymorphic sequence Chloroform-Isoamyl Alcohol Cetyltrimethylammonium Bromide Double distilled water Deoxyribonucleic Acid Deoxynucleotide triphosphate Ethylenediamine Tetraacetic Acid Express Sequences Tags Ethidium Bromide Generalized Stepwise Model Heterozygosity Hardy-Weinberg Equilibrium Infinite Allele Model Initiating Primer 1 Initiating Primer 2 Initiating Primer 3 Isopropyl,B-D-thiogalactopyranoside Inter-retrotransposon Amplified Polymorphism Inter-Simple Sequence Repeat Inter-Simple Sequence Repeat-Polymerase Chain Reaction K-allele Model Luria Broth Marker-assisted Selection Magnesium Chloride Sodium Chloride Sodium Acetate Polymerase Chain Reaction Polymorphic Information Content Proportional Slippage Polyvinylpyrrolidone Quantitative Traits Loci Randomly Amplified Polymorphic DNAs REtransposon-Microsatellite Amplified Polymorphism Restriction Fragment Length Polymorphisms Ribonuclease Seed Production Areas Sarawak Forestry Corporation Stepwise Mutation Model Simple Sequence Repeat

Xlll

SSRPs STS TBE TPM UTR UV X-gal

Simple Sequence Repeat Polymorphisms Sequence-Tagged-Site Tris-Borate-EDTA Two Phase Model Untranslated Regions Ultraviolet 5-bromo-4cloro-indolyl-p-D-galactoside

XIV

CHAPTER I

INTRODUCTION

Genetic diversity is the foundation of the ability of organisms to adapt to changes in their

environment through natural selection (Krutovskii and Neale, 2001). It became the base

of sustainable, efficient and multifunctional forestry due to its ability to contribute to the

adaptability and hence to the conservation of species and ecosystem diversity and

development (Paul et at., 2005). As reported by Krutovskii and Neale (2001), populations

with little genetic variation are not able to adapt to the environmental changing

conditions effectively. As a result, this will greatly increase the risk of extinction of

particular popUlations since they are more susceptible to the advent of new pests or

diseases, changes in climate, pollution, and habitat destruction.

In order to ensure that the needs of future generations can be met, gene

conservation managements should be carried out to conserve adaptive genetic diversity

based on the knowledge of the genetic basis of adaptation. However, reliable information

on the distribution of genetic variation is a crucial point for applicability and efficiency of

any breeding, preservation and conservation programmes for forest trees. Such genetic

variation information of a species is assessable either through measurement of

morphological and metric characters in the field or through the study of molecular

markers in the laboratory (Butcher et at., 1999).

1

The emergence of DNA marker technologies such as restriction fragment length

polymorphism (RFLPs), random amplified polymorphic DNA (RAPDs), amplified

fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs) have

revolutionized the field of plant genetics by providing new tools for rapid genetic

analysis, fingerprinting and studying relatedness among cultivars of many forest tree

species (Martinez et ai., 2006). As explained by Butcher et ai. (1999), the development of

DNA markers not only has overcome limitations on the number of variable loci but at the

same time also provide new techniques to study variation in coding, non-coding, and

highly variable regions of both nuclear or organelle genomes. Furthennore, the use of the

different genotypes in breeding programmes and the design of new crosses also can be

improVed via studies of genetic diversity and genetic relatedness assisted by DNA

markers (Perez et ai., 2005).

Recently, development of an ideal DNA marker system which is genetically co­

dominant and multiallelic is becoming a major concern due to the genetic complexity of

breeder's populations and high levels of heterozygosity in individual genotypes (Jones et

aI.,2003). Simple sequence repeat (SSR) marker is a polymerase chain reaction (PCR)­

based marker system of this kind, allowing high-throughput genotyping and genetic map

construction. According to Tamura et ai. (2005), simple sequence repeat (SSR) or

microsatellite is a small segment of DNA, consist of an array of short nucleotide motifs

1-4 bp in length which repeats itself a number of times.

2

The nature of SSR offers them a number of advantages over other molecular

markers. They show co-dominant inheritance and multi-allelism. In addition to their high

information content and widespread distribution, they have advantages of high sensitivity

and reproducibility. Because of these attributes, SSRs loci are currently the excellent

markers of choice for comparative genetic and genomic analysis, individual genotyping

and studies of gene flow in forest trees.

Neolamarckia cadamba (Roxb.) Bosser or locally known as Kelampayan was

chosen in the present study due to its high commercial value and fast growing ability.

Although N cadamba is important in tropical forestry and becoming one of the most

frequently planted trees in the tropics, but genetic information about this species is still

limited. To date, none of the DNA markers has been developed from N cadamba,

compared with that from other economically important tropical tree species.

Therefore, in order to discover, investigate and determine the genetic diversity

and structure of N cadamba, the objectives of this study are 1). to develop simple

sequence repeat (SSR) markers for genotyping N cadamba trees and 2). to evaluate the

cross species transferability of the newly developed SSR markers to other Rubiaceae

specles.

3

CHAPTER II

LITERATURE REVIEW

2.1 Selection of Species Studied

Neolamarckia cadamba (Roxb.) Bosser belongs to the Rubiaceae family. It is commonly

known as Kadam (Indian, French and trade name); common bur-flower tree (Eng.);

kaatoan bangkal (Philippines); mai sa kho (Laos); kalempajan, jabon (Indonesia);

kelempayan (Peninsular Malaysia); thkoow (Cambodia); Laran (Sabah) and Kelampayan

(Sarawak).

Joker (2000) reported that N cadamba is originated from India and Nepal. He

mentioned that through Thailand, Indo-China and eastward in the Malaysian

Archipelago, it started to spread to Papua New Guinea and had been introduced

successfully to Africa and Central America. In his explanation, N cadamba can be found

below 1,000 m altitude within the area of natural distribution and commonly where there

is more than 1,500 mm rain/year. It also can grow in dry condition such as 200 mm

rain/year. He further explained that N cadamba can grow on different kind of soils,

tolerant to periodic flooding but intolerant to frost.

N cadamba can be raised by either planting out nursery raised seedlings or

planting of bare root nursery stock. Direct sowing is not practicable due to its small-sized

seeds and sensitivity to drought, excessive moisture and direct sun. It is a light demander

but the saplings require protection from the hot sun. In addition, it should be weeded

regularly since young seedlings are highly susceptible to weeds. To ensure successful

4

us t Klii mat Jd at Akademik tJN1VE mMALAYSIA SARAWAK

establishment, seedlings should be planted out with their balls of earth. The growth ofN

cadamba is commonly fast for the first 6-8 years and at the age of 10-15 years the trees

can be felled (World Agroforestry Centre, 2004).

Figure 1.1. Seedling ofNeolamarckia cadamba (Roxb.) Bosser

As explained by Joker (2000), N cadamba can grow up to 45m tall and up to 100­

160cm in diameter, with umbrella-shaped crown and extensive branches are form and

arranged in tiers. The bark is gray and smooth in young trees. However, it is rough and

IoDgitudinally fissured in old trees. The leaves are glossy green, opposite, simple more or

sessile to petiolate, ovate to elliptical and 13-32cm long (World Agroforestry Centre,

20(4).

5

The flowers of N cadamba are orange, small, densely and globose heads. They

are characterized as bisexual, 5-merous, calyx tube funnel-shaped, corolla gamopetalous

saucer-shaped with a narrow tube and the narrow lobes imbricate in bud. The stamens are

inserted on the corolla tube and contain short filaments and basifixed anthers.

Furthermore, the flowers also contain an inferior ovary which is either bi-Iocular or

sometimes 4-locular in the upper part and consists of an exserted style and a spindle-

shaped stigma. Fruitlets numerous with their upper parts containing four hollow or solid

structures and the seed is either trigonal or irregularly shaped (World Agroforestry

Centre, 2004).

I 3

re 2.2. Neolamarckia cadamba (Roxb.)Bosser; 1) Habit of young tree; 2) Twig with iaftorescence; 3) Flower; and 4) Infructescence

(Taken from Plant Resources of South-East Asia 5:1 cited in Danida Forest Seed Centre, 2000)

6

Joker (2000) revealed that N cadamba fruit usually has small capsules and

packed closely to form an infructescence which is fleshy, yellow or orange. The

infructescence contains approximately 8000 seeds. He reported that the small capsules

will split into four parts in order to release the seed at maturity. It usually initiates

flowering when the tree is 4-5 years old. The seeds can be dispersed by wind, rain, floods

or rivers.

N cadamba is a lightweight hardwood with poor durability. The wood has a

density of 290-560 kg/cu m at 15% moisture content. It has a fine to medium texture;

straight grain; low luster and has no characteristic odour or taste. It is easy to work with

hand and machine tools by giving a good surface and cuts cleanly. However, the wood

has an average life in contact with the ground of less than 1.5 years (World Agroforestry

Centre, 2004). It is easy to preserve the N cadamba wood using either open tank or

pressure-vacuum systems. The wood also can be easily impregnated with synthetic resins

in order to increase its density and compressive strength (World Agroforestry Centre,

2004). Joker (2000) reported that N cadamba wood is mainly used for pulp, producing

low- and medium quality paper. The wood can be used only for indoors' light

construction since it is perishable in contact with ground. As the result, N cadamba is

becoming one of the most frequently planted trees in the tropics.

7