Suspension Culture of Aquilaria Beccariana Van Teigh

Faculty of Resources Science and Technology

SUSPENSION CULTURE OF AQUILARIA BECCARIANA VAN TEIGH.

Khairunisa Binti Ab Aziz

Bachelor of Science with Honours (Plant Resource Science and Management) 2013

SUSPENSION CULTURE OF AQUILARIA BECCARIANA VAN TEIGH

KHAIRUNISA BINTI AB AZIZ

26604

This project is submitted in fulfilment of the requirements for the Degree of Bachelor of Science with Honours

(Plant Science Resource and Management)

Faculty of Resource Science & Technology

UNIVERSITI MALAYSIA SARAWAK

2013

APPROVAL SHEET

Name of candidate : Khairunisa Binti Ab Aziz

Title of dissertation : Suspension Culture of Aquilaria beccariana Van Teigh

______

(Prof. Dr. Hamsawi Sani)

Supervisor

______

(Dr Rebicca Edward)

Coordinator of Plant Resource Science and Management Programme

Department of Plant Science and Environmental Ecology

Faculty of Resource Science amd Technology

Universiti Malaysia Sarawak (UNIMAS)

DECLARATION

I hereby declare that this Final Year Project 2013 is based on my original work except for quotations and citations, which have been duly declared that it has not been or concurrently submitted for any degrees at UNIMAS or other institutions of high education.

______

Khairunisa Binti Ab Aziz

Plant Resource Science and Management

Department of Plant Science and Environmental Ecology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak (UNIMAS)

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ACKNOWLEDGEMENTS

First of all, I would like to express my deepest appreciation to my supervisor, Prof. Dr.

Hamsawi Sani, for his guidance, support, advice, and help in complete my Final Year

Project (FYP) on suspension culture.

I also would like to thank Mr Zul Helmey Mohamad Shabdin and Ms.Cherrie Lim Han

Rou, the postgraduate students in our laboratory for the guidance, suggestion of essential references and giving me plenty useful information and technique about tissue culture. A special thanks to my laboratory mate, Ms Noor Izzieana Ibrahim for her full support and assistance in finishing my project. Thus, I also would like to express my gratitude to Ms

Nor Emeylia Md Zain for her help and encouragement. A special thanks to my laboratory mates Ms.Siti Zalikha Mohd Ridzuan Jamil and Ms.NurAmalia Kamal Abidin Shah.

Last but not least, deepest thank to my parents and other family members for their care, love, support and understanding me in completion this study.

Suspension Culture of Aquilaria beccariana Van Teigh

Khairunisa Binti Ab.Aziz

Plant Resources and Management Programme Faculty of Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Aquilaria beccariana van Teigh (Thymelaceae), is a non timber forest product. The tree is valuable for its fragrant wood. Agarwood which is important for incense, traditional medicine, perfumery and others products. The population of Aquilaria has been reduced drastically due to the indiscriminate collection, over exploitation and lacking of rapid natural regeneration. The population of this tree became declined and the production of gaharu is less compared to highly demand of the user. Thus, in vitro technique was selected because it offer rapid regeneration and conservation of these endangered species and this study, attempts were done to produce callus from Aquilaria sp. Works were done to induce callus from lamina explants on MS solid and MS liquid medium supplemented with different combination of hormone (2,4-D, picloram and NAA) and varied concentration of sucrose (20-50 g/L). The callus culture was successfully established in MS solid media supplemented with combination of 2.0 mg/L 2,4-D plus 0.5 mg/L BAP. While in MS liquid medium, the suspension culture successfully established in combination of 3 mg/L NAA plus 0.5 mg/L BAP by obtaining highest frequency of fresh weight (2.09g). Thus, 40 g /L found to be the best sucrose concentration with (1.95 g ) of fresh weight. While, combination of 0.25 mg/L picloram with 2.0 mg/L 2,4-D and 0.5 mg/L BAP gave high frequency of fresh weight ( 1.06 g) in Picloram effect. Suspension culture of callus gave friable and compact in texture and globular and irregular in shape.

Keywords: Aquilaria beccariana van Teigh, in vitro technique, suspension culture, callus induction, fresh weight

ABSTRAK

Aquilaria beccariana van Teigh (Thymelaceae), bukan produk hutan kayu. Pokok ini bernilai untuk kayu wangi itu. Gaharu penting untuk kemenyan, ubat tradisional, produk minyak wangi dan lain- lain produk. Populasi Aquilaria telah berkurang secara drastik kerana koleksi sembarangan, lebih eksploitasi dan kekurangan pertumbuhan semula yang pesat. Populasi pokok ini menjadi berkurangan dan pengeluaran gaharu adalah kurang berbanding dengan permintaan pengguna. Oleh itu, teknik vitro menawarkan kaedah cepat dan pemuliharaan spesis terancam dan kajian ini, usaha telah dilakukan untuk menghasilkan kalus daripada pokok karas. Kerja-kerja telah dilakukan untuk kalus daripada eksplan lamina pada MS pepejal dan MS medium cecair ditambah dengan kombinasi yang hormo yang berbeza (2,4-D, picloram dan NAA) dan kepekatan pelbagai sukrosa (20-50 g / L). Pertumbuhan kalus telah berjaya dalam MS media pepejal ditambah dengan gabungan 2.0 mg / L 2,4-D serta 0.5 mg / L BAP. Dalam MS medium cecair, pertubuhan kalus berjaya dalam gabungan 3 mg / L NAA serta 0.5 BAP dengan mendapatkan kekerapan tertinggi berat basah (2.09g.)Dalam, 40 g / L mendapati kepekatan sukrosa terbaik diperolehi (1.95 g) berat basah. .Gabungan 0.25 mg / L picloram dengan 2.0 2,4-D mg/L dan 0.5 mg / L BAP memberikan frekuensi tinggi berat badan segar (1.06 g) berkuat kuasa Picloram. Budaya penggantungan kalus memberikan rapuh dan padat dalam tekstur dan bulat dan tidak teratur dalam bentuk.

Kata kunci: Aquilaria beccariana van Teigh, in vitro teknik, cecair kultur,induksi kalus,berat basah

TABLE OF CONTENTS

TITLE PAGE i

APPROVAL SHEET ii

DECLARATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT/ABSTRAK v

TABLE OF CONTENT vi

APPENDICES ix

LIST OF FIGURES x

LIST OF TABLES xii

LIST OF ABBREAVIATION xiv

1.0 INTRODUCTION

1.1. Introduction 1

1.2. Objective of the study 2

2.0 LITERATURE REVIEW

2.1. Aquilaria sp 3

2.1.1. Taxonomic classification and nomenclature of Gaharu 3

2.1.2. Distribution and ecology 6

2.1.3. Production of gaharu 6

2.1.4. Chemistry of gaharu 8

2.1.5. Uses of gaharu 9

2.1.5.1. Incense 9

2.1.5.2. Medicine 10

2.1.5.3. Perfumery 10

2.1.5.4. Agarwood Oil 11

2.1.6. Trade and Importance of gaharu 11

2.2. Propagation of gaharu 12

2.2.1. In vitro propagation of Aquilaria sp 13

2.2.2. In vitro callus induction of Aquilaria sp 15

2.2.3. Suspension culture of Aquilaria sp 15

2.3. Effect of picloram in tissue culture 17

2.4. Effect of NAA in tissue culture 18

2.5. Effect of sucrose in tissue culture 19

3.0 MATERIALS AND METHODS

3.1. Stock plant 20

3.2. Subculture of A.beccariana 20

3.3. Preparation of Murashige and Skoog Media Stock solution 20

3.4. Preparation of culture medium using MS stock medium. 21

3.4.1. Preparation of culture media supplemented with Plant Growth 22 Hormone (PGRs)

3.5. Establishment of callus induction

3.5.1. Effect of different hormone combination on callus formation 24

3.5.2. Induction of callus formation via 2, 4- Dichlorophenoxyaceticacid 24 (2, 4-D) supplemented with 6-benzylaminopurine (BAP).

3.6. Establishment of Callus in suspension culture

3.6.1. Effect of 1-Naphthaleneaceticacid (NAA) supplemented with 6- 25 bezylaminepurine (BAP) on callus culture

3.6.2.Effect of sucrose supplemented with, 4- Dicholorophenoxylaceticacid (2, 4-D) and 6-bezylaminepurine on 25

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callus culture

3.6.3. Effect of 4-amino-3, 5, 6-trichloropicnolic acid (Picloram) 25 supplemented with 2,4-Dicholorophenoxylaceticacid (2,4-D) and 6-bezylaminepurine on callus culture

3.7. Parameter 27

3.8. Experiment design and statistical analysis 27

4.0 RESULTS AND DISCUSSION

4.1. Establishment of callus induction in MS solid medium 28

4.1.1. Effect of different hormone combination on callus formation 28

4.1.2. Establishment of callus in 2, 4-Dichlrophenoxyaceticacid (2,4-D) 33 supplemented with 6-benzyaminepurine (BAP)

4.2. Establishment of Cell suspension culture 38

4.2.1 Effect of different concentration Naphthaleneaceticacid (NAA) 38 supplemented with 6-bezylaminepurine (BAP) on callus culture

4.2.2. Effect of different concentration sucrose supplemented with, 4- 42 Dicholorophenoxylaceticacid (2, 4-D) and 6-bezylaminepurine on callus culture

4.2.3. Effect of 4-amino-3, 5, 6-trichloropicnolic acid (Picloram) 45 supplemented with 2,4-Dicholorophenoxylaceticacid (2,4-D) and 6-bezylaminepurine on callus culture

4.2.4. Identification of callus structure 49

5.0 CONCLUSION AND RECOMMENDATION 52

6.0 REFERENCES 54

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LIST OF APPENDICES

Appendix Page

Appendix A Murashige & Skoog (MS) medium formulation 60

Appendix B Tukey’s HSD Post Hoc Test for callus induction in MS solid 61 medium Appendix C Tukey’s HSD Post Hoc Test for callus area induction in MS 62 solid medium Appendix D Tukey’s HSD Post Hoc Test callus formation in Batch 1 & 2 63

Appendix E Tukey’s HSD Post Hoc Test for Fresh Weight of different 64 NAA concentration Appendix F Tukey’s HSD Post Hoc Test for Weight of different sucrose 65 concentration

LIST OF FIGURES

Figures Pages

1 A.beccariana tree 5

2 A.beccariana in culture room 20

3 Preparation of full strength MS medium stock 21

4 Methodology of 1 L MS stock medium 21

5 Axenic leaf of A.beccariana 23

6 Suspension culture method 26

7a,b Callus morphology in 2.0 mg/L 2,4-D + 0.5 mg/L BAP 30

7c,d Callus morphology in 2.0 mg/L 2,4-D + 0.5 mg/L BAP + 30

0.5 mg/L picloram

7e,f Callus morphology in 1.0 mg/L picloram 31

8 Area of callus between 3 treatment of 2.0 mg/L 2,4-D + 0.5 mg/L 33

BAP 2.0 mg/L 2,4-D + 0.5 mg/L BAP + 0.5 mg/L picloram

1.0 mg/L picloram

9a No of explants forming callus in batch 1 35

9b No of explants forming callus in batch 2 35

10a Partially developed callus in week 3 in batch 2 36

10b Fully developed callus in week 7 in batch 2 36

11a Callus contamination in batch 2 37

11b Browning of callus in batch 37

12a,b Growth of callus in week 3 and week 7 NAA in 3.0 mg/L NAA + 47

0.5 mg/L BAP

12c,d Growth of callus in week 3 and week 7 NAA in 40g/L of sucrose 47

12e,f + 2.0 mg/L 2,4-D+ 0.5 mg/L BAP

Growth of callus in week 3 and week 7 NAA in 0.25mg/L 48

picloram+ 2.0 mg/L 2,4-D+ 0.5 mg/L BAP

13 Shape of callus in cell suspension culture 40

14 Dry weight of callus in (NAA) effect 42

15 Dry weight of callus in sucrose effect 44

16 Dry weight of callus in Picloram effect 46

17a,b Cell structure in MS solid media 50

18a,b Cell structure in MS liquid media 50

19 Comparison between Fresh Weight among NAA, Sucrose and 51

Picloram

LIST OF TABLES

Table Pages

1 Taxonomic classification of A. beccariana 4

2 Treatment of callus induction in MS solid medium 24

3 Number of replicate in Batch 1 and Batch 2 24

4a Statistical analysis of callus induction in different treatment after 28 week 7

4b Comparison of mean for callus formation per replicate after 30 culturing in MS Media supplemented with different concentration of hormone combination, recorded after 7 week treatment

5a Statistical analysis of callus area in different treatment after week 7 32

5b Comparison of mean for callus area formation per replicate after 32 culturing in MS Media supplemented with different concentration of hormone combination in week 7

6a Statistical analysis of callus formation after 7 week cultured 34 (Batch 1)

6b Statistical analysis of callus formation after 7 week cultured 34 (Batch 2)

7a Comparison of mean for callus formation per replicate in Batch 34 1after culturing in MS Media supplemented with 2.0 2, 4-D + 0.5 BAP

7b Comparison of mean for callus formation per replicate in Batch2 35 after culturing in MS Media supplemented with 2.0 2, 4-D + 0.5 BAP

8a Statistical analysis of callus fresh weight formation after 7 week 39 cultured (NAA)

8b Mean comparison for of fresh weight of callus culture in MS 39 Liquid media per replicate in MS Media supplemented with different concentration of hormone.(NAA)

9 Texture and shape of callus in NAA effects 40

10a Statistical analysis of callus fresh weight formation after 7 week 42 cultured (Sucrose)

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10b Comparison of mean for of fresh weight of callus culture in MS 44 Liquid media per replicate in MS Media supplemented with different concentration of sucrose

11 Texture and shape of callus (Sucrose) 44

12 Texture and shape of callus of picloram concentration 45

13 Fresh weight of callus in different concentration of picloram 45

14a Degree of callus growth in NAA effect 49

14b Degree of callus growth in sucrose effect 49

14c Degree of callus growth in picloram effect 49

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LIST OF ABBREVIATIONS

2,4-D 2,4-Dichlropenoxacetic acid

ANOVA Analysis of variance

BAP 6-benzylaminopurine

IBA Indole 3-butyric acid

MS Murashige and Skoog medium

NAA 1-Napthaleneacetic acid

Picloram 4-amino-3,5,6-trichloropicolinic acid

FW Fresh weight

DW Dry weight

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1.0 INTRODUCTION

1.1 Introduction

Agarwood, locally known as gaharu is belongs to the Genus Aquilaria under family

Thymeleaceae. Agarwood is a natural forest product that obtained from Aquilaria spp.

There are 20 species of Aquilaria distributed in tropical and subtropical Asia.

According to Lau & Chua (2011), they are five known species in Malaysia which are

A. beccariana, A. malaccensis, A. macrocarpa, A. hirta and A. rostrata. The tree is

valuable for its fragrant wood which is used for perfumery, incense (Soehartano &

Newton, 2000) and medicinal purpose. It’s produced resin with aromatic smell.

Previous study by Wetwitayaklung et al., (2009), state that gaharu is naturally

produced by infection of fungi, bacterial, insects, physical cut and chemical

stimulation. However, the resin production could be achieved by applying tissue

culture technique. The callus from in vitro technique will infect by some fungus for

resin production. This method tends to be popular in order to avoid long period in resin

production.

In recent years, the demand for agarwood has been increased, so that this species are

critically endangered because of indiscriminately cut down in natural forest. As a

result, the population of natural gaharu tress has been drastically decreased (CITES,

2004). There are effort to plant gaharu in large scale; however most of the project has

been hampered by the storage of planting stock. With the shortage of seedling for large

scale planting, the next alternative is to produce gaharu by vegetative propagation such

as by cutting and in vitro.

In vitro suspension could be an alternative method to produce gaharu. This can be done

through formation of callus by suspension culture. Study by Okudera and Ito (2009),

showed the callus and suspension cell culture successfully in production of fragrant

compound contain in Aquilaria sp. The major compound in agarwood like

sesquiterpenoid was successfully found in their studies.

Thus, suspension cultures have ability to produce friable callus would determine the

success of gaharu production in vitro. Proper combination of auxin and cytikinin will

produce the friable callus in large scale production and being used for fungus

inoculation in gaharu production.

1.2 The objective of this study were

i. To establish a working protocol for callus induction by using MS ( Murashige

& Skoog, 1962)

ii. To determine the effect of different levels of 1-Naphtaleneacetic acid on cell

proliferation

iii. To examine the effect of sucrose concentration on cell proliferation and

iv. To identify the different of level of picloram on cell proliferation in MS liquid

media

2.0 LITERATURE REVIEW

2.1 Aquilaria sp

Aquilaria is one of fifteen genera of trees in Thymelaeaceae family, which is native to

Southeast Asia, Northern India and Vietnam. The tree can grow up to 40 meters in height and reach a diameter of 60 cm. The tree usually grows straight, but sometimes fluted or with thick (10cm) buttress up to two meters high and commonly found in mixed hill forests across Southeast Asia (Whitmore, 1972).

The leave of this tree is alternate, pinnately vein, without translucent glandular dots and fibrous. The flowers are 5-merous, the fruits loculicidal capsules and have smooth, pale whitish bark (Tawan, 2004). Besides that, others species in Aquilaria like A.crassana is medium size tree of 15-20 cm height and 40-50 cm diameter (dbh) at maturity (Hoang and

Nguyen, 2002). While the others species such as A.malacensis show differ by achieved 20-

40 cm in height and 60 cm (dbh) (Lata, 2007). According to Lata (2007), both of the species white in colour, light in weight and soft in density.

2.1.1 Taxonomic classification and nomenclature of Gaharu

According to Lau and Chua (2011), Aquilaria sp is also known as Malayan eaglewood, agarwood, aloeswood, gaharu, engkaras (Iban), Karas (Malay). Agarwood is classified as non wood forest product (Lata, 2007). The taxonomic classification and nomenclature of

A. beccariana is shown in Table 1.

Table 1: Taxonomic classification of A. beccariana Kingdom Plantae

Phylum Tracheophyta

Class Magnoliopsida

Order Myrtales

Family Thymeleaceae

Genus Aquilaria

Species Aquilaria beccariana van Teigh

Source: (Yusof, 2008)

Basically A. beccariana is widely distributed at Sumatra and Peninsular Malaysia. In

Malaysia, A. beccariana locally known as Karas where the species that producing gaharu.

According to Tawan (2004), this evergreen species can grow up to 20 m tall and 36 cm diameter. It can be found in mixed dipterocarp forest which altitude to 1000 m above the sea level. The leave is 7-27 × 3-8 cm with cuneate to attenuate base. The flower produced in umbel; the fruit is producing from top of calyx tube. Figure 1 showed the morphology and vegetative part of A. beccariana sp.

Figure 1: morphology and vegetative part of A. beccariana sp. Source: Tawan (2004).

(a). Flowering leafy twig (b). Open flower (c). Longitudinal section of open flower (d). Stamen (e). Fruiting leafy twig 5

2.1.2 Distribution and ecology

Aquilaria sp is widely distributed all over the world. This species could be found in

Malaysia, India, Burma and Thailand. According to (Ding, 1960), these species could be found in southern Asia from India to China and much of Indonesia. Others report also stated that, agarwood producing species found from India eastward to Island of New

Guinea which includes all Southeast Asian country and north to Hainan island where located at Southeast China. In Thailand, there are four indigenous species reported which are A. crassana, A. malaccensis, A. subintegra and A. baillonii (Wetwitayaklung et al.,

2009).

According to Nor Hasnida et al., (2011), there are total of 19 spp of agarwood native to

Malaysia (Peninsular: 13 spp, Sabah; 11 spp, Sarawak; 15 spp). Other report stated that, there are fifteen species in Aquilaria spp and eight of them are known to produce gaharu

(Ahmad Junaidy, 2008). Mean while, there are only 5 species producing gaharu reported in

Malaysia. They are A. macrocarpa, A. malaccensis, A. beccariana, A. hirta and A. rostrata

(Whitmore, 1972; Huey, 2008).

Thus, Aquilaria consists of species that are adapted to rocky, sandy or calcareous, well drained slopes and ridges and near swamps (Barden et al, 2000). According to

Chakrabarty et al., (1994), A. malaccensis generally found growing up to an elevation of

1000 m and locally in the foothills and undulating slopes of evergreen and semi-evergreen area.

2.1.3 Production of gaharu

Not all Aquilaria sp produced gaharu. Agarwood is an accumulation of resinaceous substances produced by the tree and deposited in heartwood (Rahayu, 2010). Naturally, 6

gaharu obtained from natural forest. According to Soehartano and Newton (2002), gaharu derived from tropical tree of Aquilaria Lam. According to Chakrabarty et al., (1994), agarwood frequently found in young tree about 20 years old but the infestation take time to mature. Trees about 50 years old have shown the highest concentration yield approximately 2 to 3 kg per tree.

The resinous substance derived from gaharu produced by tree that response to attack by pathogenic fungi (Ng, Chang & Kadir, 1997). The infection of the fungus spread slowly to all side with increasing and ageing of the initial infection. Generally, Aquilaria sp infected by imperfecti fungus such as Aspergillus sp, Penicillium sp and Fusarium sp (Chakrabarty et al., 1994). Other study reported that, the resin created in response to an attack from

Phialophora parasitica which is parasite fungus (Nurdiyana, 2008). Besides that, Rahayu

(2010) stated that the resin produced also part of defense reaction toward mechanical damage apart from attacking by fungus.

While, there are some features in identifying deposited agarwood in the tree. The features are classified below by Chakrabarty et al., (1994):

(a) A poor crown, decayed branches, an even bole

(b) Swelling or depression and cankers on the bole

(d) A distinctly yellowish tinge in the wood under outer bark

2.1.4 Chemistry of gaharu

Generally, gaharu oil greatly valued as perfumery ingredient and incense. Thus, agarwood oil is classified to five types which Super A, A, B, C and D. The study done by

Nor Azah et al., (2008) stated that majority of essential oil made up from sesquirterpenoids and other oxygenated derivatives. The chemical compound like 3-phenyl-butanone, α- guaiene, β-agarofuran, α-agarofuran, agarospirol and jinkoh-eremol were detected in

Malaysian gaharu.

Besides that, other study on chemical composition in gaharu showed that, selinene selina-

4,11-dien-14-al and selina-4,11-dien-14-al were present in A. crassana by applied water distillation and supercritical fluid carbon dioxide extraction ( Wetwitayaklung et al., 2009).

In addition, other study claimed that, the chemical compositions founded in A. agallocha.

Some of them are cycloheptane, 4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1- vinyl-, caryophyllene oxide and 7-isopropenyl-4a-methly-1- methylenedecahydronaphthalene (Md. Nazrul et al., 2009). These types of chemical obtained from infected plant agar (super agar) by Gas chromatography mass spectrometry

(GCMS) method.

Other study done by Zul Helmey et al., (2011a) showed that, the chemical components can be determined by GCMS method. In this study, they found some chemical composition in two species of Aquilaria which is A. malaccensis and A. microcarpa. There are varies in number of chemical composition founded. 20 compounds were founded in A. malaccensis while 16 compounds were found in A.microcarpa. However, they indicate four main chemical compounds such as sesquiterpene hydrocarbon, chromone derivative, oxygenated sesquiterpenenes and fatty acid. Thus, both of the species share same chemical compounds

like ɑ-guaiene, oxo-agarospirol, ɑ-humulene, caryophellene oxide and 2-(2-phenylethyl) chromones (Zul Helmey et al., 2011a)

2.1.5 Uses of gaharu

Gaharu is widely used because of its aromatic smell which produce due to the chemical components extracted from agarwood. Generally, the agarwood is used for incense, medical purpose, perfumery and others. According to Chua (2008), agarwood grade is not uniform as it characterized according to various grading system that differ in each country producing agarwood. Thus, in Malaysia the agarwood used as aromatic range, religious, ceremonial and decorative carves.

2.1.5.1 Incense

One of the gaharu usages is incense. The uses of gaharu as incense is widely discovered around the many countries (Yinzeng et al., 2007) as it has strong heavy scent of gaharu which is unique and complex, that is why the scent becomes stronger when it is burnt.

Application of incense done by burned a pieces of wood in order to produced pleasant aroma where used as general perfume to element of important religious occasion (Barden et al., 2000). The incense also applied in some country like Egypt, Arabia and throughout the Northeast part of Bangladesh (Md. Nazrul et al., 2009).

Besides that, the Arabic and Japanese enjoy the strong fragrant and incorporate the essence in incense product. According to Nurdiyana (2008), the smell produced from burned agarwood used as important religious occasion. In Japanese, the gaharu applied in Japanese appreciation of incense wood which called Koh doh (incense ceremony).