- UNIMAS Institutional Repository

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
i
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)
ii
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)
iii
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.
iv
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 lainlain 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
v
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
2.0
INTRODUCTION
1.1. Introduction
1
1.2. Objective of the study
2
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
vi
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
3.0
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
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
Hormone (PGRs)
22
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
(2, 4-D) supplemented with 6-benzylaminopurine (BAP).
24
3.6. Establishment of Callus in suspension culture
3.6.1. Effect of 1-Naphthaleneaceticacid (NAA) supplemented with 6bezylaminepurine (BAP) on callus culture
3.6.2.Effect
of
sucrose
supplemented
with,
4Dicholorophenoxylaceticacid (2, 4-D) and 6-bezylaminepurine on
vii
25
25
callus culture
3.6.3. Effect of 4-amino-3, 5, 6-trichloropicnolic acid (Picloram)
supplemented with 2,4-Dicholorophenoxylaceticacid (2,4-D) and
6-bezylaminepurine on callus culture
4.0
25
3.7. Parameter
27
3.8. Experiment design and statistical analysis
27
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)
supplemented with 6-benzyaminepurine (BAP)
33
4.2. Establishment of Cell suspension culture
38
4.2.1 Effect of different concentration Naphthaleneaceticacid (NAA)
supplemented with 6-bezylaminepurine (BAP) on callus culture
38
4.2.2. Effect of different concentration sucrose supplemented with, 4Dicholorophenoxylaceticacid (2, 4-D) and 6-bezylaminepurine on
callus culture
42
4.2.3. Effect of 4-amino-3, 5, 6-trichloropicnolic acid (Picloram)
supplemented with 2,4-Dicholorophenoxylaceticacid (2,4-D) and
6-bezylaminepurine on callus culture
45
4.2.4. Identification of callus structure
49
5.0
CONCLUSION AND RECOMMENDATION
52
6.0
REFERENCES
54
viii
LIST OF APPENDICES
Appendix
Page
Appendix A
Murashige & Skoog (MS) medium formulation
Appendix B
Tukey’s HSD Post Hoc Test for callus induction in MS solid 61
medium
Tukey’s HSD Post Hoc Test for callus area induction in MS
62
solid medium
Tukey’s HSD Post Hoc Test callus formation in Batch 1 & 2 63
Appendix C
Appendix D
Appendix E
Appendix F
Tukey’s HSD Post Hoc Test for Fresh Weight of different
NAA concentration
Tukey’s HSD Post Hoc Test for Weight of different sucrose
concentration
ix
60
64
65
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
x
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
xi
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
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
10a
Statistical analysis of callus fresh weight formation after 7 week 42
cultured (Sucrose)
xii
32
40
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
xiii
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
xiv
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.
1
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
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 2040 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.
3
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.
4
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
(c) The appearance of hordes of ant in the fissures
(d) A distinctly yellowish tinge in the wood under outer bark
7
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, αguaiene, 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 selina4,11-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)-1vinyl-,
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
8
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).
9