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UNIVERSITI PUTRA MALAYSIA
OPTIMIZATION OF CULTURE PARAMETERS FOR FLAVONOID PRODUCTION FROM CALLUS AND SUSPENSION CULTURES OF THE KARAS TREE
(AQUILARIA MALACCENSIS LAM.)
NURUL HAZWANI BINTI DAUD
FH 2013 6
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OPTIMIZATION OF CULTURE PARAMETERS
FOR FLAVONOID PRODUCTION FROM CALLUS
AND SUSPENSION CULTURES OF THE KARAS
TREE (AQUILARIA MALACCENSIS LAM.)
NURUL HAZWANI BINTI DAUD
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
2013
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COPYRIGHT
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OPTIMIZATION OF CULTURE PARAMETERS FOR FLAVONOID
PRODUCTION FROM CALLUS AND SUSPENSION CULTURES OF THE
KARAS TREE (AQUILARIA MALACCENSIS LAM.)
By
NURUL HAZWANI BINTI DAUD
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirements for the Degree of Master of Science
October 2013
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SPECIALLY DEDICATED TO:
My Beloved
Late Father, Mother, Brothers, Sisters
And all my friends
‘’Who have been a great source of motivation and supported me since
the beginning of my studies’’
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of
the requirement for the degree of Master of Science
OPTIMIZATION OF CULTURE PARAMETERS FOR FLAVONOID
PRODUCTION FROM CALLUS AND SUSPENSION CULTURES OF THE
KARAS TREE (AQUILARIA MALACCENSIS LAM.)
By
NURUL HAZWANI BINTI DAUD
October 2013
Chairman : Associate Professor Rozi Mohamed, PhD
Faculty : Forestry
Plant tissue culture technique has been shown to be efficient for producing flavonoids at
higher amount and lesser time period compared to intact plant. This study aimed at
producing flavonoids in elevated amounts using callus and cell suspension cultures of A.
malaccensis. Aquilaria trees not only produce secondary metabolites known as
agarwood (gaharu) but also rich in flavonoids.
To introduce plant materials into tissue culture, different sterilization regimes for leaf,
nodes and seeds of A. malaccensis, were investigated. Pre-sterilization using 0.2%
Benomyl for 15 minutes improved the number of „sterile and survived‟ individuals for
all types of explants (83-90%), after surface sterilization using mercury chloride (0.1 to
0.2 % HgCl2). The most favorable explants for producing highest flavonoid cell lines
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were determined between leaf and nodal explants by using spectrophotometer and
HPLC analysis. Result showed that flavonoid content in the leaf explants (8.85 ± 0.76
mg/g dry weight (DW)) was 7-fold higher than that of nodal explants (1.25 ± 0.53 mg/g
DW). Leaf contained high amount of quercetin (16.19 ± 1.02 µg/g DW) then rutin (0.21
± 0.31 µg/g DW). Nodes explants contained quercetin and rutin at 0.94 ± 0.07 µg/g DW
and 0.06 ± 0.01 µg/g DW, respectively. Kaempherol was not detected in any of the
explants.
Leaf was used as explant for callus induction due to high flavonoid content and placed
on different types of plant hormones to indicate the type of hormone use to induce
callus. Callus grew best on Murashige and Skoog (MS) medium supplemented with 2.0
mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg/L 6-benzylaminopurine
(BAP). Total flavonoid content in MS medium supplemented with 2.0 mg/L BAP and
0.5 mg/L was 6- fold higher than in phytohormone- free MS medium. Using this optimal
hormone combination, callus growth was observed on different strengths of MS
medium, carbon sources and pH. Full-strength MS medium supplemented with 2.0 mg/L
2,4-D and 0.5 mg/L BAP, 20 g/L sucrose, in pH 5.7, significantly yielded the highest
biomass (231 ± 0.13 mg DW /culture) of callus. These calli were used to initiate the cell
suspension cultures.
To enhance the flavonoid production in callus and cell suspension cultures, examine
phenylalanine (Phe) was used as a precursor in ranged of 20 to 100 mg/L. Cell
suspension culture added 60 mg/L Phe yielded the highest amount of total flavonoid
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(37.92 ± 0.77 mg/g DW), 4.8-fold higher than Phe-free cell culture, and 2-fold higher
than that in calli (19.11 ± 0.36 mg/g DW). The suspension culture with 60 mg/L Phe had
the highest amount of rutin (17.69 ± 1.37 µg/g DW), quercetin (3.46 ± 0.97 µg/g DW)
and kaempherol (2.28 ± 1.67 µg/g DW). When compared the cell suspension culture
with 60 mg/L Phe to Phe-free cell culture, only rutin and quercetin were detected and at
lower levels, 1/10-fold and 1/4-fold, respectively. This study demonstrated that callus
and cell suspension cultures of A. malaccensis produced flavonoids, and feeding the
cultures with Phe elevated flavonoids levels and induced production of additional
compound which is kaempherol.
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Abstrak thesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Master Sains
MENGOPTIMUMKAN KULTUR PARAMETER UNTUK PENGHASILAN
FLAVONOID DARI KALUS DAN AMPAIAN SEL POKOK KARAS
(AQUILARIA MALACCENSIS LAM.)
Oleh
NURUL HAZWANI BINTI DAUD
Oktober 2013
Pengerusi : Profesor Madya Rozi Mohamed, PhD
Fakulti : Perhutanan
Tisu kultur tumbuhan telah menunjukkan keberkesanan untuk menghasilkan flavonoid
yang tinggi dalam tempoh masa yang lebih singkat berbanding dengan pokok induk.
Kajian ini bertujuan untuk menghasilkan flavonoid pada kadar yang tinggi
menggunakan kalus dan ampaian sel Aquilaria malaccensis. Pokok Aquilaria bukan
sahaja menghasilkan metabolit sekunder yang dikenali sebagai gaharu tetapi juga kaya
dengan flavonoid.
Untuk memperkenalkan bahagian tumbuhan ke dalam tisu kultur, cara pensterilan yang
berbeza untuk daun, nodal dan biji benih A. malaccensis telah dikaji. Pra- pensterilan
menggunakan 0.2% Binomil selama 15 minit telah meningkatkan bilangan individu
yang nyah kuman dan sihat untuk semua jenis eksplan (83-90%), setelah pensterilan
permukaan menggunakan merkuri klorida (0.1 ke 0.2% HgCl2). Eksplan yang
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menghasilkan kandungan flavonoid yang tertinggi telah ditentukan di antara eksplan
daun dan nodal dengan menggunakan spektrofotometer dan analisis HPLC. Keputusan
menunjukkan, kandungan flavonoid di dalam eksplan daun (8.85 ± 0.76 mg/g berat
kering) adalah 7-kali ganda lebih tinggi daripada eksplan nodal (1.25 ± 0.53 mg/g berat
kering). Daun mengandungi jumlah kuersetin yang tinggi (16.19 ± 1.02 µg/g berat
kering) diikuti dengan rutin (0.21 ± 0.31 µg/g berat kering). Eksplan nodal mengandungi
jumlah kuersetin dan rutin yang rendah (masing-masing dengan berat kering 0.94 ± 0.07
µg/g dan 0.06 ± 0.01 µg/g). Kaempferol tidak dikenalpasti di dalam mana-mana
eksplan.
Daun telah digunakan sebagai eksplan untuk induksi kalus kerana mempunyai
kandungan flavonoid yang tinggi dan diletakkan di dalam hormon tumbuhan yang
berbeza untuk mengenal pasti jenis hormon utuk pertumbuhan kalus. Kalus hidup
dengan sihat di dalam media Murashige dan Skoog (MS) yang mengandungi 2.0 mg/L
asid 2,4-diklorofenoksi asetik (2,4-D) dan 0.5 mg/L 6-benzilamino Purina (BAP).
Kandungan flavonoid di dalam media yang dibekalkan dengan 2.0 mg/L 2,4-D dan 0.5
mg/L BAP adalah 6-kali ganda lebih tinggi daripada kalus yang tumbuh di dalam media
MS tanpa hormon (kontrol). Menggunakan kombinasi hormon yang optimum ini,
pertumbuhan kalus di dalam media MS yang berbeza kepekatan, sumber karbon dan pH
dikaji. Media MS dengan kepekatan penuh yang ditambah dengan 2.0 mg/L 2,4-D, 0.5
mg/L BAP, 20 g/L sukrosa dan pH pada 5.7, menghasilkan berat kalus yang tertinggi
(231 ± 0.13 mg berat kering /kultur). Kalus tersebut telah digunakan untuk
menghasilkan ampaian sel.
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Bagi meningkatkan pengeluaran flavonoid dalam kultur kalus dan ampaian sel,
fenilalina telah digunakan sebagai prekursor di antara 20 hingga 100 mg/L. Kultur sel
ampaian dengan 60 mg/L fenilalanina menghasilkan kandungan flavonoid yang
tertinggi (37.92 ± 0.77 mg/g berat kering), iaitu 4.8-kali ganda tinggi daripada ampaian
sel tanpa fenilalanina, dan 2- kali ganda tinggi daripada kalus (19.11 ± 0.36 mg/g berat
kering). Kultur sel ampaian dengan 60 mg/L fenilalanina mempunyai jumlah tertinggi
rutin (17.69 ± 1.37 µg/g berat kering), kuersetin (3.46 ± 0.97 µg/g berat kering) dan
kaempferol (2.28 ± 1.67 µg/g berat kering). Apabila sel ampaian yang ditambah dengan
60 mg/L fenilalanina dibandingkan dengan kultur sel ampaian tanpa fenilalanina, hanya
rutin dan kuersetin dikesan tetapi masing-masing 1/10 dan 1/4- kali ganda lebih rendah.
Kajian ini menunjukkan kultur kalus dan sel ampaian A. malaccensis menghasilkan
flavonoid, dan penambahan fenilalanina ke dalam kultur meningkatkan paras flavonoid
dan menggalakkan penghasilan sebatian flavonoid yang lain iaitu kaempferol.
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ACKNOWLEDGEMENTS
First and foremost, I would like to take this opportunity to express my heartiest gratitude
to the chairman of my supervisory committee, Assoc. Prof. Dr. Rozi Mohamed, for her
invaluable advice, excellence guidance, contribution, patient and criticism throughout of
completed my research and also this thesis. An appreciation also dedicated to my co-
supervisor, Assoc. Prof. Dr. Kamziah Abd Kudus, for her advices on data analysis,
assistance and valuable feedback.
My sincere appreciation is extended to Dr. Ahmad Selamat for his invaluable help on
statistical analysis; Dr. Arifin Abdu for his kindness to access in Soil Lab and used the
HPLC equipments; Dr. Rasmina Halis for her generosity in helping; to Mr Kamarul
Ariffin Hadithon, science officer of Chromatography Laboratory at Faculty of Food
Science and Technology, UPM for his guidance and kindness. Special thanks also go to
Chai Theam, for his advice about setting up the HPLC and analyzing my samples. With
appreciations to all my laboratory members and friends; Shashita a/p Jayaraman, Siah
Chai Har, Nurul Irdayu, Wong Mun Theng, Liong Yan Yee, Lee Shiou Yih, Diau Ya
Bing and Azzarina Mohd Anor Basah.
My hearties appreciation goes to my parents, brothers, sisters and Muhammad Rizal Bin
Mohd Rosli for their emotional support, encouragement and understanding throughout
the course of this project. These words are not enough to describe my gratitude and
appreciation for everyone‟s help. Thanks for everything.
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I certify that a Thesis Examination Committee has met on 9th
October 2013 to conduct
the final examination of Nurul Hazwani Binti Daud on her thesis entitled “Optimization
of Culture Parameters for Flavonoid Production from Callus and Suspension Cultures of
the Aquilaria malaccensis Lam.” in accordance with Universities and University
Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106]
15 March 1998. The Committee recommends that the student be awarded the degree of
Master of Science.
Members of the Thesis Examination Committee were as follows:
Maziah Mahmood, PhD
Professor
Faculty of Biotechnology and Science Biomolecules
Universiti Putra Malaysia
(Chairman)
Mohd Puad Abdullah, PhD
Associate Professor
Faculty of Biotechnology and Science Biomolecules
Universiti Putra Malaysia
(Internal Examiner)
Syahida Ahmad, PhD
Lecturer
Faculty of Biotechnology and Science Biomolecules
Universiti Putra Malaysia
(Internal Examiner)
Aziz Ahmad, PhD
Associate Professor
Faculty of Science and Technology
Universiti Malaysia Terengganu
Malaysia
(External Examiner)
SEOW HENG FONG, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Rozi Mohamed, PhD
Associate Professor
Faculty of Forestry
Universiti Putra Malaysia
(Chairman)
Kamziah Abd Kudus, PhD
Associate Professor
Faculty of Forestry
Universiti Putra Malaysia
(Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citation, which
have been duly acknowledged. I also declare that it has not been previously, and is not
concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other
institutions.
NURUL HAZWANI DAUD
Date: 9 October 2013
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TABLE OF CONTENTS
Page
DEDICATION ii
ABSTRACT iii
ABSTRAK vi
ACKNOWLEDGEMENTS ix
APPROVAL x
DECLARATION xii
LIST OF TABLES xv
LIST OF FIGURES xvi
LIST OF ABBREVIATIONS xviii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 4
2.1 Aquilaria malaccensis 4
2.1.1 Taxonomy and Botany 4
2.1.2 Ecology and Distribution 7
2.1.3 Utilization and Trade of Agarwood 7
2.2 Secondary metabolites 9
2.2.1 Flavonoid 10
2.2.2 Flavonoid Biosynthesis Pathway 13
2.2.3 Flavonoids in Aquilaria species 16
2.2.4 Properties of Flavonoids 17
2.3 Plant Cell culture 18
2.3.1 Surface Sterilization of Explants 21
2.3.2 Callus Culture 22
2.3.3 Cell Suspension Culture 23
2.4 Factors Influence the Production of Flavonoid 24
2.4.1 Effects of Explants 25
2.4.2 Effects of Culture Conditions 26
2.4.4 Precursors Feeding 31
3 AN IMPROVED SURFACE STERILIZATION TECHNIQUE FOR
INTRODUCING LEAF, NODAL AND SEED EXPLANTS OF
AQUILARIA MALACCENSIS FROM FIELD SOURCES INTO
TISSUE CULTURE
33
3.1 Introduction 33
3.2 Materials and Methods 34
3.2.1 Plant Materials 34
3.2.4 Sterilization Methods 35
3.2.5 Preparation of Explants 36
3.2.6 Statistical Analysis 37
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3.3 Results 37
3.4 Discussion 41
3.5 Conclusions 43
4 EFFECT OF PHENYLALANINE ON FLAVONOID PRODUCTION
BY CALLI AND CELL SUSPENSION CULTURES OF AQUILARIA
MALACCENSIS LAM.
44
4.1 Introduction 44
4.2 Materials and Methods 46
4.2.1 Plant Materials 46
4.2.2 Total Flavonoid Content 47
4.2.3 Callus Induction 47
4.2.4 Callus Growth 49
4.2.5 Cell Suspension Culture and Growth Assessment 49
4.2.6 Precursor Preparation 50
4.2.7 High Performance Liquid Chromatography (HPLC) Analysis 50
4.2.8 Statistical Analysis 51
4.3 Results 51
4.3.1 Flavonoid Content in Different Explants 51
4.3.2 Callus Induction and Flavonoid Content 53
4.3.3 Callus Growth 64
4.3.4 Assessment of Growth in Cell Suspension Culture 66
4.3.5 Effect of Phenylalanine (Phe) on Biomass and Flavonoid
production
69
4.4 Discussion 73
4.5 Conclusions 79
5 SUMMARY, GENERAL CONCLUSION AND
RECOMMANDATIONS FOR FUTURE RESEARCH
80
REFERENCES 83
APPENDICES 98
BIODATA OF STUDENT 108
LIST OF PUBLICATIONS 109