universiti putra malaysia optimization of culture parameters for

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.)


MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2013

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COPYRIGHT

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photographs and all other artwork, is copyright material of Universiti Putra Malaysia

unless otherwise stated. Use may be made of any material contained within the thesis for

non-commercial purposes from the copyright holder. Commercial use of material may

only be made with the express, prior, written permission of Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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OPTIMIZATION OF CULTURE PARAMETERS FOR FLAVONOID

PRODUCTION FROM CALLUS AND SUSPENSION CULTURES OF THE

KARAS TREE (AQUILARIA MALACCENSIS LAM.)

By


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


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


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



(Internal Examiner)

Syahida Ahmad, PhD

Lecturer



(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


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


(Chairman)

Kamziah Abd Kudus, PhD

Associate Professor

Faculty of Forestry


(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


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

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universiti putra malaysia optimization of culture parameters for