innaka ageng rineksanepsasir.upm.edu.my/27011/1/fp 2011 36r.pdfsebagai memenuhi keperluan untuk...

UNIVERSITI PUTRA MALAYSIA

SOMATIC EMBRYOGENESIS,ORGANOGENESIS AND ASSESSMENT OF SOMACLONAL VARIATIONS IN MANGOSTEEN

(Garcinia mangostana L.)

INNAKA AGENG RINEKSANE

FP 2011 36

© COPYRIG

HT UPM

SOMATIC EMBRYOGENESIS,

ORGANOGENESIS AND ASSESSMENT OF

SOMACLONAL VARIATIONS IN MANGOSTEEN



DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2011

© COPYRIG

HT UPM

ii

This thesis is dedicated to:

My husband, Epijanto Anggariadi

My mother, Siti Aisjah and my father, Amrie Anwar

My mother in law, Soemiani

My sons, Muhammad Ammar Aiman Majid and

Muhammad Zidni Ilman Tafsiri

© COPYRIG

HT UPM

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirements for the degree of Doctor of Philosophy

SOMATIC EMBRYOGENESIS, ORGANOGENESIS AND ASSESSMENT OF SOMACLONAL VARIATIONS IN MANGOSTEEN


By


May 2011

Chairman : Associate Professor Mihdzar Abdul Kadir, PhD

Faculty : Agriculture

Mangosteen is one of the most delicious tropical fruits which has an

increasing demand due to its wide range of uses. It is used for its medicinal

properties such as an antioxidant, antitumoral, anti-inflammatory, antiallergy,

antibacterial, antifungal and antiviral. One of the problems related to the

establishment of mangosteen plantation is to obtain seedlings throughout the

year, which can be solved by micropropagation.

In attempts to establish the embryogenic calli of G. mangostana, the

potential of uncoated and coated seed explants in forming embryogenic

callus was examined in the basal Linsmaier and Skoog (LS) medium

supplemented with different auxins at various concentrations. Combinations

of cytokinins and 2,4-D in two different media [Murashige and Skoog (MS)

and LS] were assessed to improve embryogenicity of calli in mangosteen.

© COPYRIG

HT UPM

iv

Addition of glutamine at various concentrations into MS medium containing 8

mg/L 2,4-D and 0.1 mg/L BAP was also carried out to induce embryogenic

callus of mangosteen. A study was also carried out to determine the growth

and multiplication of cells in suspension cultures and the effect of cytokinins

on the advanced formation of embryogenic stages of mangosteen. BAP and

NAA were considered for shoot regeneration and the rooting of mangosteen

shoots. The most favorable medium for acclimatization of mangosteen

plantlets was also produced. The assessment of somaclonal variations

among mangosteen plantlets by using RAPD was performed.

Uncoated seed explants produced a lower percentage of callus formation

(44.23 %), callus score (1.66), fresh weight of callus (59.98 mg) and a lower

percentage of contaminated explants (9.7 %) compared to coated seed

explants. Among the highest percentage of callus formation (93.3 %) and

callus score (3.06) were obtained when uncoated seed explants were

cultured on basal LS medium containing 8 mg/L 2,4-D. The calli were

yellowish, compact and nodular compared to the spongy loose, whitish and

yellowish calli produced on media containing IAA, IBA or NAA. The highest

percentage of callus formation (80 %) and the lowest percentage of callus

browning (53.53 %) occurred on MS medium supplemented with 8 mg/L 2,4-

D and 0.1 mg/L BAP. Although glutamine did not increase the growth of

calli, the texture (more friable) and color of callus (more yellowish) were

improved.

© COPYRIG

HT UPM

v

The cells were able to divide and proliferate eventhough cultured in half

strength MS liquid medium without 2,4-D. After six months of culture, the

heart embryogenic stage was obtained only on medium supplemented with 1

mg/L BAP. The globular and torpedo embryogenic stages were obtained on

media supplemented with 1, 3 and 9 mg/L TDZ after five months of culture.

Mass production of adventitious shoots was achieved by culturing seed

segments of mangosteen on MS solid medium supplemented with 5 mg/L

BAP + 0.1 mg/L NAA which produced the highest shoot number (31.7

shoots). Forty-one percent of shoots were successfully rooted in MS liquid

medium supplemented with 1 mg/L IBA, 60 g/L sucrose and 5 g/L activated

charcoal after 4 months. During acclimatization, plantlets grown in medium

consisting of organic matter only (A6) showed the highest height difference

(7 mm) as compared with other treatments.

Stunted shoots with narrow leaves were produced in abundance in MS solid

medium supplemented with 5 mg/L BAP + 0.1 mg/L NAA. These shoots

were morphologically and genetically different from shoots of other

treatments as detected by RAPD marker. RAPD marker effectively

recognized the genetic difference among the in vitro shoots and from their

mother plant with high level of similarity (80 %). Among the acclimatized

plantlets and in vitro shoots, 72 % level of similarity was obtained. The

lowest level of similarity (55%) was found between in vivo samples from

Serdang and Pahang.

© COPYRIG

HT UPM

vi

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

SOMATIK EMBRIOGENESIS, ORGANOGENESIS DAN PENGKAJIAN VARIASI SOMAKLONAL PADA MANGGIS (Garcinia mangostana L.)

Oleh


Mei 2011

Pengerusi : Profesor Madya Mihdzar Abdul Kadir, PhD

Fakult i : Pertanian

Manggis merupakan antara spesies buah tropika yang paling lazat,

mempunyai permintaan yang semakin bertambah kerana ianya mempunyai

keberkesanan sebagai anti oksidan, anti tumor, anti-inflamasi, anti alergi, anti

bakteria, anti jamur dan agen anti virus. Permasalahan yang berlaku adalah

tidak cukupnya anak pokok untuk penanaman skala besar. Salah satu pilihan

untuk pembiakan secara besar-besaran ialah dengan kultur tisu.

Dalam usaha untuk mendapatkan embrio somatik daripada G. mangostana,

kalus yang berpotensi daripada eksplan biji berkulit dan tidak berkulit telah

diuji dalam medium basal Linsmaier dan Skoog (LS) yang dibekalkan dengan

pelbagai jenis auksin pada kepekatan yang berbeza. Kombinasi sitokinin dan

2,4-D yang dibekalkan dalam dua media yang berbeza [Murashige dan Skoog

(MS) dan LS] telah diuji untuk merangsang kalus embriogenik pada manggis.

Penambahan glutamin pada kepekatan yang berbeza ke dalam medium MS

© COPYRIG

HT UPM

vii

yang mengandungi 8 mg/L 2,4-D dan 0.1 mg/L BAP juga dikenalpasti untuk

merangsang kalus embriogenik manggis. Penyelidikan juga telah dijalankan

untuk mengenalpasti regenerasi dan multiplikasi sel dalam kultur suspensi

dan mengenalpasti kesan daripada sitokinin untuk merangsang pembentukan

tahap embriogenik daripada manggis. BAP dan NAA diuji untuk regenerasi

pucuk dan akar. Media aklimatisasi yang paling berkesan untuk planlet

manggis juga telah dikenalpasti. Pengenalpastian variasi somaklonal di

antara plantlet manggis melalui kaedah RAPD juga telah dikaji.

Biji tidak berkulit menghasilkan peratusan lebih rendah pada regenerasi kalus

(44.23%), skor kalus (1.66), berat segar kalus (59.98 mg) dan peratusan lebih

rendah pada eksplan terkontaminasi (9.7%) apabila dibandingkan dengan biji

berkulit. Antara peratusan regenerasi kalus (93.3%) dan skor kalus paling

tinggi (3.06) telah diperolehi apabila eksplan biji tidak berkulit dikulturkan di

dalam medium basal LS yang mengandungi 8 mg/L 2,4-D. Kalus yang

dihasilkan adalah padat kekuningan dan bernodul apabila dibandingkan

dengan kalus berspan, putih dan kekuningan yang dihasilkan daripada media

yang mengandungi IAA, IBA atau NAA. Peratusan regenerasi kalus tertinggi

(80%) dan peratusan pemerangan kalus terendah (53.53%) dihasilkan

daripada medium MS yang dibekalkan dengan 8 mg/L 2,4-D dan 0.1 mg/L

BAP. Walaupun glutamin tidak merangsang regenerasi kalus, tekstur kalus

yang lebih rapuh (friable) dan warna kalus yang lebih kekuningan telah

ditingkatkan.

© COPYRIG

HT UPM

viii

Sel dapat membahagi dan mengganda walaupun dikulturkan di dalam

medium MS separuh cecair yang tidak mengandungi 2,4-D. Enam bulan

selepas pengkulturan, embrio somatik jantung telah diperolehi hanya pada

medium yang mengandungi 1 mg/L BAP. Embrio somatik bulat dan torpedo

telah diperolehi pada medium yang mengandungi 1, 3 dan 9 mg/L TDZ

selepas 5 bulan pengkulturan.

Pengeluaran pucuk adventitius secara besar-besaran telah diperolehi dengan

pengkulturan segmen biji manggis ke dalam medium padat MS yang

dibekalkan dengan 5 mg/L BAP dan 0.1 mg/L NAA yang telah menghasilkan

bilangan pucuk tertinggi (31.7 pucuk). Empat puluh satu peratus pucuk telah

berjaya menghasilkan akar pada medium cecair MS yang mengandungi 1

mg/L IBA, 60 g/L sukrosa dan 5 g/L arang teraktif selepas 4 bulan. Untuk

aklimatisasi, plantlet yang dipindahkan ke dalam medium organik (A6) sahaja

yang menghasilkan perbezaan ketinggian tertinggi (7 mm) apabila

dibandingkan dengan rawatan yang lain.

Pucuk terbantut dengan daun kecil telah dihasilkan dalam bilangan besar

daripada medium padat MS yang mengandungi 5 mg/L BAP dan 0.1 mg/L

NAA. Ianya menunjukkan perbezaan dari segi morfologi dan genetik dengan

pucuk-pucuk yang dihasilkan daripada rawatan lainnya selepas dikenalpasti

melalui kaedah RAPD. Kaedah RAPD telah berjaya mengenalpasti variasi

genetik di antara pucuk in vitro dan pokok induk dengan tahap persamaan

yang tinggi (80%). Di antara plantlet aklimatisasi dan pucuk-pucuk in vitro,

© COPYRIG

HT UPM

ix

tahap persamaan 72% telah diperolehi. Tahap persamaan terendah (55%)

telah dikenalpasti di antara aksesi in vivo dari Serdang dan Pahang.

© COPYRIG

HT UPM

x

ACKNOWLEDGEMENTS

Praise to Almighty Allah (SWT) the most Benevolent, Merciful and

Compassionate, for giving me the utmost strength, patience and guidance to

have this work completed.

I would like to express my most sincere gratitude and deepest appreciation

to Associate Professor Dr. Mihdzar Abdul Kadir, chairman of my supervisory

committee, for his dedicated efforts, support, invaluable advice and

intellectual guidance during the accomplishment of this research work. I

greatly appreciate all the help he availed to me while pursuing my studies. I

would also like to thank my supervisory committee members, Associate

Professor Dr. Saleh Kadzimin and Associate Professor Dr. Faridah Qamaruz

Zaman for their help, constant encouragement and constructive comments

throughout the period of this study.

I would like to extend my sincere thanks to the Muhammadiyah University of

Yogyakarta, Indonesia, through Dr. Gunawan Budiyanto, Ir Darmawan Suryo

Sudarsono, MP (in memoriam), Ir. Nafi Ananda Utama, MS for providing me

the financial support during my study. Thanks to Universiti Putra Malaysia

also for offering me with the Graduate Research Fellowship.

© COPYRIG

HT UPM

xi

My special thanks go to Dr Ahmad Selamat for his inputs and comments on

statistical analysis, Mr Mahmood Danae for his valuable suggestions and

kindness in the statistical analysis in this manuscript and Angela Ee De Silva

for editing this manuscript. My special thanks also go to Ir Agung Astuti,

MSi, Head of Agrotechnology Department, Faculty of Agriculture,

Muhammadiyah University of Yogyakarta, Indonesia, for her constant

support and motivation during the period of my study.

I am very grateful to Rostina Rolon, Siti Raziah, Rozila, Azwana, staffs of

Agrotechnology Laboratory and staffs of Department of Agriculture

Technology Universiti Putra Malaysia for their help and kindness during my

study. I would also like to thank all my lab mates for their warm friendship,

advice and help during the period of my study. Special thanks go to Eum

Sang Mi, Alireza Khosravi and P.K Dewi Hayati for their advice and help with

my RAPD works, Syaiful Bahri Panjaitan and Suleiman Elhory for their

advice and kindness.

Thanks to my family, my mother and father, for their love and

encouragement over the years to help me reach this point. I am especially

grateful to my husband, Epijanto Anggariadi, for his understanding,

encouragement and wholehearted support during the period of the study. A

special thanks to my sons, Ammar and Zidni, who made my life more

enjoyable.

© COPYRIG

HT UPM

xii

I certify that a Thesis Examination Committee has met on 19 May 2011 to conduct the final examination of Innaka Ageng Rineksane on her thesis entitled “Somatic Embryogenesis, Organogenesis and Assessment of Somaclonal Variation in Mangosteen (Garcinia mangostana L.)” in accordance with the 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 Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Mahmud Tengku Muda Mohamed, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Maheran Abdul Aziz, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Datin Siti Nor Akmar Abdullah, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Sompong Te-chato, PhD Associate Professor Faculty of Natural Resources Prince of Songkla University Thailand (External Examiner)

___________________________ NORITAH UMAR, PhD

Associate Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 26 July 2011

© COPYRIG

HT UPM

xiii

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Mihdzar Abdul Kadir, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Saleh Kadzimin, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member) Faridah Qamaruz Zaman, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Member)

________________________________ HASANAH MOHD. GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

© COPYRIG

HT UPM

xiv

DECLARATION

I declare that the thesis is my original work except for quotations and citations 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 institution.

___________________________


Date: 19 May 2011

© COPYRIG

HT UPM

xv

TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS x APPROVAL xii DECLARATION xiv LIST OF TABLES xx LIST OF FIGURES xxii LIST OF PLATES xxiv LIST OF ABBREVIATIONS xxvii CHAPTER 1 INTRODUCTION

1.1. Background 1

1.2. Objectives 4

2 LITERATURE REVIEW 2.1. Mangosteen 5

2.1.1 Origin and Distribution 5

2.1.2 Taxonomy and Morphology 6

2.1.3 Production and Utilization 8

2.1.4 Genetic Variability in Mangosteen 11

2.2. Mangosteen Micropropagation 13

2.2.1. Organogenesis 14

2.2.2. Somatic Embryogenesis 16

2.2.3. Callus Induction 18

2.2.4. Cell Suspension 21

2.2.5. Acclimatization 23

2.3. Biochemistry of In Vitro Cultured Plant 24

2.3.1. Auxins 25

2.3.2. Cytokinins 27

2.3.3. Activated Charcoal 28

2.3.4. Organic Nitrogen Source 29

© COPYRIG

HT UPM

xvi

2.4. Molecular Markers 30

2.4.1. RAPD as Genetic Marker 32

2.4.2. Somaclonal Variations 33

3 IN VITRO DEVELOPMENT OF EMBRYOGENIC CALLI IN MANGOSTEEN 3.1. Introduction 35

3.2. Materials and Methods 37

3.2.1. The Effects of Auxins on Callus Induction from

Mangosteen Seeds 37

Preparation of Plant Materials 37

Basic Medium 38

Treatments 38

Experimental Design 40

Parameters 40

3.2.2. The Effect of 2,4-D and Cytokinins on Callus

Induction from Mangosteen Seed Explants 41


Basic Medium 41

Treatments 42


Parameters 43

3.2.3. The Effect of Glutamine on Embryogenic Callus

Formation in Mangosteen 43


Basic Medium 44

Treatments 44


Parameters 45

3.3. Results and Discussion 45

3.3.1. The Effects of Auxins on Callus Induction from

Mangosteen Seeds 45

Percentage of Explants Forming Callus 47

© COPYRIG

HT UPM

xvii

Callus Scoring 54

Fresh Weight of Callus 55

Percentage of Contamination 56

Texture and Color of Callus 56

3.3.2. The Effect of 2,4-D and Cytokinins on Callus

Induction from Mangosteen Seed Explants 60

Percentage of Callus Formation 66

Percentage of Browning Callus 68

3.3.3. The Effect of Glutamine on Embryogenic Callus

Formation in Mangosteen 70

Callus Score and Fresh Weight 70

Detection of Embryogenic Callus 74

4 DEVELOPMENT OF EMBRYOGENIC STAGES IN CELL

SUSPENSION CULTURES OF MANGOSTEEN 4.1. Introduction 78


4.2.1. Cell Multiplication 79

Plant Materials 79

Basic Medium 80

Treatments 80


Parameters Observed 81

4.2.2. The Effect of BAP on the Development of

Embryogenic Stages in Mangosteen Cell

Suspension Cultures 81

Plant Cell Used for Culture 81

Basic Medium 82

Treatments 82



© COPYRIG

HT UPM

xviii

4.2.3. The Effect of TDZ on Development of

Embryogenic Stages in Mangosteen Cell

Suspension Cultures 83


Basic Medium 84

Treatments 84




4.3.1. Cell Multiplication 85

4.3.2. The Effect of Ctyokinins on the Development of

Embryogenic Stages from Cell Suspension

Cultures 91

4.3.3. The Effect of BAP on the Development of


Cultures 93

4.3.4. The Effect of TDZ on the Development of


Cultures 96

5 IN VITRO REGENERATION OF MANGOSTEN THROUGH

ORGANOGENESIS 5.1. Introduction 99


5.2.1. Shoot Induction 101


Basic Medium 102

Treatments 103


Experimental Design and Data Analysis 104

5.2.2. Root Induction 104


Plant Materials 106

© COPYRIG

HT UPM

xix

Acclimatization Media 106

Treatments 107


Experimental Design and Data Analysis 108


5.3.1. Shoot Induction 108

5.3.2. Root Induction 117


6 DETECTION OF SOMACLONAL VARIATIONS IN

MANGOSTEEN BY USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) 6.1. Introduction 123


6.2.1. Plant Materials, Samples Preparation and Primer

Tested 124

6.2.2. DNA Isolation and Quantification 128

6.2.3. Polymerase Chain Reaction (PCR) Optimization

and Primer Screening 129

6.2.4. Data Analysis 130


6.3.1. DNA Isolation and Quantification 131

6.3.2. Primer Screening 132

6.3.3. RAPD Analysis 134

7 SUMMARY, GENERAL CONCLUSION AND RECOMMENDATION FOR FUTURE RESEARCH 142

REFERENCES 149 APPENDIX 161 BIODATA OF STUDENT 170

innaka ageng rineksanepsasir.upm.edu.my/27011/1/fp 2011 36r.pdfsebagai memenuhi keperluan untuk...

Documents