By SREERAMANAN SUBRAMANIAM - Universiti …psasir.upm.edu.my/514/1/549738_FBSB_2005_18.pdfBy SREERAMANAN SUBRAMANIAM ... effect of various sizes of explants, ... menggunakkan kaedah

GENETIC ENGINEERING FOR TOLERANCE TO FUSARIUM WILT

IN MUSA SAPIENTUM CV RASTALI (AAB)

By

SREERAMANAN SUBRAMANIAM

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Doctor of Philosophy

April 2005

II

Dedicated To:

My Lovingly Parents: Amma, InthraniMy Lovingly Parents: Amma, InthraniMy Lovingly Parents: Amma, InthraniMy Lovingly Parents: Amma, Inthrani AndAndAndAnd

AppaAppaAppaAppa, Mr C.S.Maniam, Mr C.S.Maniam, Mr C.S.Maniam, Mr C.S.Maniam

My beloved wife: Deepa ChandramohanMy beloved wife: Deepa ChandramohanMy beloved wife: Deepa ChandramohanMy beloved wife: Deepa Chandramohan

WonderfuWonderfuWonderfuWonderful Brother and Sister: Sreeharan Subramaniaml Brother and Sister: Sreeharan Subramaniaml Brother and Sister: Sreeharan Subramaniaml Brother and Sister: Sreeharan Subramaniam Meena Meena Meena Meena

My InMy InMy InMy In----Laws: Dr and Mrs Chandramohan, LavanyaLaws: Dr and Mrs Chandramohan, LavanyaLaws: Dr and Mrs Chandramohan, LavanyaLaws: Dr and Mrs Chandramohan, Lavanya

Great Lab Friends: VanithaGreat Lab Friends: VanithaGreat Lab Friends: VanithaGreat Lab Friends: Vanitha Mariappan, Sun Mariappan, Sun Mariappan, Sun Mariappan, Sunilililil Bhalla, Bhalla, Bhalla, Bhalla, Rosli Normi, Ali Reza, Adeline Ting, Rosli Normi, Ali Reza, Adeline Ting, Rosli Normi, Ali Reza, Adeline Ting, Rosli Normi, Ali Reza, Adeline Ting, Yap Wai Sum Yap Wai Sum Yap Wai Sum Yap Wai Sum and Chewand Chewand Chewand Chew Yern Chern Yern Chern Yern Chern Yern Chern....

III

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the Degree of Doctor of Philosophy

GENETIC ENGINEERING FOR TOLERANCE TO FUSARIUM WILT

IN MUSA SAPIENTUM CV RASTALI (AAB)

By


April 2005

Chairman: Professor Maziah Mahmood, PhD

Faculty : Biotechnology and Biomolecular Sciences

This study aims at developing tolerant Pisang Rastali (AAB) to Fusarium wilt via genetic

engineering. Several considerations support that banana is a good candidate crop that

could benefit from biotechnology applications.

Single buds from multiple bud clumps (Mbcs) of Pisang Rastali (AAB) were established

to be suitable target tissues for the introduction of antifungal protein and reporter genes

using the Agrobacterium-mediated and particle bombardment transformation methods.

Multiple bud clumps (Mbcs) were easily micropropagated in vitro that provided a reliable

source of potentially regenerable single bud tissues. The effectiveness of kanamycin,

neomycin, geneticin G-418, paromomycin, basta and hygromycin as selection agents to

inhibit growth of Pisang Rastali (AAB) was evaluated in solid and liquid media. Of six

IV

antibiotic selection agents tested, hygromycin was the best, followed by basta and

geneticin G-418. In addition, the use of liquid media has been shown to be more effective

than in solid medium. Geneticin G-418 was the preferable antibiotic agent compared to

kanamycin, neomycin and paromomycin since all plasmids used in this studies confers

resistance to npt11 gene.

Chemotaxis of Agrobacterium tumefaciens strains (EHA 101 and LBA 4404) towards

wounded banana tissues has been studied using swarm agar plates. The results obtained

indicate a minor role of chemotaxis in determining host specificity and suggest that it

could not be responsible for the absence of tumorigenesis in banana under natural

conditions. A method developed for the quantification of bacterial attachment to banana,

based on the use of the gusA and gfp genes in marked Agrobacterium strains, is also

described. Its application has demonstrated that attachment may also not be a main factor

determining host specificity in Agrobacterium tumefaciens. In addition, optimization of

several factors affecting transient gusA and gfp genes expression such as preculture

period, co–cultivation period, acetosyringone concentrations, types of wounding, strains

of Agrobacterium tumefactions, influence of single bud sizes and post cultivation period

were evaluated to determine the efficiency of Agrobacterium-mediated transfer during

the early stages of transformation in banana particularly in Pisang Rastali (AAB).

Physical and biological parameters affecting DNA delivery into Pisang Rastali (AAB)

single buds have been optimised. The physical parameters tested were helium pressure,

distance from stopping plate to target tissues, vacuum pressure, multiple bombardment

V

and gold microcarrier size. The optimised biological parameters were explant types,

effect of various sizes of explants, effect of preculture treatment prior bombardment,

effect of DNA concentrations and post bombardment incubation time.

Regeneration of transgenic Pisang Rastali (AAB), molecular analyses (PCR and Southern

blot), analysis of chitinase and β-1,3-glucanase protein production via Agrobacterium-

mediated transformation were carried out. DNA samples from transgenic Pisang Rastali

(AAB) obtained only from selection media contained geneticin G-418 as a selectable

marker and were tested positive for the presence of the chitinase (RCC2) or β-1,3

glucanase (Eg) and npt11 coding sequences by PCR analyses. Genomic Southern blot

hybridization confirmed the incorporation of the the chitinase (RCC2) or β-1,3-glucanase

(Eg) gene into host genome.

Using particle bombardment system, five different treatments using different chitinases

and β-1,3-glucanase genes inserted singly or in combination were carried out. Genes

encoding either chitinases (RCC2 or Chi) or β-1,3-glucanase (Eg) or both was bombarded

together with gfp gene (pGEM.Ubi-SgfpS65T) for early transient expressions signal.

Integration of the transgenes and stablity of the particle bombardment system were

confirmed by PCR amplification of gfp, gusA, RCC2 (chitinase), Chi (chitinase), Eg (β-1,

3-glucanase) and npt11 genes. Genomic Southern blot hybridization confirmed the

incorporation of the RCC2, Chi and Eg genes in host genome between one and five

inserted copies in transformed plantlets. Similar to Agrobacterium-mediated

transformation system, the chitinase and β-1,3-glucanase enzyme activity of transgenic

VI

plantlets obtained from particle bombardment was higher than untransformed plantlets. In

addition, co-bombardment of chitinase and β-1,3 glucanase genes (Treatment 1 and 2)

gave higher enzyme activities compared with single gene insertion (Treatment 3, 4 and

5).

For Fusarium bioassay method, time course of Fusarium oxysporum f.sp. cubense (race 1

; VCG 01217) spore production in vitro and the actual number of germinated spores were

determined . The 28 days old Fusarium spores with 62% germinating capacity at 2x106

spores/ml were used for bioassay testing of transgenic Pisang Rastali (AAB) plantlets.

Hydrogen peroxide (H2O2) and phenylalanine ammonia lyase (PAL) is the most sensitive

chemical compound and enzyme to Fusarium spores inoculation. The chitinase and β-

1,3-glucanase enzyme activities of transgenic plantlets was almost four folds higher than

untransformed plantlets. The peroxidase (PER) and polyphenol oxidase (PPO) enzyme

activities in banana roots increased markedly up to 2-3 folds higher in transformed plants

compared to untransformed plantlets after 28 days of inoculation.

VII

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

KEJURUTERAAN GENETIK PISANG YANG TOLERAN TERHADAP LAYU

FUSARIUM PADA MUSA SAPIENTUM CV RASTALI (AAB).

Oleh


April 2005

Pengerusi: Profesor Maziah Mahmood, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Kajian ini bertujuan menghasilkan pisang Rastali (AAB) yang toleran terhadap penyakit

layu Fusarium melalui kejuruteraan genetik. Beberapa kenyataan menyokong bahawa

pisang adalah tanaman pertanian yang sesuai dan mendapat faedah daripada aplikasi

bioteknologi.

Pucuk tisu individu daripada kelompok tisu pucuk pisang Rastali (AAB) dihasilkan bagi

kesesuaian tisu sasaran untuk menerima protein antikulat dan gen pelapor dengan

menggunakkan kaedah transformasi perantaraan Agrobakterium dan tembakan partikel.

Kelompok pucuk tisu mudah dikulturkan secara in vitro dan berpotensi untuk regenerasi

pucuk tisu secara individu. Kesesuaian kanamycin, neomycin, geneticin G-418,

paromomycin, basta dan hygromycin sebagai agen pemilih dinilai dalam media pepejal

dan cecair. Daripada enam agen antibiotik yang diuji, hygromycin adalah yang terbaik,

diikuti dengan basta dan geneticin G-418. Selain itu, penggunaan medium cecair didapati

VIII

lebih efektif daripada medium pepejal. Geneticin G-418 merupakan agen antibiotik yang

lebih sesuai berbanding dengan kanamycin, neomycin dan paromomycin kerana semua

plasmid yang digunakan dalam penyelidikkan ini menunjukkan kerintangan terhadap gen

npt11.

Kemotaksis oleh jenis Agrobakterium tumefaciens (EHA 101 dan LBA 4404) terhadap

tisu pisang yang dilukakan telah dikaji dengan menggunakan ‘swarm agar plates’.

Keputusan yang diperolehi menunjukkan peranan kemotaksis adalah kecil dalam

menentukan kekhususan penerima dan ini menunjukkan ia tidak bertanggungjawab bagi

ketiadaan tumoregenesis pada pisang dalam keadaan semulajadi. Satu kaedah pengukuran

bagi pelekatan bakteria berdasarkan penggunaan gen gusA dan gfp yang terdapat pada

Agrobakterium juga dihasilkan. Aplikasi ini menunujukkan bahawa pelekatan bukanlah

merupakan faktor utama dalam menentukan kekhususan penerima dalam Agrobakterium

tumefaciens. Selain itu, pengoptimuman beberapa faktor yang mempengaruhi

pengekspresan gen sementara gusA dan gfp, seperti tempoh para-kultur, tempoh

pengeraman, kepekatan acetosyringone, jenis kecederaan, jenis Agrobakterium

tumefaciens, pengaruh saiz tunas individu dan jangka masa selepas subkultur dinilai

untuk menentukan keberkesanan pemindahan perantaraan Agrobakterium pada peringkat

awal transformasi pisang, khususnya pisang Rastali (AAB).

Parameter fizikal dan biologi yang mempengaruhi pemindahan DNA ke dalam individu

pucuk tisu pisang Rastali (AAB) telah dioptimumkan. Parameter fizikal yang diuji ialah

tekanan helium, jarak antara plat penghalang dengan tisu sasaran, tekanan vakum,

IX

tembakan berganda dan saiz pembawa mikro emas. Parameter biologi yang

dioptimumkan ialah jenis tisu penerima, kesan pelbagai saiz tisu asal, kesan rawatan

para-kultur sebelum penembakan, kesan kepekatan DNA dan tempoh pengeraman

selepas tembakan.

Regenerasi pisang Rastali (AAB) transgenik, analisis molekul (PCR and Southern blot),

analisis protein chitinase dan β-1,3-glucanase melalui transformasi perantaraan

Agrobakterium telah dijalankan. Sampel DNA daripada pisang Rastali (AAB) transgenik

diperolehi hanya daripada media pemilihan yang mengandungi geneticin G-418 sebagai

penanda terpilih dan telah diuji positif bagi kehadiran gen chitinase dan β-1,3-glucanase

(Eg) dan kod linear npt11 melalui analisis PCR. Hybridasi genomik Southen blot telah

membuktikan pemindahan gen chitinase (RCC2) dan β-1,3-glucanase didalam genomik

penerima.

Menggunakan sistem tembakan partikel, lima rawatan berlainan menggunakan gen

chitinase dan β-1,3-glucanase berlainan kombinasi dipindahkan secara tunggal dan

gabungan dijalankan. Gen berkod chitinase (RCC2 atau Chi) atau β-1,3-glucanase (Eg)

dan kedua-duanya sekali ditembakkan bersama dengan gene gfp (pGEM.Ubi-SgfpS65T)

bagi mendapatkan isyarat awal pengekspresan gen sementara pada peringkat awal.

Integrasi transgen dan kestabilan system tembakan partikel dibuktikan melalui

amplifikasi gen gfp, gusA, RCC2 (chitinase), Chi (chitinase), Eg (β-1,3-glucanase) dan

npt11. Hybridasi Southern blot genomik membuktikan kehadiran gene RCC2, Chi dan Eg

pada genomik penerima antara satu hingga lima salinan dimasukkan pada anak pokok

X

yang ditransformkan. Sama seperti kaedah transformasi perantaraan Agrobakterium,

aktiviti enzim chitinase dan β-1,3-glucanase pada anak pokok transgenik yang diperolehi

daripada tembakan partikel adalah lebih tinggi daripada pokok yang tidak

ditransformasikan. Selain itu, tembakan bersama gen chitinase dan β-1,3-glucanase

(Rawatan 1 dan 2) menghasikan aktiviti enzim yang lebih tinggi berbanding kemasukkan

gen tunggal (Rawatan 3, 4 dan 5).

Bagi kaedah biouji Fusarium, tempoh pengeraman spora Fusarium oxysporum f.sp.

cubense (race 1; VCG 01217) secara in vitro dan bilangan sebenar percambahan spora

telah ditentukan. Spora Fusarium pada hari ke 28 dengan keupayaan 62% percambahan

pada 2x106 spora/ml telah digunakan sebagai biouji pokok pisang Rastali (AAB)

transgenik. Hidrogen peroxide (H202) dan phenylalanine ammonia lyase (PAL) adalah

bahan kimia dan enzim yang paling sensitif kepada inokulasi spora Fusarium. Aktiviti

enzim chitinase dan β-1,3-glucanase pada anak pokok transgenik adalah hampir empat

kali ganda lebih tinggi daripada pokok yang tidak ditransformasikan. Aktiviti enzim

peroxidase (PER) dan polyphenol oxidase (PPO) pada akar pisang didapati dua hingga

tiga kali ganda lebih tinggi daripada anak pokok yang tidak ditransformasikan selepas 28

hari inokulasi.

XI

ACKNOWLEDGEMENTS

In has been a long way since 2000 in Lab 235 to the pleasant moment of writing

thesis last (first 1 lines of the thesis). This a work that started during my first year doing

Master programme in May 2000, before convert into PhD, which could have never been

alone without the help, support and contributions of those that I met along the path.

First of all, I would like to thank Professor Dr Maziah Mahmood for giving me the

opportunity, support and strength to start this journey. I’m undoubtedly grateful for the

continuous guidance of Professor Maziah Mahmood, without this thesis would not been

possible and for the invaluable contributions she has made to this work, which has been

indeed the results of a shared effort. Special thanks again to Professor Dr Maziah

Mahmood, who gave me two times opportunities to participate banana symposiums

which had been held in Belgium (September 2002) and Brazil (September 2003).

I want to thank to Dr Puad Abdullah, Professor Sariah Meon and Associate

Professor, Dr Nor’Aini Mohd Fadzillah as well the members of jury, for their corrections

and suggestions to improve the quality of this dissertation.

To everybody at lab 235, thank for your help and friendship during all these years.

Thanks to Dr Gurmit Singh, Madam Ho Yuk Wah, Adeline Ting, Deepa Chandramohan,

Vanitha Mariappan and Sunil Bhalla for raising ideas behind a cup of coffee.

XII

Special thank for providing Agrobacterium tumefaciens strains and plasmids to Dr

Richard Brettell from Australia, [LBA4404 (pCAMBIA 1304)], Mr Hikaru Tsukazaki

from Japan, [EHA 101 (pIG121-Hm), Dr Yoshikawa from Japan (pBI333-EN4-RCC2),

Dr Nakamura from Japan (pROKLa-EG), Dr Ruslan Abdullah from UKM, (pMRC

1301), and Adrian Elliot from Australia [pGEM.Ubi1-sgfps65T (GFP)], respectively.

XIII

I certify that an Examination Committee met on _____ to conduct the final examination

of Sreeramanan Subramaniam on his Doctor of Philosophy thesis entitled “Genetic

Engineering For Tolerance To Fusarium wilt in Musa sapientum cv Rastali (AAB)” in

accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti

Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that

the candidate be awarded the relevant degree. Members of the Examination Committee

are as follows:

Maziah Mahmood, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia,

(Chairman)

Puad Abdullah, PhD

Lecturer

Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia,

(Member)

Sariah Meon, PhD

Professor

Faculty of Agriculture,

Universiti Putra Malaysia

(Member)

Prof Dr N. Kumar, PhD

Professor

College of Horticulture,

Tamil Nadu Agriculture University (TNAU),

India.

(Independent Examiner)

___________________________________

GULAM RUSUL RAHMAT ALI, PhD

Professor/Deputy Dean

School of Graduate Studies


Date:

XIV

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as

fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the

Supervisory are as follows:

Maziah Mahmood, PhD

Professor

Faculty of Biotechnology and Biomolecular Science


(Chairman)

Mohd Puad Abdullah, PhD

Lecturer

Faculty of Biotechnology and Biomolecular Science


(Member)

Sariah Meon, PhD

Professor

Faculty of Agriculture


(Member)

_____________________

AINI IDERIS, PhD

Professor/Dean

School of Graduate Studies


Date:

XV

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and

citations that have been duly acknowledged. I also declare that it has not been previously

or concurrently submitted for any other degree at UPM or other institutions.

----------------------------------------------


Date:

TABLE OF CONTENTS

Page

DEDICATION II

ABSTRACT III

ABSTRAK VII

ACKNOWLEDGEMENTS XI

APPROVAL XIII

DECLARATION XV

LIST OF TABLES XXI

LIST OF FIGURES XXIV

LIST OF ABBREVATIONS XXXIIII

CHAPTER

1 INTRODUCTION 1

1.1 Botanic description and taxonomy 1

1.2 Bananas and plantains of different genomic groups 2

1.3 Worldwide production 2

1.4 Malaysia’s production 3

1.5 Fungal diseases threatening banana production 4

1.5.1 Panama disease 4

1.5.2 False Panama disorder 9

1.6 Biotechnology based banana improvement 10

1.7 Somaclonal variation and induced mutation 11

1.8 Opportunities and requirements for molecular breeding of

banana 12

1.9 Objectives 14

1.10 Thesis outline 14

2 LITERATURE REVIEW 17

2.1 Genetic transformation

17

2.2 Gene transfer methods for monocotyledonous plants 19

2.2.1 Agrobacterium-mediated transformation 19

2.2.2 Particle bombardment 29

2.2.3 Co–transformation 38

2.2.4 Transgene expression 45

2.2.5 Constitutive promoters 49

2.3 Transgenic plants for the control of fungal diseases 55

2.4 Strategies to generate plant tolerance to fungal pathogen 58

2.4.1 Antifungal proteins 58

XVII

3 PRODUCTION OF SUITABLE TARGET TISSUES

AND MINIMAL INHIBITORY CONCENTRATION

OF SELECTION AGENTS FOR TRANSFORMATION

STUDIES 68

3.1 Introduction 68

3.2 Objectives 70

3.3 Materials and methods 71

3.3.1 Plant materials 71

3.3.2 Preparation of stock solutions 71

3.3.3 Preparation of culture media 71

3.3.4 Effect of 5mg/L of BAP on shoots production

from five potential regions of corm tissues 71

3.3.5 Multiple bud clumps (Mbcs) formation from

corm slice (number 2) with different

concentration of BAP (mg/L) 72

3.3.6 Frequency of single bud converting to shoot in

1 mg/L of BAP 72

3.3.7 Minimal inhibitory concentration of selective

agents 73

3.3.8 Statistical Analysis 73

3.4 Results and discussion 74

3.4.1 Effect of 5mg/L of BAP on shoots production

from five potential regions of corm tissues 74

3.4.2 Multiple bud clumps (Mbcs) formation from

corm slice (number 2) with different

concentration of BAP (mg/L) 77

3.4.3 Frequency of single bud converting to shoot

in 1mg/L of BAP 82

3.4.4 Determining the minimal inhibitory

concentration of selection agents 86

3.5 Conclusions 103

4 CHEMOTAXIS, ATTACHMENT AND TRANSIENT

GENE EXPRESSION IN AGROBACTERIUM

– MEDIATED BANANA TRANSFORMATION 104

4.1 Introduction 104

4.2 Objectives 108



4.3.2 Bacterial strains 109

4.3.3 Chemotaxis assays 110

4.3.4 Quantification of bacterial attachment 111

4.3.5 Transient gene expression in Agrobacterium

– mediated banana transformation 113

XVIII

4.3.6 Co-cultivation of Agrobacterium and culture

conditions 113

4.3.7 Optimization of parameters 113

4.3.8 Histochemical GUS staining and

fluorescence microscopy 114



4.4.1 Chemotaxis of Agrobacterium tumefaciens 115

4.4.2 Quantification of bacterial attachment 118

4.4.3 Preculture period 121

4.4.4 Co–cultivation period 126

4.4.5 Acetosyringone treatment 129

4.4.6 Agrobacterium strains 135

4.4.7 Wounding effect 138

4.4.8 Size of single buds 140

4.4.9 Post cultivation periods 142

4.5 Conclusions 145

5 OPTIMISATION OF PHYSICAL AND BIOLIGICAL

PARAMETERS AFFECTING TRANSIENT gus A AND gfp

GENES EXPRESSION USING PARTICLE BOMBARDMENT 146


5.2 Objectives 151


5.3.1 Establishment of explants 151

5.3.2 Plasmid DNA preparation 152

5.3.3 Plasmid constructs 153

5.3.4 Preparation of DNA and particle bombardment153

5.3.5 Histochemical GUS staining and fluorescence

microscopy 155



5.4.1 Helium pressure 156

5.4.2 Distance from stopping plate to target tissue 161

5.4.3 Vacuum pressure 163

5.4.4 Multiple bombardment 166

5.4.5 Gold microcarrier size 170

5.4.6 Explant types 172

5.4.7 Influence of explant size 177

5.4.8 Preculture treatment prior bombardment 179

5.4.9 DNA concentration 181

5.4.10 Effect of post-bombardment incubation time 185

5.5 Conclusions 187

XIX

6 GENERATION OF TRANSGENIC PISANG RASTALI VIA

AGROBACTERIUM-MEDIATED TRANSFORMATION WITH

CHITINASE and β-1,-3 GLUCANASE 188


6.2 Objectives 190


6.3.1 Plant material 191

6.3.2 Bacterial strains and binary vectors 191

6.3.3 Heat shock of Agrobacterium transformation

method 191

6.3.4 Agrobacterium cultures 192

6.3.5 Plasmid DNA purification 193

6.3.6 Explant and bacterial toxic effects of

carbenicillin and cefotaxime 194

6.3.7 Transformation protocols of single meristematic

buds 194

6.3.8 Total genomic DNA extraction 195

6.3.9 Polymerase chain reactions (PCR) 197

6.3.10 Southern blot analyses 199

6.3.11 Extraction and determination of total

acid soluble proteins 202

6.3.12 Chitinase activity assay 202

6.3.13 β-1,3-glucanase activity assay 203



6.4.1 Toxicity of carbenicillin and cefotaxime on

single buds and Agrobacterium growth 205

6.4.2 Transformation of single meristematic buds

and plant regeneration 210

6.4.3 Analysis of T-DNA integration via PCR

amplification 218

6.4.4 Southern blot hybridization analysis 223

6.4.5 Analysis of chitinase and β-1,3 glucanase

protein production in transgenic plantlets 227

6.5 Conclusions 231

7 PARTICLE BOMBARDMENT-MEDIATED CO-

TRANSFORMATION OF CHITINASE AND β-1,-3

GLUCANASE GENES 232


7.2 Objectives 236



7.3.2 Plasmid constructs 237

XX

7.3.3 Plasmid DNA preparation 238

7.3.4 Preparation of DNA and particle bombardment239

7.3.5 Co-bombardment 240

7.3.6 Selection and regeneration procedure 242

7.3.7 Histochemical GUS Staining 242

7.3.8 Visualisation of GFP 242

7.3.9 Total genomic DNA extraction 243

7.3.10 Polymerase chain reactions (PCR) 244

7.3.11 Southern blot analyses 246

7.3.12 Extraction and determination of total acid

soluble proteins 250

7.3.13 Chitinase activity assay 250

7.3.14 β-1,3-glucanase activity assay 251



7.4.1 Evaluation of GFP and GUS constructs as

reporter gene using co-bombardment 252

7.4.2 Selection of stable transformation 257

7.4.3 PCR analysis of transformants 271

7.4.4 Genomic southern blot hybridisation analysis 279

7.4.5 Analysis of chitinase and β-1,3 glucanase

protein production in transgenic plantlets 288

7.5 Conclusions 292

8 BIOASSAY METHOD FOR TESTING FUSARIUM WILT

DISEASE TOLERANCE IN TRANSGENIC BANANA 293


8.2 Objectives 298


8.3.1 Source of fungal isolate 298

8.3.2 Induction of sporulation 299

8.3.3 Spore production and germination test 299

8.3.4 Planting materials 299

8.3.5 The development of the Fusarium wilt bioassay300

8.3.6 Extraction and determination of total basic


8.32.7 Extraction and determination of total acid


8.3.8 Chemical compound and enzymatic analysis 303

8.3.9 Statistical analysis 307


8.4.1 Induction of sporulation and germination of

Fusarium oxysporum f.sp. cubense race 1

(FocR1) 308

XXI

8.4.2 Effect of inoculum density of Fusarium

oxysporum f.sp. cubense (Race 1) on the

magnitude of infection in control and

transgenic Pisang Rastali (AAB) plantlets 310

8.4.3 Hydrogen peroxide (H2O2) 313

8.4.4 Phenylalanine ammonia lyase (PAL) 317

8.4.5 Transgenic plantlets obtained from

Agrobacterium-mediated transformation 321

8.4.6 Transgenic plantlets obtained from particle

bombardment-mediated transformation 324

8.4.7 Comparison of chitinase, β -1,3-glucanases,

peroxidase (PER) and polyphenol oxidase

(PPO) activities during pre and post

inoculation with Fusarium oxysporum f.sp

cubense race 1 (FocR1) in transgenic banana

plantlets 327

8.5 Conclusions 334

9 GENERAL CONCLUSIONS AND PERSPECTIVES 335

BIBLIOGRAPHY 340

APPENDICES 401

A 402

B 403

C 405

BIODATA OF THE AUTHOR 409

Documents

By SREERAMANAN SUBRAMANIAM - Universiti …psasir.upm.edu.my/514/1/549738_FBSB_2005_18.pdfBy SREERAMANAN SUBRAMANIAM ... effect of various sizes of explants, ... menggunakkan kaedah