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Genetic Transformation of Groundnut for Resistance to Tikka Disease” Submitted by Mr GENETIC TRANSFORMATION OF GROUNDNUT FOR RESISTANCE TO TIKKA DISEASE MAHMOOD UL HASSAN 95-arid-52 Department of Plant Breeding and Genetics Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2013 GENETIC TRANSFORMATION OF GROUNDNUT FOR RESISTANCE TO TIKKA DISEASE by MAHMOOD UL HASSAN (95-arid-52) A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Breeding and Genetics Department of Plant Breeding and Genetics Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2013 ii CERTIFICATION I hereby undertake that this research is an original one and no part of this thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences. Student’s Name: Mahmood ul Hassan Signature: ___________________ Registration No: _____95-arid-52_____ Date: _______________________ Certified that the contents and form of thesis entitled “Genetic Transformation of Groundnut for Resistance to Tikka Disease” submitted by Mr. Mahmood ul Hassan have been found satisfactory for the requirement of the degree. Supervisor: ___________________________ (Dr. Zahid Akram) Co-Supervisor: ___________________________ (Dr. Yusuf Zafar) Member: __________________________ (Dr. Ghulam Shabbir) Member: __________________________ (Dr. Tariq Mukhtar) Chairperson: _________________________ Dean: _______________________________ Director, Advanced Studies: _____________________________ iii "In the Name of Allah, the most Beneficent, the most Merciful" iv DEDICATED TO UNFATHOMABLE LOVE OF MY PARENTS v CONTENTS Page List of Tables xi List of Figures xiii List of Abbreviations xvi Acknowledgements xviii ABSTRACT 1 1 GENERAL INTRODUCTION 3 2 IN VITRO REGENERATION FROM COTYLEDONS 7 2.1 INTRODUCTION 7 2.2 REVIEW OF LITERATURE 9 2.3 MATERIALS AND METHODS 12 2.3.1 Explant Preparation 12 2.3.2 Data analysis 14 2.4 RESULTS AND DISCUSSION 14 2.4.1 Number of Responding Explants (%) 14 2.4.2 No. of shoots/responding explants (%) 15 2.4.3 Rooting percentage 15 3 STANDARDIZATION OF IN VITRO CULTURE SYSTEM FROM 26 LEAF DISCS 3.1 INTRODUCTION 26 3.2 REVIEW OF LITERATURE 27 3.3 MATERIALS AND METHODS 30 3.3.1 Explant preparation 30 vi 3.3.2 Medium and Culture Conditions 31 3.3.3 Data analysis 33 3.4 RESULTS AND DISCUSSION 33 3.4.1 Number of Responding Explants (%) 33 3.4.2 No. of shoots/responding explants 34 3.4.3 Rooting percentage 34 4 STANDARDIZATION OF IN VITRO REGENERATION VIA CALLUS 41 INDUCTION 4.1 INTRODUCTION 41 4.2 REVIEW OF LITERATURE 42 4.3 MATERIALS AND METHODS 45 4.3.1 Explant Preparation 45 4.3.2 Culture Medium and Conditions 46 4.3.3 Data Analysis 46 4.4 RESULTS AND DISCUSSION 47 4.4.1 Number of Responding Explants (%) 47 4.4.2 Number of Differentiated Embryos/ Responding Explant 48 4.4.3 Germination Percentage of Embryos 48 5 AGROBACTERIUM MEDIATED TRANSFORMATION 55 5.1 INTRODUCTION 55 5.2 REVIEW OF LITERATURE 56 5.3 MATERIALS AND METHODS 72 5.3.1 Explant Preparation 72 5.3.2 Bacterial Strain and Vector 73 vii 5.3.3 Determination of Lethal Dose of Hygromycin for In Vitro 73 Grown Shoots 5.3.4 Selection and Rooting of Putative Transformed Shoots 74 5.3.5 PCR Analysis of Putative Transgenic Plants 74 5.3.6 Gel Electrophoresis 75 5.3.7 Southern Blot 75 5.3.8 Determination of Lethal Dose of Hygromycin for Field Grown 77 T1 Plants 5.3.9 Selection of T1 Plants 77 5.3.10 Pathogenicity Test 77 5.3.11 RT-PCR Analysis 78 5.4 RESULTS AND DISCUSSION 78 5.4.1 Effect of Co-Cultivation Period and Acetosyringone 78 Concentration 5.4.1.1 Co-cultivation Period 78 5.4.1.2 Acetosyringone 79 5.4.2 Determination Of Lethal Dose Of Hygromycin For In Vitro 80 Grown Shoots 5.4.3 PCR analysis of Putative Transgenic Plants 80 5.4.4 Southern Blot 80 5.4.5 T1 Generation 80 5.4.6 Determination of Lethal Dose of Hygromycin for Field Grown 85 Plants 5.4.7 Selection of T1 Plants 85 viii 5.4.8 Pathogenicity Test for Intact Plants 85 5.4.9 RT-PCR Analysis 85 6 SILICON CARBIDE WHISKER MEDIATED TRANSFORMATION 91 6.1 INTRODUCTION 91 6.2 REVIEW OF LITERATURE 92 6.3 MATERIALS AND METHODS 96 6.3.1 Production of Embryogenic Callus 96 6.3.2 Preparation of Silicon Carbide Whiskers and DNA Delivery 96 6.3.3 Selection for Stable Transformants 96 6.3.4 PCR analysis 97 6.3.5 Selection of T1 Plants 97 6.3.6 Pathogenicity test for intact plants 97 6.3.7 RT-PCR analysis 98 6.4 RESULTS AND DISCUSSION 98 6.4.1 T0 generation 100 6.4.2 T1 generation 100 6.4.3 Pathogenicity test 100 6.4.4 RT-PCR analysis 104 7 TRANSFORMATION USING GENE GUN 106 7.1 INTRODUCTION 106 7.2 REVIEW OF LITEATURE 108 7.3 MATERIALS AND METHODS 111 7.3.1 Production of Embryogenic Callus 111 7.3.2 Preparation of Micro-carriers 112 ix 7.3.3 Coating DNA onto Micro Particles 112 7.3.4 Selection of Transformants 113 7.3.5 PCR analysis 113 7.3.6 Selection of T1 Plants 114 7.3.7 Pathogenicity test for intact plants 114 7.3.8 RT-PCR analysis 114 7.4 RESULTS AND DISCUSSION 115 7.4.1 T0 generation 115 7.4.2 T1 generation 117 7.4.3 Pathogenicity test 117 7.4.4 RT-PCR analysis 120 8 GENERAL DISCUSSION 124 SUMMARY 135 LITERATURE CITED 139 x LIST OF TABLES Table No. Page 2.1 Treatments (Hormone Combination Used) 16 2.2 Hormone concentrations for root induction 16 2.3 Analysis of variance for number responding explants (%) 16 2.4 Analysis of variance for No. of shoots/responding explants (%). 17 2.5 Analysis of variance for rooting percentage 19 2.6 Duncan's Multiple Range Test for ranking of treatments with 20 respect to rooting percentage 2.7 Duncan's Multiple Range Test for ranking of varieties with rooted 20 plants/explant (% 3.1 Treatments (Hormone Combination Used) 32 3.2 Analysis of variance for number responding explants (%) 35 3.3 Analysis of variance for No. of shoots/responding explants 37 3.4 Analysis of variance for rooting percentage 38 3.5 Duncan's Multiple Range Test for ranking of treatments with 38 respect to rooting percentage 3.6 Duncan's Multiple Range Test for ranking of varieties with rooting 38 percentage. 4.1 Analysis of variance for number responding explants (%) 49 4.2 Analysis of variance for No. of embryos/responding explants 50 4.3 Analysis of variance for germination percentage 52 4.4 Duncan's Multiple Range Test for ranking of treatments with 52 respect to germination percentage xi 4.5 Duncan's Multiple Range Test for ranking of varieties with respect 52 to germination percentage 5.1 Quantities of different reagents in PCR mixture 76 5.2 Analysis of variance for factors affecting transformation efficiency 81 5.3 Mean values of transformation efficiency at different co- 81 cultivation periods ranked by Duncun’s Multiple Range Test 5.4 Mean values of transformation efficiency at different 82 acetosyringone concentrations ranked by Duncun’s Multiple Range Test 5.5 Table of means for Infection frequency (IF, number of lesions/cm2 85 leaf area), incubation period (IP, number of days from inoculation to appearance of first lesion), lesion diameter (LD), leaf area damage (LAD) and disease score of transgenic and control plant 5.6 Modified 9-point scale used for screening groundnut genotypes for 90 resistance to late leaf spot disease 6.1 Analysis of variance for factors affecting transformation efficiency 99 7.1 Analysis of variance for factors affecting transformation efficiency 116 7.2 Mean values of transformation efficiency at different distances as 116 ranked by Duncan's Multiple Range Test 7.3 Mean values of transformation efficiency at different particle size 116 as ranked by Duncan's Multiple Range Test xii LIST OF FIGURES Fig. No Page 2.1 Duncan's Multiple Range Test for ranking of interaction means with 17 respect to number of responding explants (%). 2.2 Duncan's Multiple Range Test for ranking of interaction means with 19 respect to No. of shoots/responding explants (%) 2.3 Bunch of spontaneous roots emerged from point of contact with 20 solid surface due to mechanical stress 2.4 Grafting in groundnut 23 2.5 Different steps in peanut tissue culture 24 3.1 Duncan's Multiple Range Test for ranking of interaction means with 35 respect to number of responding explants (%) 3.2 Duncan's Multiple Range Test for ranking of interaction means with 37 respect to No. of shoots/responding explants 3.3 Different steps of shoot induction from leaf discs 40 4.1 Duncan's Multiple Range Test for ranking of interaction means with 49 respect to number of responding explants (%) 4.2 Duncan's Multiple Range Test for ranking of interaction means with 50 respect to No. of embryos/responding explants 4.3 Different steps in peanut somatic embryogensis 54 5.1 Diagram for vector containing RCG-3 under EN4 promoter 76 5.2 Effect of acetosyringone and length of co-cultivation period on 82 production of hygromycin resistant shoots (%) xiii 5.3 Amplification of RCG 3 gene fragment by PCR in 4 surviving T0 83 plants. 5.4 Southern blot analysis of four surviving T0 plants 83 5.5 Cut twigs of plants on different concentrations of hygromycin 84 5.6 Amplification of RCG 3 gene fragment by PCR in T1 plants 84 5.7 Pathogenicity test of intact plants for susceptibility to late leaf spot 87 disease 5.8 Means for Infection frequency, incubation period, lesion diameter, 87 leaf area damage and disease score of transgenic and control plants 5.9 RT-PCR for RCG-3 gene in T1 plants.
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