Transfer of rol genes and Evaluation of Artemisinin Synthesis in Transgenic Artemisia annua L. and Artemisia dubia Wall. By BUSHRA HAFEEZ KIANI Department of Biochemistry Quaid-i-Azam University Islamabad, Pakistan 2012 Transfer of rol genes and Evaluation of Artemisinin Synthesis in Transgenic Artemisia annua L. and Artemisia dubia Wall. Submitted by BUSHRA HAFEEZ KIANI Thesis Submitted to Department of Biochemistry Quaid-i-Azam University, Islamabad In the partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry Department of Biochemistry Quaid-i-Azam University Islamabad, Pakistan 2012 In the name of ALLAH who is the Beneficent and The most Merciful, Guardian of faith, The Majestic, The Bestower and The Forgiver, Whose help and guidance I always importune at every step. CERTIFICATE The department of Biochemistry, Quaid-i-Azam University, Islamabad accepts this thesis submitted by Bushra Hafeez Kiani in its present form, as satisfying the thesis requirement for the Degree of Doctor of Philosophy (Ph.D.) in Biochemistry and Molecular Biology. Supervisor: _________________ Prof. Dr. Bushra Mirza External Examiner: _________________ External Examiner: _________________ Chairman: _________________ Prof. Dr. Bushra Mirza Dated: __________________ Declaration I hereby declare that the work presented in this thesis is my own effort except where others acknowledged and that the thesis is my own composition. No part of this thesis has previously been presented for any other degree. Bushra Hafeez Kiani DEDICATION This humble effort is sincerely dedicated to My Abbu Ji whose wishes and devotions Made me capable of achieving such a Success and to my sweet, ever-loving Ammi Ji whose hands always rose for my Success, And My Family (Words are futile to that They have done for me) Contents Acknowledgements i List of Tables iii List of Figures iv List of Abbreviations x Abstract xiii Chapter 1 Introduction and review of Literature 1 1.1 Genus Artemisia, a natural source of artemisinin 2 1.1.1 Evidence of presence of artemisinin in some Artemisia 2 species other than A. annua 1.1.2 Evidence of antimalarial activity of some Artemisia species 3 other than A. annua 1.1.3 Taxonomic classification of genus Artemisia 3 1.1.4 History of Artemisia 4 1.1.5 Morphology 4 1.1.6 Distribution 6 1.2 Morphology of Artemisia annua 6 1.3 Morphology of Artemisia dubia 8 1.3.1 Selection of Artemisia dubia for artemisinin enhancement 9 1.4 Secondary metabolites and Medicinal Constituents of 10 Artemisia 1.5 Medicinal Importance of genus Artemisia 11 1.5.1 Non medicinal importance of the genus Artemisia 11 1.5.2 Toxic effects of Artemisia 12 1.6 Artemisinin 12 1.6.1 Artemisinin Synthesis and Storage 13 1.6.2 Derivatives of artemisinin 14 1.6.3 Mode of action of artemisinin 15 1.6.4 Biological activities of artemisinin 16 1.6.4.1 Malaria 17 1.6.4.2 Schistosomiasis 18 1.6.4.3 Hepatitis B 18 1.6.4.4 Cancer 19 1.6.4.4.1 Effect of artemisinin and its derivatives on different types 19 of cancer 1.6.4.5 Herbicides 20 1.7 Improvement of Artemisinin Content 20 1.7.1 Selection of best cultivar 21 1.7.2 Selection of different stages 21 1.7.3 Tissue Culture of Artemisia 22 1.7.4 Transformation 24 1.7.4.1 Agrobacterium-mediated Transformation 24 1.7.4.1.1 Classification of Agrobacterium 24 1.7.4.1.2 Agrobacterium tumefaciens 24 1.7.4.1.3 Agrobacterium rhizogenes 25 1.7.4.2 Molecular mechanism of Agrobacterium tumefacienes 26 mediated transformation 1.7.4.2.1 The binary vector strategy 28 1.7.4.2.2 The cointegration strategy 28 1.7.4.3 Production of transformed plants with Ti plasmid 29 1.7.4.4 Mechanism of A. rhizogenes infection 30 1.8 Agrobacterium tumefacienes mediated transformation in 31 Artemisia species 1.9 Agrobacterium rhizogenes mediated transformation in 32 Artemisia species 1.10 Role of rol genes in Plants 33 1.10.1 Synergistic effects of rol genes 37 1.11 Extraction of artemisinin 38 1.12 Analysis of artemisinin 39 1.13 Biosynthetic pathway of artemisinin 40 1.13.1 Post-IPP terpene biosynthesis 41 1.13.2 Committed steps in artemisinin biosynthesis 42 1.13.3 Regulation of the artemisinin biosynthetic pathway 44 1.14 Aims and objective 46 Chapter 2 Genetic Transformation of Artemisia annua and 47 Artemisia dubia with rol ABC genes through Agrobacterium tumefacienes and Agrobacterium rhizogenes 2.1 Introduction 47 2.2 Materials and methods 48 2.2.1 Glassware and chemicals 48 2.2.1.2 Medium 48 2.2.3 Inoculation area and manipulation tools 48 2.2.4 Culturing of tissues 49 2.2.5 Collection of Plant Material 49 2.2.6 Seed Germination 49 2.2.7 Culture environment 49 2.2.8 Transformation of Artemisia species with rol ABC genes 50 2.2.8.1 Agrobacterium tumefaciens mediated transformation of 50 Artemisia dubia and Artemisia annua with rol ABC Genes 2.2.8.2 Plant material 50 2.2.8.3 Bacterial strain and plasmid construction 50 2.2.9 Transformation procedure 51 2.2.9.1 Preparation of Explants 51 2.2.10 Co cultivation 51 2.2.10.1 Co cultivation medium 51 2.2.10.2 Co cultivation 51 2.2.11 Selection and regeneration 52 2.2.12 Rooting 52 2.2.13 Transplantations to pots and acclimatization 53 2.2.14 Agrobacterium rhizogenes mediated transformation of 53 Artemisia dubia and Artemisia annua with rol ABC Genes 2.2.15 In-vitro plant production and sterilization 53 2.2.16 Media used for establishment of hairy root cultures 54 2.2.16.1 Preparation of MS shooting medium 54 2.2.16.2 Preparation of ½MS rooting medium 54 2.2.16.3 Preparation of nutrient agar medium 54 2.2.16.4 Preparation of B5 solid medium 54 2.2.16.5 Preparation of B5 selection medium 54 2.2.16.6 Preparation of B5 liquid medium 54 2.2.17 Induction of Hairy Roots 55 2.2.17.1 Preparation of A. rhizogenes strain 55 2.2.17.2 A. rhizogenes infection on Artemisia annua and Artemisia 55 dubia 2.2.17.3 Transfer of hairy root cultures on B5 solid medium 55 2.2.17.4 Transfer of hairy roots in to B5 liquid medium 56 2.2.18 Molecular analysis 56 2.2.18.1 Isolation of genomic DNA from plant leaves 56 2.2.18.1.1 Composition of CTAB buffer 57 2.2.18.2 Extraction and purification of plasmid DNA 57 62.2.18.2.1 Solutions 58 2.2.19 Polymerase chain reaction 58 2.2.19.1 Primers used during PCR 59 2.2.19.2 Agarose gel electrophoresis 59 2.2.20 Southern blot analysis 59 2.2.20.1 DNA restriction 60 2.2.20.2 Agarose gel electrophoresis 60 2.2.20.3 Transfer of restriction fragments to membrane 60 2.2.20.4 Labeling of DNA 60 2.2.20.5 Hybridization process 61 2.3 Results 61 2.3.2 Seed Surface Sterilization 61 2.3.3 Medium for Seed Germination 62 2.3.4 Types of explants 65 2.3.5 Transformation 66 2.3.5.1 Effect of Co-cultivation Period on Transformation 66 2.3.5.2 Regeneration of Transgenic Plants 66 Effect of Antibiotics on Agrobacterium in Regeneration 66 2.3.5.2.1 Medium 2.3.5.2.2 Regeneration of Transgenic Plants 66 2.3.5.3 Rooting of Transformed Plants 71 2.3.6 Morphological Analysis of Transformed Plants 72 2.3.7 In-vitro plant production and sterilization 74 2.3.8 Production of Hairy Roots 75 2.3.9 Molecular Analysis of Transformed Plants 75 2.3.9.1 Genomic DNA Extraction 75 2.3.9.2 Polymerase Chain Reaction (PCR) 77 2.3.10 Southern Blotting 79 2.3.11 Acclimatization 81 2.4 Conclusion 83 Chapter 3 Analysis of Artemisinin and its Derivatives and their comparison in transformed and un-transformed plants of Artemisia annua and Artemisia dubia 3.1 Introduction 84 3.2 Materials and methods 85 3.2.1 Extraction of artemisinin 85 3.2.2 Reagents used 85 3.2.2.1 Solvents used 85 Solvent No.1: Mixture of Ethyl acetate and Hexane in the 85 3.2.2.1.1 ratio of 5:95 3.2.2.1.2 Solvent No.2: Mixture of Acetonitrile and Water in the ratio 86 of 50:50 3.2.2.1.3 Biomass and Solvent Ratio 86 3.2.3 Analysis of artemisinin content 86 3.2.3.1 High performance liquid chromatography 86 3.2.3.1.1 Preparation of mobile phase 86 3.2.3.1.2 Preparation of dilutions of standard 87 3.2.3.1.3 Column used for HPLC 87 3.2.3.1.4 Flow of mobile phase through HPLC system 87 3.2.3.1.5 Detector 87 3.2.3.1.6 Retention time of peak 87 3.2.3.1.7 Injection volume 87 3.2.3.1.8 Calibration curve 88 3.2.3.1.9 Quantification of artemisinin and its derivatives in sample 88 3.2.3.1.10 Verification of artemisinin and derivatives of artemisinin 88 concentrations 3.2.4 Statistical analysis 89 3.3 Results 89 3.3.1 Analysis of artemisinin content in transformed and 89 untransformed Artemisia annua and Artemisia dubia 3.3.2 Analysis of derivatives of artemisinin in transformed and 93 untransformed Artemisia annua and Artemisia dubia plants concentrations 3.3.3 Statistical Analysis 108 3.3.3.1 Comparative analysis of artemisinin and its derivatives in 108 different tissues of Artemisia annua and Artemisia dubia 3.4 Conclusion 109 Chapter 4 Analysis of Metabolic pathway and Trichome development and their comparison in transformed and un-transformed plants of Artemisia annua and Artemisia dubia 4.1 Introduction 111 4.2 Materials and methods 112 4.2.1 Analysis of Metabolic Pathway 112 4.2.1.1 RNA Extraction 112 4.2.1.2 cDNA synthesis 113 4.2.2 Quantitative Real Time Polymerase chain reaction (qRT- 115 PCR) 4.2.3 Analysis of Trichome density 116 4.2.3.1 Environmental Scanning Electron Microscope (ESEM) 117 setup 4.3 Results 117 4.3.1 Analysis of Metabolic pathway and Trichome development 117 4.3.2 Relative expression of genes involved in metabolic pathway 117 of artemisinin biosynthesis 4.3.3 Trichome Development 119 4.3.4 Statistical Analysis 122 4.3.4.1 Analysis of different genes involved in artemisinin 122 production pathway in different tissues of Artemisia