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Molecular Analyses of Salvia Africana- Lutea L. Transgenic Root Clones For MOLECULAR ANALYSES OF SALVIA AFRICANA- LUTEA L. TRANSGENIC ROOT CLONES FOR SECONDARY BIOACTIVES WATSIE PRINCESS NEO RAMOGOLA BSc (Hons) Submitted in fulfilment of the academic requirements for the degree of MASTER OF SCIENCE in the Institute for Plant Biotechnology, Faculty of AgriSciences, Department of Genetics Stellenbosch University DECEMBER 2009 i DECLARATION I, the undersigned, hereby declare that the work contained in this thesis is my own original work and that I have not previously submitted it in its entirety or in part, at any university for a degree. ____________________________________ Watsie Princess Neo Ramogola I declare that the above statement is correct. _________________________________ Doctor Nokwanda Makunga (SUPERVISOR) ______/_____/__________ Date ii DEDICATION This thesis is dedicated to my Mother; Matshediso Ramogola “Mama ke a leboga” iii SCIENTIFIC OUTPUTS (i) PUBLICATIONS IN PEER-REVIEWED JOURNALS Kamatou GPP, Makunga NP, Ramogola WPN and Viljoen AM (2008) South African Salvia species: A review of biological activities and phytochemistry. Journal of Ethnopharmacology 119: 664-672 (ii) CONFERENCE OUTPUTS Ramogola WPN , Makunga NP and van Staden J (2008) Molecular analysis of Salvia africana-lutea L. organ cultures. Joint South African Association for Botanists (34 th ) and Southern African Society for Systematic Biology Annual Congress (University of Johannesburg). South African Journal of Botany 74: 376 Makunga NP, Colling J, Horsthemke HR, Ramogola WPN and Van Staden J (2007) Changing the chemical mosaic of South African medicinal plants through biotechnological strategies. South African Association for Botanists 33 rd Annual Congress (University of Cape Town). South African Journal of Botany 73: 299 Makunga NP, Colling J, Horsthemke HR, Ramogola WPN and van Staden J (2007) Adopting biotechnological strategies-plants from South Africa as targets. PSE Congress: Plants for Human Health in the Post-Genome Era (Helsinki, Finland). iv TABLE OF CONTENTS DECLARATION ............................................................................................................... i DEDICATION ................................................................................................................. iii SCIENTIFIC OUTPUTS ................................................................................................. iv TABLE OF CONTENTS .................................................................................................. v LIST OF TABLES .......................................................................................................... ix LIST OF FIGURES .......................................................................................................... x ABBREVIATIONS......................................................................................................... xii ACKNOWLEDGEMENTS ............................................................................................. xx ABSTRAK .................................................................................................................. xxii ABSTRACT ................................................................................................................ xxv CHAPTER 1 INTRODUCTION ........................................................................................ 1 1.1 REFERENCES ...................................................................................................... 8 CHAPTER 2 LITERATURE REVIEW ............................................................................ 12 2.1 BACKGROUND INFORMATION ON GENUS SALVIA ....................................... 12 2.1.1 Botany and geographical distribution of S. africana-lutea ....................... 12 2.1.2 Biological activity of Salvia ........................................................................ 14 2.1.3 Phytochemistry of Salvia ............................................................................ 20 2.1.4 Biosynthesis of terpenes in Salvia ............................................................. 21 2.2 BIOTECHNOLOGICAL APPLICATIONS ON SALVIA SPECIES ........................ 28 2.2.1 Plant tissue culture ..................................................................................... 29 v 2.2.2 Plant tissue cultures of Salvia species ...................................................... 30 2.2.3 Recombinant DNA technology ................................................................... 42 2.2.4 Agrobacterium -mediated genetic modification ......................................... 43 2.2.5 Transgenic plants with rol genes ............................................................... 47 2.2.6 Secondary metabolism in rol -transgenic plants ....................................... 49 2.2.7 Transformation of Salvia species ............................................................... 50 2.3 CURRENT BIOTECHNOLOGICAL CHALLENGES AND FUTURE PROSPECTS .................................................................................................................................. 53 2.4 AIMS AND OBJECTIVES .................................................................................... 54 2.5 REFERENCES .................................................................................................... 55 CHAPTER 3 MICROPLANT PROPAGATION AND HAIRY ROOT CULTURE OF SALVIA AFRICANA-LUTEA ........................................................................................ 71 3.1 INTRODUCTION ................................................................................................. 71 3.1.1 In vitro plantlet culture ................................................................................... 71 3.1.2 In vitro hairy root culture ................................................................................ 72 3.2 MATERIALS AND METHODS ............................................................................ 73 3.2.1 Micropropagation of plantlets .................................................................... 73 3.2.2. Rooting of plantlets .................................................................................... 73 3.2.3. Acclimatisation of plantlets ....................................................................... 74 3.2.4 Liquid hairy root culture ............................................................................. 74 3.2.5 Effect of different basal media on hairy root growth ................................ 75 3.2.6. Data collection and statistical analyses ................................................... 75 3.3 RESULTS AND DISCUSSION ............................................................................ 76 vi 3.3.1. Micropropagation of plantlets ................................................................... 76 3.3.2. Rooting of plantlets .................................................................................... 77 3.3.3 Acclimatisation of plantlets ........................................................................ 79 3.3.4 Liquid hairy root culture ............................................................................. 80 3.3.5 Effect of different basal media on hairy root growth ................................ 81 3.4 REFERENCES .................................................................................................... 85 CHAPTER 4 MOLECULAR ANALYSIS OF S. AFRICANA-LUTEA HAIRY ROOTS ... 89 4.1 INTRODUCTION ................................................................................................. 89 4.2 MOLECULAR ANALYSIS OF HAIRY ROOTS .................................................... 90 4.2.1 Polymerase chain reaction ......................................................................... 91 4.2.2 Southern hybridisation ............................................................................... 92 4.3 MATERIALS AND METHODS ............................................................................ 93 4.3.1 Genomic DNA extraction from hairy roots ................................................ 93 4.3.2 PCR amplification of rol and ags genes in the root DNA .......................... 96 4.3.3 Probe preparation and labelling ................................................................. 99 4.3.4 Southern hybridisation analysis .............................................................. 100 4.4 RESULTS AND DISCUSSION .......................................................................... 102 4.4.1 Genomic DNA isolation ............................................................................. 102 4.4.2 Optimisation of the PCR amplification ..................................................... 106 4.4.3 Validation of trangenesis of the hairy roots ............................................ 109 4.4.4 Probe preparation ..................................................................................... 110 4.4.5 Southern hybridisation ............................................................................. 111 vii 4.5 REFERENCES .................................................................................................. 112 CHAPTER 5 PHYTOCHEMISTRY AND PHARMACOLOGY OF S. AFRICANA-LUTEA CLONES ..................................................................................................................... 116 5.1 STRATEGIES FOR THE DISCOVERY OF BIOACTIVE PHYTOCHEMICALS . 116 5.2 MATERIALS AND METHODS .......................................................................... 119 5.2.1 Preparation of plant extracts (Non-volatile compounds) .......................
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