TABLE OF CONTENTS. Acknowledgements Table of contents List of abbreviations Executive summary. Chapter 1: Introduction

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1 TABLE OF CONTENTS Acknowledgements Table of contents List of abbreviations Executive summary Molecular aspects of drought tolerance Objectives and major results Tobacco Potatoes Cotton Maize Conclusion Recommendation for future research Technology transfer Publication list from drought research Postgraduate studies MSc. dissertations Ph.D. theses Research papers Conference proceedings Papers Posters iii iv x xiv xv xvi xvii xviii xvix ii ii v Chapter 1: Introduction 1.1 Effect of drought Literature survey Physiological processes involved Screening methods Stress related protein synthesis Abscisicacid Chlorophyll fluorescence 8 IV

2 Enzymes from the antioxidative system Changes in free proline concentrations and polyamine levels ,3,5-Triphenyltetrazolium chloride reduction Enhanced drought tolerance in transgenic plants Differential screening RNA isolation, cdna library construction and differential plaque cultivarization Sequence analysis of two of the drought-related clones Characterisation of isolated genes Two-cultivar analysis of water-stress induced genes from maize Principle of the two-cultivar assay 21 Chapter 2: Materials and Methods 2.1 Plant material Potatoes Cotton Tobacco Maize Screening methods Drought related protein synthesis Heat shock protein synthesis Protein extraction and autoradiography Protein-DNA binding study Efficiency of protein synthesis Incorporation of 14 C-proline Water gradient study Soil water potential and dry weight measurement in maize Water relations Proline and abscisic acid Chlorophyll fluorescence Leaf water potential Cu/Zn superoxide dismutase, glutathione reductase and ascorbate

3 Cu/Zn superoxide dismutase, glutathione reductase and ascorbate peroxidase levels Free proline determination Polyamine analysis Analysis of polyamine biosynthetic enzymes 34 ' Enzyme assays of ADC and ODC Method of estimation of ADC and ODC ,3,5-Triphenyltetrazolium chloride reduction Succinic dehydrogenase and malic dehydrogenase enzyme assays Potato transformation Maintenance of in vitro plantlets Leaf disc regeneration : Kanamycin tolerance experiments Cloning of a Arabidopsis thaliana Cu/Zn SOD cdna into transformation vectors Growth ofagrobacterium tumefaciens Leaf disk transformation and regeneration Molecular confirmation of transformed potatoes Hardening off and drought stress of transformed plants Differential screening RNA isolations and cdna synthesis cdna probe synthesis and plaque isolation Sequence analysis of two of the drought related clones Clone selection Sequencing Sequence analysis Characterisation of isolated genes RNA slot blots Probe synthesis and cultivarization Poly(A)+ RNA qualification /// situ cultivarization Two-cultivar analysis of water-stress induced genes Maize transformation Plant material Surface sterilisation of young cobs 52 VI

4 2.5.3 Initiation of embryogenic tissue Prepare embryogenic tissue for bombardment PPT selective medium Particle gun bombardment Coating of particles DNA preparation Manipulation of the superoxide dismutase gene for transformation Transformation of the maize with the plasmid pahc25sod Confirmation of transformation of pahc25sod into maize calli 55 Chapter 3. Results and Discussion 3.1 Potatoes Screening methods Drought related protein synthesis is cultivar and organ specific in potato : Chlorophyll fluorescence as measure of drought tolerance Cu/Zn superoxide dismutase, gtutathione reductase and ascorbate peroxidase levels during drought stress The effect of drought on proline and polyamine levels ,3,5-Triphenyltetrazolium chloride reduction as a measurement of drought tolerance Summary Potato transformation in an attempt to enhance drought tolerance Cu/Zn SOD activity of transformants ,3,5-Triphenyltetrazolium chloride reduction assays Conclusion Cotton Screening methods Drought related protein synthesis Analysis of heat tolerance in cotton Heat related protein synthesis Efficiency of protein synthesis 84 2 i i n i Vll

5 Viability assay Conclusion Chlorophyll fluorescence as measure of drought and heat tolerance Enzymes from the antioxidative system in response to drought and heat stress Leaf water potential The effect of drought and heat on proline levels The effect of drought and heat on polyamine levels ,3,5-TriphenyltetrazoIium chloride viability assay Summary Heritabtlity and combining ability studies in cotton Tobacco Screening methods Water gradient study as a screening method for drought tolerance C 14 - proline incorporation Chlorophyll fluorescence as a possible screening method Proline accumulation Potyamine titres during a drought treatment ,3,5-Triphenyltetrazolium chloride viability assay Conclusion Maize Screening methods Triphenyltetrazolium chloride reduction assay on maize Physiological differences between drought resistant and drought sensitive maize inbred lines Conclusion Differential screening RNA isolation, cdna library construction and differential plaque cultivarization Sequence analysis of two of the drought-related genes Characterisation of isolated genes Conclusion Two-cultivar analysis of water-stressed induced genes 137 Mil

6 3.4.3 Transformation of embryos with the Cu/Zn SOD gene Embryogenic tissue Manipulation of the superoxide dismutase gene for maize transformation Particle gun bombardment Transformation of the maize with the plasmid pahc25sod Confirmation of transformation of pahc25sod into maize calli Conclusion 147 Chapter 4: Conclusion 149 Chapter 5: References 154 IX