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Physiological and Molecular Effects of Thidiazuron and Ethylene on Leaf Yellowing and Rooting of Pelargonium (Pelargonium Zonale Hybrids) Cuttings

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Physiological and Molecular Effects of Thidiazuron and Ethylene on Leaf Yellowing and Rooting of Pelargonium (Pelargonium Zonale Hybrids) Cuttings Physiological and Molecular effects of Thidiazuron and Ethylene on Leaf Yellowing and Rooting of Pelargonium (Pelargonium zonale hybrids) Cuttings Von Naturwissenschaftliche Fakultät der Universität Hannover zur Erlangung des akademischen Grades eines Doktors der Gartenbauwissenschaften -Dr. rer. hort.- genehmigte Dissertation von Theophilus Mwendwa Mutui, M. Sc. Hort. (Nairobi) geboren am 21. May 1969 in Kitui, Kenya 2005 Referent: Prof. Dr. Margrethe Serek Korreferent: Prof. Dr. Sridevy Sriskandarajah Tag der Promotion: 14.09.2005 Dedication To my Younger Brothers, Wife, and Children to be. This thesis is made to inspire them to aspire to strive to achieve the highest level of education possible. AMEN i Acknowledgements First and foremost, I would like to extend my special thanks to Prof. Dr. Margrethe Serek, a distinguished professor in the field of post harvest physiology of ornamental crops with special interest in ethylene biology and physiology for her invaluable guidance and tireless supervision throughout the course of this study. I also thank Dr. Heiko Mibus-Schoppe for introducing me to the field of molecular biology that enabled me to gain insights into these novel and dynamic tools in all aspects of study in biology. The author expresses his sincere appreciation to other staff members of the Floriculture Section for their untiring support and technical advice on various (physiological and molecular) methods throughout the course this study. Moreover, I thank my fellow Ph. D students for providing a good working environment both in the laboratory and in our office. I would also like to thank the Vice-Chancellor, Moi University, Eldoret, Kenya for granting me study leave for four years which enabled me to successfully complete this doctoral study. Special thanks go to the German Academic Exchange Service (DAAD) for financially supporting six months’ Deutsch course and this Ph. D. project. Additionally, I would like to thank Selecta Klemm GmbH & Co. KG, Stuttgart, Germany, a commercial breeder for generously supplying Pelargonium stock plants used in the investigations reported here. My heartfelt thanks and gratitude goes to my special friend Miss Jennifer Kasanda Sesabo for the assistance she offered me with data entry into Excel computer program, support, prayers and encouragement during both the most difficult and happy times of this work. Lastly, but not least I would like to thank my parents Joseph Mutui Lemba and Esther Kanono Mutui together with my brothers for the encouragement and moral support throughout both high and low periods during this study. ii Table of Contents Acknowledgements.............................................................................................ii List of Tables..................................................................................................... iv List of Figures.................................................................................................... v List of Appendices............................................................................................. vi Abbreviations ................................................................................................... vii Abstract .............................................................................................................. x 1.0 General Introduction ...................................................................................... 1 2.0 Effects of Thidiazuron, Ethylene, Abscisic acid and dark Storage on leaf yellowing and rooting of Pelargonium zonale hybrid cuttings......................... 5 Abstract .............................................................................................................. 5 2.1 Introduction.................................................................................................. 6 2.2 Materials and Methods .............................................................................. 15 2.3 Results ........................................................................................................ 20 2.4 Discussion.................................................................................................. 35 2.5 Conclusion ................................................................................................. 41 3.0 Isolation, Characterization and Expression studies on ACC synthase and Ethylene Receptor (ETR) genes in Pelargonium ............................................ 42 Abstract ............................................................................................................ 42 3.1 Introduction................................................................................................ 43 3.2 Materials and Methods .............................................................................. 51 3.3. Results ....................................................................................................... 57 3.4 Discussion.................................................................................................. 65 3.5 Conclusion ................................................................................................. 76 Summary............................................................................................................. 77 References .......................................................................................................... 79 Appendices ......................................................................................................... 98 iii List of Tables Page Table 1. Effect of continuous exposure to 0.5, 1 and 2 µl l-1 ethylene for 4 days on root induction and growth in cuttings of 5 Pelargonium cultivars.................................................... 29 Table 2. Effect of application of 25, 50 and 100 µM ABA for 4 days on root induction and growth in cuttings of 5 Pelargonium cultivars................................................................................30 Table 3. Effect of dark storage for 4 days on root induction and growth in cuttings of 5 Pelargonium cultivars........................................................................................................................31 Table 4. Effect of application of 4, 8 and 12 µl l-1 IBA in the rooting solution for 28 days on root induction and growth for 5 Pelargonium cultivars. ................................................................33 Table 5. Effect of treating cuttings with 5 µM TDZ followed by application of 4, 8 and 12 µl l-1 IBA in rooting solution for 28 days on root induction and growth for 5 Pelargonium cultivars.. .............................................................................................................................................34 Table 6. Degenerate primer pairs (ACSd2s and ACSdas) constructed after alignments of homologous sequence areas and specific gene primer ...................................................................53 iv List of Figures Page Fig. 1. Schematic presentation of treatment application and rooting of Pelargonium cuttings19 Fig. 2. Appearance of representative Pelargonium cv ‘Katinka’ leaves after treatments.........20 Fig. 3. Effects of treatments for 4 days on leaf chroma of 5 Pelargonium cultivars. ...............21 Fig. 4. Effects of application of TDZ at 5, 10 and 20 µM for 4 days on leaf chroma, leaf hue and total leaf chlorophyll content of 5 Pelargonium cultivars.................................................23 Fig. 5. Effects of treatments for 4 days on leaf hue of 5 Pelargonium cultivars.......................24 Fig. 6. Effects of treatments for 4 days on total leaf chlorophyll content of 5 Pelargonium cultivars. ........................................................................................................................................26 Fig. 7. Appearance of representative Pelargonium cv ‘ Katinka’ roots after the cuttings were continuously exposed to various treatments for 4 days followed by application of 4 µl l-1 IBA in rooting solution for 28 days. .....................................................................................28 Fig. 8. A model for the role of ethylene receptors in ethylene signaling. ..................................46 Fig. 9. Ethylene signal transduction pathway model in Arabidopsis..........................................47 Fig. 10. Ethylene production in Pelargonium zonale ‘Katinka’ cuttings after treatments. ......57 Fig. 11. Amplification patterns of putative ACC synthase genes of Pelargonium zonale ‘Katinka’ with ACSd2 primer pair and different annealing temperatures (44oC to 50oC)....58 Fig. 12. Base pairs sequence of (A) PzACS3 and (B) PzACS4)..................................................59 Fig. 13. Schematic representation of the new Pelargonium zonale putative ACC synthase genes (PzACS3 and PzACS4). .....................................................................................................60 Fig. 14. Alignment of deduced amino acid sequences for partial putative PzACS3, PzACS4 and CsACS1...................................................................................................................................60 Fig. 15. Dendrogram of various nucleotide sequences of ACC synthase genes from different plant species. .................................................................................................................................61 Fig. 16. Expression of ACC synthase (PzACS1, PzACS2, PzACS3, PzACS4) and ETR (PzETR1 and PzETR2) mRNAs in various tissues of ‘Katinka’..............................................62
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