Molecular cytogenetic studies and technology development for creating aroid (Araceae) asymmetric somatic hybrids Prabhu Shankar Lakshmanan Know the self to be sitting in the chariot, the body to be the chariot, the intellect the charioteer, and the mind the reins. (Katha Upanishad, ~ 1400 BC) Promoters: Prof. Erik Van Bockstaele Department of Plant Production Faculty of Bioscience Engineering Ghent University, Belgium Prof. Ludmila Khrustaleva Center of Molecular Biotechnology Department of Genetics and Biotechnology Russian State Agrarian University-Timiryazev Agricultural Academy, Russia Dean: Prof. Guido Van Huylenbroeck Rector: Prof. Anne De Paepe Molecular cytogenetic studies and technology development for creating aroid (Araceae) asymmetric somatic hybrids Prabhu Shankar Lakshmanan Thesis submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Applied Biological Sciences The title in Dutch: Moleculair cytogenetische studies en technologieontwikkeling voor het creëren van asymmetrische somatische hybriden binnen de aronskelkfamilie (Araceae) Cover pictures: A Zantedeschia aethiopica ‘spadix’ covered by colorless ‘spathe’ as an example for aroid flower morphology, a DAPI stained chromosome spread of Spathiphyllum wallisii, a mix of FDA stained Z. rehmanii (green) and RITC stained S. wallisii (red) protoplasts prior to fusion and DAPI stained micronucleated S. wallisii microspores. All the microscopic pictures were captured under a fluorescence microscope using UV irradiation. To refer to this thesis: Lakshmanan, P. S. (2013). Molecular cytogenetic studies and technology development for creating aroid (Araceae) asymmetric somatic hybrids. Ph.D. thesis. Faculty of Bioscience Engineering, Ghent University, Belgium. ISBN: 978-90-5989-661-1 The author and promoters give the permission to consult and copy parts of the work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in the work should be obtained from the author. Jury members Promoter: Prof. Erik Van Bockstaele Department of Plant Production Faculty of Bioscience Engineering Ghent University, Belgium Co-promoter: Prof. Ludmila Khrustaleva Center of Molecular Biotechnology Department of Genetics and Biotechnology Russian State Agrarian University-Timiryazev Agricultural Academy, Russia Board of examiners: Prof. Els Van Damme Department of Molecular Biotechnology Faculty of Bioscience Engineering Ghent University, Belgium Prof. Stefaan Werbrouck Department of Applied Biosciences Faculty of Bioscience Engineering Ghent University, Belgium Prof. Marie-Christine Van Labeke Department of Plant Production Faculty of Bioscience Engineering Ghent University, Belgium Dr. Pascal Geerts Department of Life Science, Bio-engineering Walloon Agricultural Research Center Gembloux, Belgium Dr. Tom Eeckhaut Institute for Agricultural and Fisheries Research Plant Sciences Unit-Applied Genetics and Breeding Melle, Belgium Preface It was dusk on the Graslei. I saw people enjoying their talks, drinks and puffs. Watching that scene made me happy. I strolled through the city streets and ate a warm waffle... So began my first day in Ghent. I loved it then and have loved it ever since. In October 2009, under the magical influence of those enchanting moments in Ghent, I officially started my joint PhD between UGent and Moscow State Agrarian University. I joined ILVO’s ‘protoplast team’ with Tom Eeckhaut, Dieter Deryckere and Ronald van den Oord. Ronald was patient when teaching me in vitro techniques. Technical discussion with Dieter and Ronald about improving protocols helped me work more efficiently. To my surprise, even Pepijn De Raeymaecker (who seemed to be cold at first) started to warm up to me. Joking and laughing with Dieter and Pepijn was fun. I went to Moscow in 2010 for cytogenetic experiments as a part of this PhD program. There I worked with a Russian version of Dieter and Pepijn - Michel Bodelin and Dimitry Romanov. (When I came here for a PhD, I sure didn’t expect my life to be filled with this kind of people! A Mediterranean version of these guys would be Panagiotis Theocharis and Luca Pipino; the Indian version would be Hemant Pratap Singh and Joji John.) I thank you all for the fun we had! The city of Moscow was pleasant to explore and our other colleagues were very warm- hearted. During the winter of 2011, I was “cold-treated” and sent back to Belgium. Now when I miss Moscow, I just go to the big freezer at ILVO and stay there for a while, full of nostalgia. I am very lucky to have marvelous teachers, tuition masters, mentors, guides, yoga masters and supporters. I have always wondered how they could help a person without having any expectations and with such great patience. I remember every one of you and thank you all. In particular, I thank Prof. Van Bockstaele for his supervision. I thank Prof. Khrustaleva for her guidance and her efforts to start this collaboration in spite of all the tough administrative procedures. I also thank the board of examiners: Prof. Van Damme, Prof. Werbrouck, Prof. Van Labeke and Dr. Geerts for their valuable comments on this doctoral thesis. My most heartfelt thanks go to Tom for everything he has taught me and his fine mentorship throughout my stay at ILVO. Special thanks go to Johan Van Huylenbroeck for his training on critical writing and guidance on publishing and presenting the results. I would like to thank Katrijn Van Laere and Leen Leus for their help in cytogenetic and cytological analysis, Jorien Oomen for her technical assistance in cytogenetic experiments and Miriam Levenson for her proof reading efforts. I am also grateful to Kristiaan Van Laecke, Emmy Dhooghe, Prof. Elena Kalashnikova, Prof. Alexander Soloviev, Irina Lapitskaya, Ilya Kirov, Laurence Desmet, Anne-Marie Van Exter, Kristien Janssens and all of my other colleagues from ILVO and Moscow State Agrarian University. vii Fortunately or unfortunately, I also have to thank the UZ hospital emergency units, orthopedics and general practitioners for treating me after the multiple accidents and illnesses I suffered during my stay in Belgium. My friends (Mary, Arthi, Linda, Vasia, Vidhya, Fenila, Jacob, Karthik, Giacomo,…) were always there for me whenever I was in need. Thanks for the food you cooked for me when I was sick, even when your food made me sicker . It was the thought that counted… I give thanks to my mother for teaching me feelings and emotions. I am grateful to my father for showing me how to be straight-forward and honest. My three sisters have cared for me to the extreme, which has spoilt me rotten. I thank them for their continued love and care. I love my mom, I like my dad and I adore my sisters. My nephews, nieces, cousins and my brothers-in-law love me the most and I love them too. Some wise old men have told me that I am always surrounded by the best, especially when it comes to people. They were right. I give thanks and respect to these wise elders who have shown up out of nowhere to give me the taste to appreciate life, my own being and my connection to nature. The journey has been wonderful so far and will continue to be! 25∙11∙2013, Melle, Belgium.