Unicentre CH-1015 Lausanne http://serval.unil.ch Year : 2019 CHARACTERIZATION OF BRACHYPODIUM DISTACHYON ROOT DEVELOPMENT Van Der Schuren Alja Van Der Schuren Alja, 2019, CHARACTERIZATION OF BRACHYPODIUM DISTACHYON ROOT DEVELOPMENT Originally published at : Thesis, University of Lausanne Posted at the University of Lausanne Open Archive http://serval.unil.ch Document URN : urn:nbn:ch:serval-BIB_9493436ADAFB5 Droits d’auteur L'Université de Lausanne attire expressément l'attention des utilisateurs sur le fait que tous les documents publiés dans l'Archive SERVAL sont protégés par le droit d'auteur, conformément à la loi fédérale sur le droit d'auteur et les droits voisins (LDA). A ce titre, il est indispensable d'obtenir le consentement préalable de l'auteur et/ou de l’éditeur avant toute utilisation d'une oeuvre ou d'une partie d'une oeuvre ne relevant pas d'une utilisation à des fins personnelles au sens de la LDA (art. 19, al. 1 lettre a). 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I Département de Biologie Moléculaire Végétale CHARACTERIZATION OF BRACHYPODIUM DISTACHYON ROOT DEVELOPMENT Thèse de doctorat ès sciences de la vie (PhD) présentée à la Faculté de biologie et de médecine de l’Université de Lausanne par Alja VAN DER SCHUREN Master Biotechnology de Wageningen University and Research Jury Prof. Paul FRANKEN, Président Prof. Christian S. HARDTKE, Directeur de thèse Prof. Julia SANTIAGO CUELLAR, expert Prof. Lars ØSTERGAARD, expert Lausanne 2019 I I Département de Biologie Moléculaire Végétale CHARACTERIZATION OF BRACHYPODIUM DISTACHYON ROOT DEVELOPMENT Thèse de doctorat ès sciences de la vie (PhD) présentée à la Faculté de biologie et de médecine de l’Université de Lausanne par Alja VAN DER SCHUREN Master Biotechnology de Wageningen University and Research Jury Prof. Paul FRANKEN, Président Prof. Christian S. HARDTKE, Directeur de thèse Prof. Julia SANTIAGO CUELLAR, expert Prof. Lars ØSTERGAARD, expert Lausanne 2019 I I Table of Contents TABLE OF CONTENTS ...................................................................................................................... I ACKNOWLEDGEMENTS ................................................................................................................. II ABSTRACT ........................................................................................................................................ III RÉSUMÉ ............................................................................................................................................... V RÉSUMÉ VULGARISÉ - CARACTÉRISATION DU DÉVELOPPEMENT RACINAIRE CHEZ BRACHYPODIUM DISTACHYON .................................................................................................. VII 1. INTRODUCTION ....................................................................................................................... 1 1.1 AN INTRODUCTION TO PLANT DEVELOPMENT .................................................................................. 1 1.2 DICOTYLEDON VERSUS MONOCOTYLEDON ROOT DEVELOPMENT ................................................. 2 1.3 BRACHYPODIUM DISTACHYON AS A MONOCOTYLEDON MODEL SPECIES ....................................... 3 1.4 THE PLANT HORMONE AUXIN (IAA) ................................................................................................... 6 1.5 THE AUXIN IMPORTER AUX1 ............................................................................................................... 7 1.6 AUXIN SIGNALING IN MONOCOTS AND DICOTS .................................................................................. 9 1.7 THE IMPORTANCE OF PHLOEM FOR THE PLANT ............................................................................. 11 1.8 REGULATORS INVOLVED IN THE DISTURBED PROTOPHLOEM SYNDROME ................................ 14 1.9 TOOLS FOR RESEARCH IN MONOCOTYLEDON PROTOPHLOEM DEVELOPMENT ......................... 21 1.10 RESEARCH OUTLINE ......................................................................................................................... 24 2. HOW TO WORK WITH BRACHYPODIUM DISTACHYON ............................................. 26 2.1 GROWTH CONDITIONS: FROM SEED TO NEXT GENERATION SEEDS ............................................ 26 2.2 CROSSES ................................................................................................................................................ 29 2.3 TRANSFORMATION OF IMMATURE EMBRYOS WITH AGROBACTERIUM TUMEFACIENS ............. 30 2.4 PCR OR GENOTYPING .......................................................................................................................... 35 2.5 OBSERVATION OF BRACHYPODIUM ROOT VIA MICROSCOPY ......................................................... 37 2.6 TRANSVERSAL SECTIONING OF EMBEDDED ROOTS ....................................................................... 39 2.7 IN SITU HYBRIDIZATION ...................................................................................................................... 40 3. BROAD SPECTRUM DEVELOPMENTAL ROLE OF BRACHYPODIUM AUX1 ........... 48 3.1 FOLLOW-UP EXPERIMENTS .................................................................................................................... 57 4. DEVELOPMENT OF A FUNCTIONAL CRISPR-CAS GENOME EDITING SYSTEM FOR BRACHYPODIUM DISTACHYON ........................................................................................ 61 4.1 OPTIMIZING CRISPR-CAS ................................................................................................................. 61 4.1 BRX ........................................................................................................................................................ 63 4.2 OPS ........................................................................................................................................................ 65 4.3 BRI1 ...................................................................................................................................................... 66 4.4 APL, CLE45 AND BAM3 .................................................................................................................. 68 4.5 OFF-TARGET ANALYSIS ....................................................................................................................... 69 5. DISCUSSION AND FUTURE PERSPECTIVES .................................................................. 70 6. REFERENCES .......................................................................................................................... 80 7. SUPPLEMENTARY DATA .................................................................................................... 90 7.1 THE EFFECTS OF HIGH STEADY STATE AUXIN LEVELS ON ROOT CELL ELONGATION IN BRACHYPODIUM ............................................................................................................................................... 90 7.2 MATERIALS AND METHODS .................................................................................................................. 106 7.3 SUPPLEMENTARY FIGURES ................................................................................................................... 108 7.4 SUPPLEMENTARY TABLES .................................................................................................................... 109 7.5 MEDIA USED DURING THIS THESIS ...................................................................................................... 110 7.6 SEQUENCES USED DURING THIS THESIS ............................................................................................. 116 I II Acknowledgements I would like to thank Professor Christian S. Hardtke for the opportunity to do my PhD in his laboratory. Also the other Hardtke members for their support, the great atmosphere and the helpful discussions. Especially Amelia Amiguet Vercher for her help in working with Brachypodium distachyon. Also Dr. Pauline Anne and Dr. Mortiz Graeff for their excellent comments on my thesis report. Special thanks to Amandine Masson, Matthieu Leclerc and my family for mental support. Many thanks to Chulmin Kim and the Dolan laboratory for teaching me how to perform crosses in Brachypodium and Zhongjuan Zhang and Cecilia Aliaga for their input in in situ hybridizations. Furthermore I would like to thank our collaborators in the Karin Ljung laboratory and Markus Pauly laboratory and last but not least, the thesis committee for taking the time to read this report and commenting on my work. II III Abstract Crops like maize, rice and wheat are economically of high importance, however current yield will not sustain the world’s demands in the long run. Plant roots are crucial for uptake and transport of minerals, hormones and water via their vasculature and are therefore of interest for yield improvement.