CITRUS BIOTECHNOLOGY IN THE 21ST CENTURY By SHAUN PAUL JENSEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017 0 © 2017 Shaun Jensen To my wife, daughter, and mother ACKNOWLEDGMENTS I thank my wife for all the time, effort, and love she has spent in helping me get to this point in my career and education. I thank my parents for always being supportive of my decisions and my life goals. I thank my all my sisters for helping me learn valuable life skills without having to experience them. Truly, my whole family is wonderful. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 Citrus on the Global Stage ...................................................................................... 17 Citrus Characteristics .............................................................................................. 19 Citrus Plant Structure ....................................................................................... 19 Citrus Flowering: Timing, Morphology, and Juvenility ...................................... 20 Citrus Fruits and Seed Development ................................................................ 21 Primary Citrus Diseases ......................................................................................... 22 Citrus Greening/HLB ........................................................................................ 23 Citrus Canker ................................................................................................... 25 Citrus Black Spot and Sweet Orange Scab ...................................................... 25 Citrus Technologies: A Response to Citrus Diseases and Industry Concerns ........ 27 Conventional Breeding of Genetic Improvement in Citrus ................................ 28 Citrus Transformation ....................................................................................... 29 New and Prospective Citrus Biotechnologies ................................................... 32 2 CELL-PENETRATING PEPTIDES: A MOLECULAR DELIVERY SYSTEM IN CITRUS .................................................................................................................. 33 CPP Literature Review............................................................................................ 33 Characteristics of Cell-Penetrating Peptides .................................................... 33 Cell-penetrating peptide types ..............................................................................34 Cell-penetrating peptide uses ...............................................................................35 Translocation Mechanisms of Cell-Penetrating Peptides ................................. 36 Direct translocation ..............................................................................................36 Macropinocytosis .................................................................................................37 Receptor-mediated endocytosis ...........................................................................37 Cargoes of Cell-Penetrating Peptides for Use in Plants ................................... 38 Materials and Methods............................................................................................ 40 Materials ........................................................................................................... 40 Cell-penetrating peptides .....................................................................................40 Genetic material and sequence data ....................................................................40 Citrus cultivars and other plant tissue used ..........................................................41 Methods............................................................................................................ 42 Large scale purification of plasmid DNA ...............................................................42 5 CPP protein delivery in citrus ...............................................................................44 CPP transient expression method in citrus ...........................................................44 CPP transformation protocol ................................................................................45 CPP tracking in citrus ...........................................................................................46 Development of a relative GUS scale ...................................................................46 Results and Discussion........................................................................................... 48 Protein Delivery ................................................................................................ 48 Systemic Tracking of CPPs .............................................................................. 49 CPP-mediated Transient Expression of Plasmid DNA ..................................... 50 Cell-Penetrating Peptide Conclusions .................................................................... 53 Tables ..................................................................................................................... 55 Figures .................................................................................................................... 58 3 NANOPARTICLES IN CITRUS: A TARGETED DELIVERY SYSTEM.................... 69 Nanoparticle Literature Review ............................................................................... 69 Materials and Methods............................................................................................ 73 Materials ........................................................................................................... 73 Synthesis of Polysuccinimide (PSI) .................................................................. 73 Preparation of Functionalized Nanoparticles .................................................... 74 Preparation of Germination Medium for the Plant Toxicity Assay ..................... 74 Preparation of Citrus Seeds ............................................................................. 74 Culture of Citrus Seeds .................................................................................... 75 Preparation of MSBC Medium .......................................................................... 75 Toxicity Assessment by Tissue Culture ............................................................ 76 Results and Discussion........................................................................................... 76 pH Responsiveness of Nanoparticles ............................................................... 76 Plant Toxicity .................................................................................................... 78 Plant Cell Viability Assay .................................................................................. 79 Nanoparticle Conclusions ....................................................................................... 80 Figures .................................................................................................................... 82 4 CRISPR/CAS9 TRANSCRIPTIONAL REGULATION IN CITRUS: A MOLECULAR SWITCH FOR EARLY FLOWERING .............................................. 86 CRISPR/Cas9 Literature Review ............................................................................ 86 Genome Targeting Technologies ..................................................................... 86 Overview of the CRISPR/Cas9 System ............................................................ 88 Transcriptional Regulation of Target Genes Using CRISPR/Cas9 ................... 91 Physiology of Flowering in Citrus ..................................................................... 92 Materials and Methods............................................................................................ 93 Plant Materials .................................................................................................. 93 Plasmid Construction ....................................................................................... 93 pCAMBIA-2201-Cas9m4 and derivatives .............................................................93 Construction of pIDT:SMART::AtU6p:sgRNA::Sp_term and insertion of sgRNAs .95 Agroinfiltration of Citrus .................................................................................... 96 RNA Extraction ................................................................................................. 97 RNA Purification ............................................................................................... 98 6 Generation of cDNA ......................................................................................... 98 Quantitative Real-Time PCR (qRT-PCR) ......................................................... 98 Results and Discussion..........................................................................................