Propagation Studies of Sugar Maple (Acer saccharum Marsh.) BY TERESA ALEXANDRA SUTANTO A Thesis Submitted to the Faculty of Graduate Studies in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Plant Science University of Manitoba Winnipeg, Manitoba © Copyright by Teresa Alexandra Sutanto 2010 ii ACKNOLEDGEMENTS I would like to acknowledge the help of many people during my graduate study: Dr. Claudio Stasolla for his encouragement, guidance and support throughout the program. Dr. Philip Ronald and Dr. Muhammad Tahir, for serving on my advisory committee and Dr. Ronald for his time and guidance, particularly on the cutting experiment. Dr. Rob Gulden and Dr. Gary Crow for the generous time helping me on the statistical analysis of the data. Bert Luit for the assistance in the lab and the companion during the trips to Morden and Portage La Prairie for sample collections. Jeffries Nurseries Ltd. for supplying the plant materials for the project. Rob and Sue MacLeod of Glenlea Greenhouses and Dave Klassen of Shannon Oaks Tree Farm for providing their time and for contributing space in their greenhouses for the cutting experiment. Kevin Baron for helping with seed collection, Mohamed Elhiti for teaching the techniques for cell fixation and staining, and every lab members for making the lab enjoyable! Lorne Adam for the valuable insights and help on the digital image analysis using the computer software. Ian Brown for the setting the propagation box and helping with selecting rooting medium for the cutting experiment. Rhanissa Hirawan for drawing the illustration materials used in the thesis. Fellow graduate students for their friendship and support. Committee members of Agri-Food Research and Development Initiative (ARDI) for the financial support, as well as Province of Manitoba for awarding Manitoba Graduate Scholarship. And lastly, my parents, Hartono Sutanto and Fonny Hadi Gunawan, and my brother, Vito, for their continued support and love. Thanks to you all! iii TABLE OF CONTENTS ACKNOLEDGEMENTS .................................................................................................................................... ii LIST OF TABLES .............................................................................................................................................. v LIST OF FIGURES ........................................................................................................................................... vi LIST OF ABBREVIATIONS ............................................................................................................................ viii ABSTRACT ..................................................................................................................................................... ix 1.0 INTRODUCTION ................................................................................................................................. 1 2.0 LITERATURE REVIEW ......................................................................................................................... 4 2.1 Introduction to sugar maple (Acer saccharum Marsh.) ................................................................ 4 2.2 Plant propagation: an overview .................................................................................................... 6 2.2.1 Sexual propagation ............................................................................................................... 7 2.2.2 Asexual or vegetative propagation ..................................................................................... 13 2.3 Role of plant growth regulators in propagation ......................................................................... 23 2.3.1 Auxins .................................................................................................................................. 24 2.3.2 Cytokinins ............................................................................................................................ 25 2.3.3 Gibberellins ......................................................................................................................... 26 2.3.4 Abscisic Acid ........................................................................................................................ 27 2.3.5 Ethylene .............................................................................................................................. 27 2.3.6 Brassinosteroids .................................................................................................................. 28 3.0 MATERIALS AND METHODS ............................................................................................................ 30 3.1 Embryo Conversion ..................................................................................................................... 30 3.1.1 Plant materials .................................................................................................................... 30 3.1.2 Culturing procedures .......................................................................................................... 31 3.1.3 Data collection and analysis ................................................................................................ 32 3.2 Shoot organogenesis ................................................................................................................... 35 3.2.1 Plant materials .................................................................................................................... 35 3.2.2 Culturing procedures .......................................................................................................... 35 3.3 Rooting of softwood cuttings ...................................................................................................... 40 3.3.1 Experimental year 2008 ...................................................................................................... 40 3.3.2 Experimental year 2009 ...................................................................................................... 45 iv 4.0 CHAPTER 1: EMBRYO CONVERSION ................................................................................................ 53 4.1 Results ......................................................................................................................................... 53 4.1.1 Effects by genotypic differences ......................................................................................... 53 4.1.2 Effects of plant growth regulators ...................................................................................... 55 4.2 Discussion .................................................................................................................................... 61 5.0 CHAPTER 2: IN VITRO SHOOT ORGANOGENESIS ............................................................................ 69 5.1 Results ......................................................................................................................................... 69 5.1.1 Bud explants ........................................................................................................................ 69 5.1.2 Embryo explants ................................................................................................................. 70 5.1.3 Shoot tips and petioles ....................................................................................................... 71 5.2 Discussion .................................................................................................................................... 74 6.0 CHAPTER 3: ROOTING OF STEM CUTTINGS .................................................................................... 80 6.1 Results ......................................................................................................................................... 80 6.1.1 Experimental year 2008 ...................................................................................................... 80 6.1.2 Experimental year 2009 ...................................................................................................... 92 6.1.3 Effects of variation between years ................................................................................... 106 6.2 Discussion .................................................................................................................................. 107 6.2.1 Effects of cutting type based on the presence of apical meristem .................................. 107 6.2.2 Effects of genotypic variation among cultivars ................................................................. 108 6.2.3 Effects of collection timing................................................................................................ 110 6.2.4 Effects of rooting hormone application ............................................................................ 113 6.2.5 Effects of rooting medium and conditions ....................................................................... 116 6.2.6 Effects of age and growing environmental conditions of stock plants ............................. 120 6.2.7 Relationship between rooting responses and leaf size of the second-youngest node .... 121 6.2.8 Relationship between rooting responses and leaf color of the second-youngest node, as measured by color saturation and intensity ....................................................................................