UNIVERSITY OF HAWAII LIBRARY DENDROBIUMFLOWER COLOR: mSTOLOGY AND GENETIC MANIPULATION A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PIDLOSOPHY IN HORTICULTURE MAY 2003 By Rasika Geethanjali Mudalige Dissertation Committee: Adelheid R. Kuehnle, ChailJleTSon Robert E. Paull Richard M. Manshardt Chung-Shih Tang David T. Webb © Copyright 2003 by Rasika G. Mudalige All Rights Reserved 111 I dedicate this dissertation to my most wonderful mother, who taught me courage, resilience and dedication, and to the memory ofmy father, my biggest admirer and the strongest critic, who convinced me luck is in favor ofthose who strive the most. IV ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my advisor, Dr. Adelheid R. Kuehnle, for her advice and support throughout my studies, heartiest thanks to my dissertation committee, Drs. Robert Paull, Richard Manshardt, C.-S. Tang, and David Webb for their support and encouragement. My most sincere thanks are given to the following persons without whom this work would not have been possible; Drs. David Hieber and Robert Bugos for their good humor, advice and help in molecular biology techniques; Dr. Tessie Amore for her help in histology work and providing valuable information about orchid species and hybrids; Nellie Sugii for advice on biolistic bombardment and other laboratory techniques; Sue Gamer and Stacie Aragon for their help in screening the transgenic plants, and Dr. Michele Champagne for her initial work on isolation ofcolor genes, Dr. H. Yamamoto for kindly allowing me to use his laboratory and equipment; all current and past members ofour laboratory for their help and friendship; office staff, Ms. Lynne Horiuchi and Ms. Susan Takahashi for their help, and all other friends for their encouragement. I would like to thank the Florigene Ltd., Australia, for their help in editing the last chapter and providing the color genes for this research, scientists (Drs. S. Deroles, M.R. Boase, K. Davies, and D.H. Lewis) at the Crop & Food Research, New Zealand, for gene constructs and their kind support, and Sanford Scientific for some ofthe gene constructs. v My thanks are also due to Nancy Craig, Lynne and Ed for providing the most valuable support during difficult times, and to the Sri Lankan community in Hawaii and to my friends, Anokha and Lisa, for providing a family away from home. I am also grateful to my family, my mother for giving me courage and determination to finish my studies despite many obstacles, my late father who always believed in me, and my most wonderful brother and his family, for their love and support throughout the long years ofmy education. VI ABSTRACT Dendrobium is the most important cut flower orchid in the world. Understanding the chemical, histological and molecular aspects offlower color is crucial for the development ofbreeding strategies for novel colors. The objectives of this research were to examine the histology offlower color, cloning and characterization offlavonoid biosynthetic genes, and metabolic engineering of Dendrobium flavonoid pathway to obtain new colors. In Dendrobium, anthocyanins can be confined to a single layer ofcells (epidermal or suepidermal) in pale flowers. More intensely colored flowers had anthocyanin in several cell layers. Striped patterns on the perianth were due to the restriction ofpigment to cells surrounding the vascular bundles. Color perception is markedly influenced by the presence or absence ofcarotenoids. Four types ofepidermal cells were found in Dendrobium: flat, dome, elongated dome, and papillate. Epidermal cell shape and cell packing in the mesophyll affected the visual texture. Perianth parts with flat cells and a tightly packed mesophyll had a glossy texture, whereas dome cells and loosely packed mesophyll contributed a velvety texture. The labella in the majority offlowers examined had a complex epidermis with more than one epidermal cell shape, predominantly papillate epidermal cells. We were able to isolate a full clone ofDendrobium dihydroflavonol 4-reductase (dfr), and partial clones ofchalcone synthase (chs), flavonoid 3'· hydroxylase (j3'h) and flavonoid 3', 5'-hydroxylase (j3'5'h), from Dendrobium VJl Jaquelyn Thomas 'Uniwai Prince' (URS03). Expression data indicated that dfr and chs were expressed to the greatest degree in unopened buds. Amount ofj3'h and j3'5'h mRNA was too small to detect. Southern analysis has shown thatj3'h and j3'5'h is represented by 2 copies each in URS03. These clones will be extremely useful in future for flower color manipulation. Two different color genes, dfr andj3'5'h from two non-orchid plants, under the constitutive promoter ubiquitin3, were inserted into Dendrobium Icy Pink 'Sakura' with the intention ofcreating orange-red and blue shades, which are absent in commercial Dendrobium. Presence ofthe transgene in two sets oftransformants was confirmed by PCR. Expression ofthe transgene from a few plants was indicated by RT-PCR and northern analyses. Vlll TABLE OF CONTENTS Acknowledgements . ._ .. .. _.. __ .__ .. __ __ _ v Abstract .. " __ __ vii List oftables _ xiv List offigures __ __ xvi List ofabbreviations xix Chapter 1: Orchid biotechnology in production and improvement... I 1.1 Introduction 1 1.2 Functions ofsome cloned orchid genes 2 1.2.2 Flower color (flavonoid biosynthesis) 8 1.2.3 Flower senescence 11 1.2.4 Disease defense 14 1.3 Orchid genetic engineering and commercially applicable traits 16 1.3.1 Methods oftransgenesis 16 1.3.2 Engineering disease resistance 20 1.3.3 Engineering novel flower colors 21 1.3.4 Engineering increased vase life 22 1.4 Other biotehnology applications in production and improvement.. 22 1.4.1 Virus detection '" 22 IX 1.4.2 Antiviral strategies 23 1.4.3 Flower color gene activity test 24 1.4.4 DNA markers to improve breeding strategy 25 1.5 Literature cited 27 Chapter 2: Pigment distribution and epidermal cell shape ofsome Dendrobium species and hybrids 35 2. I Introduction 35 2.2 Materials and methods 36 2.2.1 Plant materia1. 36 2.2.2 Preparation oftissue sections and analysis 37 2.3 Results 38 2.3.1 Spatial localization ofanthocyanins 38 2.3.2 Flowers with striped patterns .42 2.3.3 Distribution ofpigments in purple/yellow combinations ..42 2.3.4. Cell shapes ofthe upper epidermis .44 2.4 Discussion 48 2.5 Literature cited 53 Chapter 3: Cloning and characterizaton ofanthocyanin biosynthetic genes from Dendrobium 55 3.1 Introduction 55 3.1.1 Flavonoid pathway 55 3.1.2 Anthocyanin skeleton 57 3.1.3 Chalcone synthase 57 x 3.1.4 DihydroflavonoI4-reductase 59 3.1.5 Flavonoid hydroxylases 60 3.2 Objetive 61 3.3 Materials and methods 62 3.3.1 RNA extraction and cDNA synthesis 62 3.3.2 PCR with degenerate primers to amplify flavonoid genes 63 3.3.3 3'RACE and 5'RACE to obtain the full/partial sequences 65 3.3.4 Radioactive probe synthesis for northern and Southern hybridization ............................................................................................................67 3.3.5 Northern blot hybridization 67 3.3.4 Genomic DNA extraction and Southern hybridization 69 3.4 Results 70 3.4.1 Cloning and sequence analysis offlavonoid biosynthetic genes ....... 70 3.4.2 Gene expression by northern analyses 77 3.4.3 Genomic southern analysis ofDendrobium (UH503 & K1224) DNA .. ............................................................................................................ 86 3.5 Discussion 91 3.6 Conclusion 94 3.7 Literature cited 96 Chapter 4: Metabolic engineering ofDendrobium orchids to modify flower color102 4.1 Introduction 102 4.1.1 New colors through introduction ofbiosynthetic genes 102 4.1.2 Sense or antisense suppression ofthe flavonoid biosynthetic genes I05 XI 4.1.3 Introduction ofregulatory genes to activate the native biosynthetic gene(s) 106 4.2 Objective 107 4.3 Materials and methods 109 4.3.1 Plant material 109 4.3.2 Color genes, plasmid DNA and particle bombardment 109 4.3.3 Selection and regenemtion oftransformants 112 4.3.4 PCR and RT-PCR analyses 112 4.3.5 Northern blot analysis 117 4.3.6 Probe synthesis for northern and Southern hybridization 117 4.3.7 Southern hybridization and development... 118 4.3.8 Potting, acclimatization and maintenance ofPCR positive plants... 119 4.4 Results 119 4.4.1 Selection and regenemtion 119 4.4.2 PCR analyses 121 4.4.3 RT-PCR and northern analyses 127 4.5 Discussion 137 4.5.1 PCR results and selection oftmnsformed plants 137 4.5.2 Transgene expression and silencing 138 4.5.3 Other bombardment experiments and their status 142 Appendix A 144 Appendix B 145 xu Appendix C 146 Appendix D 147 4.6 Literature cited 149 xiii LIST OF TABLES 1.1 Isolated orchid genes or eDNA clones and their presumptive functions 3 1.2 Examples ofgenetically engineered orchids: Means oftransgenesis .... 17 1.3 Examples ofgenetically engineered orchids: Potential commercial traits 18 2.1 Pigment distribution in the epidermal, subepidermal, and mesophyll cells ofthe perianth ofDendrobium 39 2.2 Shapes ofthe adaxial epidermal cells ofthe perianth ofDendrobium 45 3.1 Degenerate and oligo dT primers, reverse transcription conditions and PCR conditions used fOT amplification offlavonoid genes from Dendrobium 64 3.2 Primers and PCR conditions used in 3' RACE and 5' RACE reactions to amplifY flavonoid genes __ 66 3.3 Description ofthe bud and flower stages ofDendrobium Jaquelyn Thomas 'Uniwai Prince' used in temporal expression of flower color genes 68 3.4 Comparison ofpartial clone Den-chs-II