DENDROBIUM FLOWER COLOR: HISTOLOGY AND GENETIC MANIPULATION A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAIT IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HORTICULTURE MAY 2003 By Rasika Geethanjali Mudalige Dissertation Committee: Adelheid R. Kuehnle, Chairperson Robert E. Pauli Richard M. Manshardt Chung-Shih Tang David T. Webb We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Horticulture. DISSERTATION COMMITTEE /f. 11 © 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 of my father, my biggest admirer and the strongest critic, who convinced me luck is in favor of those 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 Pauli, 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 of color genes. Dr. H. Yamamoto for kindly allowing me to use his laboratory and equipment; all current and past members of our 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 constmcts and their kind support, and Sanford Scientific for some of the gene constructs. 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 of my education. VI ABSTRACT Dendrobium is the most important cut flower orchid in the world. Understanding the chemical, histological and molecular aspects of flower color is crucial for the development of breeding strategies for novel colors. The objectives of this research were to examine the histology of flower color, cloning and characterization of flavonoid biosynthetic genes, and metabolic engineering of Dendrobium flavonoid pathway to obtain new colors. In Dendrobium, anthocyanins can be confined to a single layer of cells (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 of pigment to cells surrounding the vascular bundles. Color perception is markedly influenced by the presence or absence of carotenoids. Four types of epidermal 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 of flowers examined had a complex epidermis with more than one epidermal cell shape, predominantly papillate epidermal cells. We were able to isolate a full clone of Dendrobium dihydroflavonol 4-reductase (dfr), and partial clones of chalcone synthase {chs), flavonoid 3'- hydroxylase {J3'h) and flavonoid 3', 5'-hydroxylase {f3'5'h), from Dendrobium vii Jaquelyn Thomas ‘Uniwai Prince’ (UH503). Expression data indicated that dfr and chs were expressed to the greatest degree in unopened buds. Amount offS'h and fS ’5'h mRNA was too small to detect. Southern analysis has shown that f3'h and f3'5’h is represented by 2 copies each in UH503. These clones will be extremely useful in future for flower color manipulation. Two different color genes, dfr and fi'5 'h from two non-orchid plants, under the constitutive promoter ubiquitin3, were inserted into Dendrobium Icy Pink ‘Sakura’ with the intention of creating orange-red and blue shades, which are absent in commercial Dendrobium. Presence of the transgene in two sets of transformants was confirmed by PCR. Expression of the transgene from a few plants was indicated by RT-PCR and northern analyses. Vlll TABLE OF CONTENTS Acknowledgements...........................................................................................................v Abstract .................................................................................................................... vii List of tables................................................................................................................... xiv List of figures................................................................................................................. xvi List of abbreviations.......................................................................................................xix Chapter 1: Orchid biotechnology in production and improvement...............................1 1.1 Introduction........................................................................................................1 1.2 F unctions of some 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 of transgenesis..........................................................................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 of some Dendrobium species and hybrids.......................................................................................35 2.1 Introduction......................................................................................................35 2.2 Materials and methods....................................................................................36 2.2.1 Plant material............................................................................................36 2.2.2 Preparation of tissue sections and analysis............................................ 37 2.3 Results.............................................................................................................. 38 2.3.1 Spatial localization of anthocyanins.......................................................38 2.3.2 Flowers with striped patterns.................................................................42 2.3.3 Distribution of pigments in purple/yellow combinations.....................42 2.3.4. Cell shapes of the upper epidermis.........................................................44 2.4 Discussion........................................................................................................ 48 2.5 Literature cited.................................................................................................53 Chapter 3: Cloning and characterizaton of anthocyanin biosynthetic genes from Dendrobium...................................................................................................55 3.1 Introduction...................................................................................................... 55 3.1.1 Flavonoid pathway.................................................................................. 55 3.1.2 Anthocyanin skeleton.............................................................................