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Tissue Culture of Western Hemlock Dwarf Mistletoe and Its Application to Studies on Biological Control

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Tissue Culture of Western Hemlock Dwarf Mistletoe and Its Application to Studies on Biological Control TISSUE CULTURE OF WESTERN HEMLOCK DWARF MISTLETOE AND ITS APPLICATION TO STUDIES ON BIOLOGICAL CONTROL Shannon J. Deeks B.Sc. (with Distinction), University of Victoria, 1995 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biological Sciences O Shannon J. Deeks 2000 SIMON FRASER ONIVERSITY MI rights remcd This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 WdIiStreet 305, rue Wellingtm ûttawaON K1AON4 Oi(awa0N K1AON4 Canecfa Canade The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Libraq of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seii reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT Dwarf mistletoes (Arceuthobiurn spp.) are parasitic flowering plants that attack commercially valuable conifers. The biology of this genus along with 22 other geneni of parasitic flowering plants that have been cultured in vitro is reviewed in detail with respect to distribution, host range. and tissue culture procedures. A procedure for in vitro culture of western hemlock dwarfmistletoe (A. tsugense Rosend. G.N. Jones subsp. tsugenre) is described. A factorial experiment evaluated the effects of media, temperatures, presence or absence of light, and plant growth regulators on the production of radicles, holdfasts, and callus. Optimal conditions for radicle elongation were White's medium at 20°C in the presence of light and without plant growth regulators. Holdfasts developed fkom tips of radicles or fiom swollen radicles, and were maximally produced with Harvey's medium, iight, 2,4-dichlorophenoxyaceticacid (2,4-D) at 1 mg 1-', and 6- benzy laminopurine (BAP) at 0.1 mg 1-' . Callus arose fiom split radicles and hold fasts and optimal development was on White's medium with 0.5 mg 1-' 2,4-D and 1 mg T' BAP at 20°C in the dark. The tissue culture procedure was used for in vitro screening of calli and germinated seeds of A. tsugense subsp. tsugense with potential fungal biological control agents (Cy findrocurpon cylindroides and Colletohichum gloeosporioides). Mistletoe tissues were prepared for light microscopy 1,2,3,7 and 14 days post-contact with fiingi. The process of pathogenesis was investigated, and specimens were rated for extent of colonization. Cushion development, ce11 wall degradation, and both intercellular and intracellular colonization were evident for germinated seeds challenged with both fûngi. Gmwth of dwarf mistietoe callus was reduced by both fun@, and ce11 iii wall degradation dong with intercellular and intracellular colonization was evident. Cells infected with Cylindrocarpon cyfindroideswere disorganized and appeared plasmolysed. The in vitro screening method was useful to elucidate the host-pathogen interactions, and sensitive enough to determine that Cylindrocarpon cylindroides was more aggressive at colonization than Colletohichum gloeosporioides. 1 would like to thank the following people for making this thesis a redity: Dr. Simon Shamoun of the Pacific Forestry Centre for his advice, encouragement, and patience, Dr. Zarnir hinja for his excellent reviews and for securing bding to send me to Montreal for the CPS meeting, and Dr. Patrick von Aderkas of the University of Victoria for his fiiendship and encouragement. 1 also wish to thank Ed Wass for assistance with seed collections, Hugh Barclay for statistical advice, and Lesley Manning and Terry Holmes for microtechnique assistance. I also gratefully acknowledge the funding provided by Forest Renewal B.C., and the Natural Sciences and Engineering Research Cowicil of Canada. I also wish to thank my lab mates, Tod Ramsfield and Carmen Oleskevich, for their constant support during my studies. Finally, 1 thank Mom, Dad, Missy, Peeps, Wendy, and Von for their endless encouragement. TABLE OF CONTENTS .. APPROVAL............................................................................................................... 11 ... AB STMCT................................................................................................................... 111 ACKNOWLEDGMENTS ............................................................................................. v TABLE OF CONTENTS ............................. .... .............................................................. vi LIST OF TABLES ........................................................................................................ ix LIST OF FIGURES ........................................................................................................ x CHAPTER 1 .................................................................................................................. 1 1 . iNTRODUCTION...................................................................................................... 1 1 .0. INTRODUCTION .............................................. ........................................ 1 1.1 . General information ........................................................................ 1 1 .2. Hosts and distribution ..................................................................... 1 1.4. Biology and life cycle ..................................................................... 3 1 .5. Detection ..................................................................................... 5 1 .6 . Control methods ............................................................................. 6 1. 7. Tissue culture.................................................................... 9 . 1 .8. Researc h objectives .........................................*.**.......**..................10 CHAPTER 2 .................................................................................................................. 11 2. TISSUE CULTURE OF PARASITIC FLOWERING PLANTS:METHODS AND APPLICATIONS IN AGRICULTURE AND FORESTRY ............................... .. ....... 11 2.0 INTRODUCTION ........................................................................................11 2.1 TISSUE CULTURE: METHODS AND RESULTS .................................. 16 2.1 .1 Convolvulaceae .................................... ... .........*.......................16 2.1.3 Loranthaceae ................................................................................. 18 2.1.4 ûrobanchaceae ........................................................................ 20 2.1 .5 . Santalaceae................................................................................... 24 2.1.6. Scrophulariaceae ......................................................................... 28 2.1.7. Viscaceae ................................................................................. 37 2.2. APPLICATIONS OF TISSUE CULTURE:PRESENT AND FUTURE ... 4 1 CHAPTER 3 ............................................................................................................... 47 3 . IrN VITRO GERMINATION AND DEVELOPMENT OF WESTERN HEMLOCK DWARF MISTLETOE (ARCEUTHOBIUIM TSUGENSE SUBSP . TSUGENSE.......... 47 3.0. INTRODUCTION..................................................................................... 47 3.1. MATERIALS AND METHODS ................................................................ 50 3.1 .1. Seed collection ............................ .., .......................................... 50 3.1 .2 . Pre-saeening seeds for viability .................................................. 50 3.1.3. Media ........................................................................................... 51 3.1.4. Experimental design ................................................................. 51 3.1 .5 . Light rnicroscopy ........................................................................ 52 3.1.6. Statistical analysis ........................................................................ 52 3.2. RESULTS AND DISCUSSION ................................................................ 53 3.2.1. Radicle development .................................................................... 53 3.2.2. Holdfast development ............................................................... 55 3.2.3. Callus development ............. ... .................................................. 61 3.3. CONCLUSIONS ....................................................................................... 65 CHAPTE2 4 ..................................................................................................................67 A HISTOPATHOLOGICAL STUDY OF INFECTION OF GERMINATED SEEDS AND CALLUS OF WESTERN HEMLOCK DWARF MISTLETOE BY CYLIhllROCARPON CYLI1VDROLUE.S AND COLLETOTRICHOM GLOEOSPORIOLDES IN DUAL CULTURE .............................................................. 67 4.0. WIRODUCTION.....................................................................................
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