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Determination of Nuclear Dna Content in Orchids by Flow

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Determination of Nuclear Dna Content in Orchids by Flow DETERMINATION OF NUCLEAR DNA CONTENT IN ORCHIDS BY FLOW CYTOMETRY A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN HORTICULTURE MAY 1996 By Wendy E. Jones Thesis Committee: Adelheid R. Kuehnle, Chaiq^erson Chifumi Nagai Sterling Keeley 11 We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Mcister of Science in Horticulture. THESIS COMMITTEE (Chairperson) Ill © Copyright 1996 by Wendy E. Jones All Rights Reserved IV Acknowledgments I would like to thank the following people for their assistance: Drs. H. Kamemoto, T. Amore of the Department of Horticulture at the University of Hawaii, for sharing their expertise in orchid breeding with me, as well as identifying orchid specimens; Mr. R. Tokunaga at H & R Nurseries and Dr. Y. Sagawa of the Department of Horticulture at the University of Hawaii, for providing additional specimens; Dr. K. Arumuganathum at the University of Nebraska Biotechnology Center for allowing me to train in his laboratory; and the people at the Marine Sciences Department at the University of Hawaii, especially Mr. H. Nolla, for their technical expertise and the use of their FCM equipment. Abstract Flow cytometric analysis was found useful for determining ploidy and DNA content values for orchid species and hybrids. Immature leaf tissue yielded the most reliable results for all orchids sampled. Leaf endopolyploidy was common, with levels as high as 16N found in mature D. gouldii leaves. DNA content values for 37 Dendrobium species, 11 Dendrobium hybrids, 33 additional orchid species and 8 hybrids were determined. Values for Dendrobium species ranged from 1.53 pg to 4.23 pg/2C nuclei. C-values for the remaining orchid species ranged from 1.91 pg to 15.19 pg/2C nuclei, with those of Cattleya alone ranging from 3.29 pg to 9.29 pg/2C nuclei. The highest variation within Cattleya corresponded to different specimens of the same species, suggesting polyploidy. An analysis of Dendrobium hybrids and parent species showed that DNA content values could be reliably predicted in cases where parentage was indefinite. VI Table of Contents Acknowledgments IV Abstract List of Tables..............................................................................................................................................................ix List o f Figures List of Abbreviations................................................................................................................................................ xi Chapter 1: Literature Review .................................................................................................................................. 1 I* Introduction 1 n. Orchid Breeding A. History B. Breeding Compatibility.................................. 3 C. Breeding Objectives...............................................................................................................................4 D. Production of Polyploids......................................................................................................................5 E. Dendrobium Distribution and Taxonomy •••••••■•••••••••■■•••••••••••••••■••••••■■•••■•••••■•••■•••■•••■•••••••••••••a 6 F. Genome Breeding Strategies for Dendrobium in. DNA Content and Ploidy Analysis: General M ethods................................................................... 8 IV. Introduction to Flow Cytometry.. A. General Principles.................... V. Modern Flow Cytometry................................................................................................ 13 A. Instrumentation 13 B. M ethodologies.......................................................................................................................................14 C. Plant Nuclei Extraction Procedures.................................................................................................16 VI. Application of FCM to Orchids and Other Horticultural Crops................................................ 18 Vn. Specific Research Goals •••••••••••••••••••••••••••••••••••••••••••••••••••••••••♦•••••••••••••••••••••••••••••••••••••••••••••••a 19 VIII. Tables...................................................................................................................................................... 23 IX. Literature Cited........................................................................................................................................33 Vll Chapter!: Characterization of Dendrobium Tissues and Identification ofPloidy Levels Using F CM .................................................................................................................................................................38 I. Introduction J8 II. Materials and Methods • ••••••••••••••••••■•■••••■•••■■•••••••••••••••••••••••••••••••••••••••■•••♦••••••••••••■•••••••••••••••••••A .40 A. Plant Material 40 B. Nuclei Extraction Methods 41 C. FCM and Data Analysis .41 D. Interpretation of Histograms 42 m. Results. 42 IV. Discussion ....45 V. Summary...................................................................................................................................................... 49 VI. Tables...................................................................... 50 A^II. Tigures ......................................................................................................................................51 Vni. Literature Cited.................................................................................................................................... 57 Chapter!: Nuclear DNA Content of 25 Orchid Genera with emphasis on Dendrobium ......................60 I. Introduction 60 II. M aterials and M ethods ...............................................................................................................................................................................................................................................................................6 1 A. Plant Material 6 1 B. Nuclei Extraction Methods 6 2 111* R e s u lts ••••••••••••••••«••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••«••••«•••••••••••••••••••• 6 3 IV. Discussion 63 V . S u m m a r y ................................................................................................................................................................................................................................................................................... \ ^ I . Tu b l e s . 4 V II. L ite ra tu re C ite d ........M .....M ............................................................4 ........................................4 7 6 Chapter 4: Evaluation of Dendrobium species and Hybrids Using FCM .................................................................................................................. 7 9 I. In tro d u ctio n ................... .44.44444.••4444V .....* ..4444.44444444.444.4444444444.4444444444444444444444444.44..44444.444.444..44444444.4..4.444.4444.79 II. M aterials and M ethods ......................................................................................................................................................................................................................................................................................8 0 Vlll A. Plant Material ...M B. Nuclei Extraction Methods^ .... 80 C. FCM and Data Analysis 81 in. R esults.... 81 IV. Discussion ••••••••••••••• ...............82 V. Summary. ...83 VI. Tables. ,..85 Vn. F igures........ .86 Vni. Literature Cited............................................ .90 Chapter 5: Perspectives on Orchid FCM Analysis... ...............91 I. Introduction '•♦••••••••••••••••••••••I 91 n. Material and Methods ••••••••••••••••••••••••••••I ,.91 III. Results and Discussion — .92 IV. Future Applications of FCM to Orchidology...... 94 V. Tables................................................................................ 97 VI. Literature Cited.... ...98 Appendix: Protocol for Isolation of Plant Nuclei.... .101 IX List of Tables Table £aS£ 1.1 N um ber of offspring of differing ploidy levels •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••I ................. 23 1.2 Seed propagated triploid Dendrobium cultivars used commercially 24 U C ellular param eters m easurable by flow cytom etry • •••••••••••••••••••••••••••••••••••••••••••••••••••••■•••■•■•a aeaaaaaa 25 1.4 Fluorochrom es used in flow cytom etry for nucleic acid staining aaaaaaeaaaaaaaaaaaaaaaaaaaaaaaaeaaaaaaaaaaaaaaaaaae 26 1.5 C om ponents of different buffers used to extract plant nuclei aaaaaaaaaeaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 27 1.6 DNA content of orchid species with m ethod of C-value determ ination aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaeaaaaaa 28 2.1 Distribution of nuclei at different ploidy levels found in Dendrobium tissues................................ 50 3.1 Mean DNA content for species of 25 orchid genera, excluding Dendrobium .....................................69 3 2 Mean nuclear DNA content for Dendrobium sp ecies............... 72 3.2 Range of DNA content values found within each DendroWi/m section .............................................. 75 4.1 Listing of Dendrobium hybrids and the parental species showing actual and expected DNA content values........................................ 85 5.1 Mean DNA content
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