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(Lodd.) and the Pol Yploidi UNIVERSITY OF HAWAII LIBRARY THE IDENTIFICATION OF A SUITABLE IRRADIATION DOSAGE FOR MUTATION INDUCTION IN ZAMIOCULCAS ZAMlIFOLIA (LODD.) AND THE POL YPLOIDIZATION OF Z ZAMlIFOLIA AND MARSDENIA FLORIBUNDA A THESIS SUBMITIED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN HORTICULTURE DECEMBER 2006 By Susana D. Vanzie-Canton Thesis Committee: Kenneth Leonhardt, Chairperson Richard CrUey Yoneo Sagawa Kheng-Tuan Cheah 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 Master of Science in Horticulture. THESIS COMMITIEE ii ACKNOWLDEGEMENTS I am grateful to my committee chairman Dr. Kenneth Leonhardt for his support His knowledge, experiences, and technical assistance were of great value throughout the entire process of this research project. I would also like to express my sincere appreciation to Drs. Richard CriIey, Yoneo Sagawa, and Kbeng-Tuan Cheah who served on my thesis committee. I had great help from all Magoon Research Facility personnel, namely Craig Okazaki and Ronald Matsuda. In addition, my appreciation extends to Susan Takahashi, Shirley Ishihara, and Elsie Sun in the Department office, who have always been kind and helpful. I wish to express my sincere appreciation to Dr. Mark Wright, who provided statistical support; Dr. Ania Wieczorek, Mr. Jaco Le Roux, and Ms. Carol Tran who kindly provided assistance when needed; and Dr. Karen Selph for her time and helpful suggestions during flow cytometric analysis. Sandy Kasman (Hawaiian Sunshine Nursery) also contributed to my knowledge of Zamioculcas zamiiJolia. I would also like to thank my labmates, namely Emily Teng for her help, and Tilden Miguel for his helpful suggestion of the callus inducing media recipe utilized in Chapter 2. Finally, special thanks are expressed to my husband for his patience, emotional support, encouragement, and understanding throughout my graduate education. Thanks to my parents, my brothers and my sisters for all their words of encouragement. iii ABSTRACT Radiation mutation has been successfully used to create a great variety of ornamental crop cultivars by supplementing existing germplasm and improving existing cultivars, and chemical mutagens such as colchicine and oryza1in have been used to create new plant cultivars by doubling the chromosome number of the treated plant material to produce tetraploids. The main objective of this thesis research was to develop protocols to create tetraploid plants of Zamioculcas zamii/olia (Lodd.) Engl., an important foliage plant, and Marsdenia jloribunda (Brongn.), an important lei flower plant, and to determine the LDso of ZZ leaflets. ZZ leaflets and M. jloribunda seeds were treated with colchicine at various concentrations and durations in order to induce ploidy changes and regenerate polyploids. Five ZZ tetraploids and one M. jloribunda tetraploid were produced using colchicine. A tissue culture protocol was also developed for the oryza1in treatment of ZZ callus for the in vitro polyploidy induction of ZZ. The LDso of ZZ leaflets irradiated with x-rays was calculated as 20±1 Gy. A ZZ germplasm collection was also initiated to provide ZZ plant material for use in future breeding studies. iv TABLE OF CONTENTS ACKNOWLDEGEMENTS ............................................................................................... iii ABSlRACT ........................................................................................... iv . TABLE OF CONTENTS .................................................................................................... v LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii LIST OF ABBREVIATIONS ............................................................................................. x LITERATURE REVIEW ................................................................................................... 1 SECTION 1- ZAMIOCULCAS ZAMIIFOLIA .................................................................... 6 Introduction ................................................................................................................. 7 CHAPTER 1: COLCHICINE TREATMENT OF ZZ LEAFLETS IN VIVO ............ 9 Background Information ......................................................................................... 9 Materials and Methods ............................................................................................ 9 Results and Discussion ......................................................................................... 13 Tables and Figures ................................................................................................ 21 CHAPTER 2: POL YPLOIDIZATION OF ZAMIOCULCAS ZAMIIFOLIA VIA ORYZALlN TREATMENT OF CALLUS .............................................................. 31 Background Information ....................................................................................... 31 Materials and Methods .......................................................................................... 32 Results and Discussion ......................................................................................... 36 Figures ................................................................................................................... 39 CHAPTER 3: IDENTIFICATION OF A SUITABLE IRRADIATION DOSAGE FOR ZAMIOCULCAS ZAMIIFOLIA ........................................................................ 41 Background Infonnation ....................................................................................... 41 Materials and Methods .......................................................................................... 42 Results and Discussion ......................................................................................... 43 Tables and Figures ................................................................................................ 46 CHAPTER 4: GERMPLASM COLLECTION OF ZAMIOCULCAS ZAMIIFOLIA50 Figures ................................................................................................................... 53 . SECTION 2 - MARSDENIA FLORIBUNDA .................................................................... 63 CHAPTER 5: POL YPLOIDIZATION OF MARSDENIA FLORIBUNDA .............. 64 Background Information ....................................................................................... 64 Materials and Methods .......................................................................................... 65 Results and Discussion ......................................................................................... 68 Tables and Figures ................................................................................................ 71 v APPENDIX. SASS DATA OUTPUT FOR PROBIT ANALySIS ......................... 77 LITERATURE CITED ..................................................................................................... 81 vi LIST OF TABLES 1.1 Guard cell measurements of colchicine treated ZZ leaflets before rhizome development ......................................•...........•...........21 1.2 Guard cell measurements of colchicine treated ZZ leaflets after rhizome development ........................•.............•........................ 21 1.3 Number of ZZ tetraploids, mixoploids, and DNA aneuploids identified via flow cytometIy ..........................................•............•.....22 3.1 Number ofleaflets irradiated and dosages used to identifY LDloo and LDso ..................................................•...............•...................................46 3.2 Results of ZZ leaflets irradiated in March 2006 .....................•..............•...46 3.3 Results of ZZ leaflets irradiated in June 2006 ..........................................47 3.4 Results ofZZ leaflets irradiated in August 2006 .......................................47 5.1 Number of seedHngs and seedHng height of M. j/oribunda after colchicine treatment of seeds .....................................•.................67 5.2 Guard cell measurements of M. j/oribunda seedHngs visually identified as possible polyploids .........................................................68 vii LIST OF FIGURES Figure ~ 1.1 Vegetative propagation of ZZ via leaflet cuttings .....................................23 1.2 Actively growing ZZ root tips harvested for chromosome counts ........................................................................23 1.3 Sample preparation for flow cytometric analysis ......................................24 1.4 ZZ leaflet morphology of diploid and suspected polyploids .........................25 1.5 Stained ZZ chromosomes isolated from single cells ..................................26 1.6 Distribution of DNA content of diploid ZZ .............................................27 I.7 Distribution of DNA content of tetraploid ZZ ..........................................28 1.8 Distribution of DNA content ofmixoploid ZZ .........................................29 1.9 Distribution of DNA content of a ZZ DNA aneuploid ................................30 1.10 Identified debris in samples to be analyzed by flow cytometry .............................30 2.1 Tissue culture protocol developed for callus induction followed by plantiet regeneration
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