A s p e c t s o f Z y g o t ic a n d So m a t ic E mbryogenesis IN ANTHURIUMS A THESIS SUBMITTED TOTHE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULHLLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN HORTICULTURE DECEMBER 1994 By Tracie K. Matsumoto Thesis Committee: Adelheid Kuehnle, Chairperson Richard Criley William Sakai 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 Masto- of Science in Horticulture THESIS COMMITTEE Chairperson © Copyright 1994 by Trade Matsumoto m AKNOWLEDGEMENTS In addition to my advisor, Dr. Adelheid Kuehnle and my committee members, Dr. Richard Criley and Dr. William Sakai, I would like to thank the following people for their assistance in my research; Dr. David Webb for the use of the histological supplies and ultramicrotome equipment; Nellie Sugii for acquainting me with the microtechnique procedures; Dr. Haruyuki Kamemoto and Dr. Tessie Amore for sharing their expertise in breeding and caring for anthurium plants; Dr. Michael Tanabe, John Kunisaki and Joanne Imamura-Lichty for the anthurium plants used in this study; Jean Takabayashi for review of this manuscript; and Mach Fukada for help with "computer problems". I would also like to thank Nuttha, Gillian, Katrina, Vicky, Koon-Hui, Wendy, and Chen for their help and support during my time at UHM. IV TABLE OF CONTENTS Acknowledgements................................................................................................................. iv List of Tables ......................................................................................................................... ix List of F ig u re s.........................................................................................................................x Chapter 1 ; Literature Review and Questions to be Answered...........................................1 A raceae.............................................................................................................................. 1 Anthurium ....................................................................................................................... 3 Zygotic Embryogenesis...................................................................................................5 Microscopy...................................................................................................................5 Histochemistry, Cytochemistry and Autoradiography............................................6 Tissue Culture............................................................................................................ 6 Molecular Techniques..................................................................................................7 Morphological Description of Zygotic Embryogenesis..........................................7 Embryogenesis in Araceae.........................................................................................9 Embryogenesis in A nthurium .................................................................................9 Somatic Embryogenesis...................................................................................................10 Factors Influencing Somatic Embryogensis..........................................................11 Applications of Somatic Embryogenesis................................................................12 Propagation of Anthurium .............................................................................................13 S e e d s ..........................................................................................................................13 D iv isio n ..................................................................................................................... 13 Cuttings...................................................................................................................... 14 Tissue Culture of A nthurium ...................................................................................... 14 Establishment of Cultures.......................................................................................14 Seeds......................................................................................................................14 Leaf, Spathe and Spadix.....................................................................................15 Axillary Buds.......................................................................................................16 Multiplication and Plant Regeneration...................................................................17 C allus....................................................................................................................17 Enhanced Axillary Branching........................................................................... 17 Somatic Embryogenesis..................................................................................18 Factors Affecting Multiplication............................................................................19 G enotype............................................................................................................. 19 V Explant Source....................................................................................................20 Commercial V alue ...................................................................................................20 Questions to be Answered..............................................................................................22 Improvement of Microtechnique Procedures for Anthurium ...............................22 Anthurium Zygotic Embryogenesis.......................................................................22 Somatic Embryogenesis in Greenhouse-Grown Anthurium.................................22 Histology of the Somatic Embryo........................................................................... 23 Aim and Scope of W ork........................................................................................... 23 References..........................................................................................................................25 Chapter 2 : Microtechnique Improvement for Anthurium............................................... 33 Introduction......................................................................................................................33 Materials and Methods...................................................................................................35 Basic Procedure...................................................................................................... 35 Methods to Remove or Fix M ucilage...................................................................35 Methods to Overcome a Hardened Carpel Wall and Seed Coat..........................36 Results and Discussion.................................................................................................. 37 Methods to Overcome Mucilage.............................................................................37 Methods to Overcome a Hardened Carpel Wall and Seed Coat..........................44 C onclusion......................................................................................................................45 R eferences..................................................................................................................... 46 Chapter 3 : Morphology and Anatomy of Anthurium Zygotic Embryogenesis.................48 Introduction.................................................................................................................... 48 Materials and M ethods................................................................................................. 48 Pollination and Harvest............................................................................................49 Microtechnique........................................................................................................... 51 Ovule / Embryo Culture..........................................................................................52 Results and Discussion.................................................................................................. 53 Embryo development................................................................................................53 Week 4 .................................................................................................................53 Week 6 .................................................................................................................56 Week 8 .................................................................................................................56 Week 1 0 .............................................................................................................56 Week 12 / Week 14 .......................................................................................... 60 vi Embryo Maturation.................................................................................................. 60 Week 1 6 .............................................................................................................. 60 Week 1 8 .............................................................................................................65 Week 2 0 .............................................................................................................65 Week 2 2 .............................................................................................................69