CONSERVATION OF HAWAIIAN LOBELIOIDS — IN VITRO AND MOLECULAR STUDIES A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW ATI IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HORTICULTURE MAY 1996 By Gregory A. Koob Dissertation Committee: Yoneo Sagawa, Co-Chairperson Sterling Keeley, Co-Chairperson Adelheid Kuehnle Fred Rauch Clifford Smith 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 I (^Chairperson^ !^-Chairperson AkjJU^jA ■ UilU 11 © Copyright 1996 by Gregory A. Koob All Rights Reserved 111 Acknowledgments I would like to thank the staff and volunteers at the Harold L. Lyon Arboretum and the Lyon Arboretum Association for their support of the in vitro research. The staff of the National Tropical Botanical Garden, the State of Hawai'i Division of Forestry and Wildlife, the Nature Conservancy of Hawai'i, the U.S. Fish and Wildlife Service, John Obata, and Rick Palmer are appreciated for supplying plant material. Partial funding was supplied by the U.S. Fish and Wildlife Service, the State of Hawai'i Division of Forestry and Wildlife, the Center for Plant Conservation, the University of Hawai'i Foundation, and Sigma Xi Grants-in-Aid of Research. Thank you to Dave Lorence, Kay Lynch, Loyal Mehrhoff, Carol Nakamura, John Obata, Rick Palmer, Joshlyn Sands, and Alvin Yoshinaga for information used in this report. Special thanks to Sterling Keeley for the use of her lab and supplies and support for the RAPDs work and to Yoneo Sagawa for his knowledgeable support of the in vitro research. IV Abstract With over 25% of Hawai'i’s Campanulaceae already extinct and many more on the verge of extinction, research in the propagation and the genetics of the remaining populations is greatly needed. In vitro propagation of 58 Hawaiian Campanulaceae species were attempted, through in vitro germination, organogenesis, or micropropagation. More than 80% of the species received as seeds were successfully germinated. No differences in germination rate or percentage was found between immature and mature seeds. Leaf explants produced viable shoots in 43% of the species, and 29% of wild-collected bud explants were successfully grown into plants. RAPDs was used to detect variability of the seedling populations of two bottlenecked species, Cyanea asarifolia St. John (original wild population of 15 plants) and Delissea undulata ssp. undulata Gaud, (original wild population of one plant). DNA was extracted from each species using small amounts of leaf tissue produced in vitro and used for the RAPDs studies. No detectable variation was found within these populations (indicating the impoverished remaining genetic information). The value of in vitro propagation and molecular studies of reduced populations is discussed. Table of Contents Acknowledgments.............................................................................................................. iv Abstract .................................................................................................................................. v List of Tables........................................................................................................................vii List of Figures .................................................................................................................... viii Chapter 1. Introduction ..........................................................................................................1 Hawaiian Campanulaceae.......................................................................................... 2 In vitro Propagation ...................................................................................................2 RAPDs..........................................................................................................................5 Chapter 2. In Vitro Propagation of Hawaiian Campanulaceae .........................................10 Introduction................................................................................................................10 Materials and Methods .............................................................................................11 Plant Material..................................................................................................... 11 M edia..................................................................................................................12 Growth Conditions.............................................................................................13 Disinfestation..................................................................................................... 14 Records ............................................................................................................. 21 R esults........................................................................................................................22 Seed Germination...............................................................................................22 Organogenesis ...................................................................................................29 Micropropagation...............................................................................................36 Transfer to Greenhouse .................................................................................... 37 Discussion..................................................................................................................38 Chapter 3. Seedling Variability of Cyanea asarifolia St. John and Delissea undulata ssp. undulata Gaud. (Campanulaceae) as Determined by R A PD s.......................................... 43 Introduction............................................................................................................... 43 Materials and Methods ............................................................................................ 44 Plant M aterial.....................................................................................................44 DNA Extraction.................................................................................................45 RAPDs PC R .......................................................................................................49 R esults........................................................................................................................50 Discussion..................................................................................................................52 Chapter 4. General Discussion.............................................................................................65 Appendix A. Additional in Vitro Propagation Data Tables.............................................. 69 Appendix B. RAPDs Data T ab les.....................................................................................126 Literature Cited ..................................................................................................................145 VI List of Tables Tablg Page 2.1 Hawaiian Campanulaceae evaluated for in vitro propagation and covered by the Endangered Species Act (ESA) ............................................................................ 15 2.2 Hawaiian Campanulaceae evaluated for in vitro propagation but not covered by the Endangered Species Act (ESA) ......................................................................17 2.3 Germination of Hawaiian Campanulaceae, immature (I) versus mature (M) seed s........................................................................................................................24 2.4 Species successfully germinated in vitro............................................................... 25 2.5 Species successfully propagated from leaf explants..............................................32 2.6 BA and 2,4-D combinations tested for shoot production from leaf explants. 33 2.7 BA and NAA combinations tested for shoot production from leaf explants .. 33 2.8 Micropropagation results of Hawaiian Campanulaceae showing number of shoots produced and production rate ................................................................... 34 A. 1 In vitro treatments used on Hawaiian Campanulaceae........................................ 69 A.2 Hawaiian Campanulaceae attempted in vitro at Harold L. Lyon Arboretum Micropropagation Facility .................................................................................... 85 A.3 In vitro seed germination trials and results......................................................... 110 A.4 Leaf explants in 0.5X MS with plant growth regulator combinations (in m g/L)................................................................................................................121 B.l Operon™ primers screened and species............................................................. 126 B.2 PCR results using RAPDs with Cyanea 133 B.3 PCR results using RAPDs with Delissea undulata ssp. undulata................... 140 Vll List of Figures P^ge 3.1 DNA cleaned with cesium chloride (top of gel) is notably cleaner than DNA extracted without a cesium-gradient step (bottom of gel) .............................56 3.2 RAPDs markers resulting from using primer OPA-5 with DNA from Cyanea asarifolia ...........................................................................................................58 3.3 RAPDs markers