Implications of Embryo Desiccation Tolerance, Seed Dormancy
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IMPLICATIONS OF EMBRYO DESICCATION TOLERANCE, SEED DORMANCY, AND SEED DAMAGE FOR CONSERVATION OF PRITCHARDIA PALMS ENDEMIC TO HA WAIT A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HA WAIT IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN TROPICAL PLANT AND SOIL SCIENCES (ECOLOGY, EVOLUTION, AND CONSERVATION BIOLOGY) MAY 2006 By: Hector E. Perez Dissertation Committee: Richard Criley, Chairperson Kent Kobayahsi Carol Baskin Mike Maunder Christina Walters Donald Drake DEDICATION This dissertation is dedicated to the memory of Amparo Claramunt. Her blessings allowed me to pursue my ambitions. Ill ACKNOWLEDGMENTS I would like to thank the National Science Foundation Grades K-12 Teaching Fellowship Program; the Ecology, Evolution, and Conservation interdisciplinary specialization; the ARCS Foundation; and the Haruyuki Kamemoto Scholarship for funding for my research and academic endeavors, I am thankful to all my committee members for their mentoring and support during my studies. I am grateful for the invaluable technical and logistical support provided by Lisa Hill, Jennifer Crane, and John Waddell of the National Center for Germplasm Preservation Research (USDA- ARS, Ft. Collins, CO). I thank Drs. Nancy Chen, Sung-Eun Lee, Qingxiao Li, Loren Gautz, Robert Pauli, Yoneo Sagawa, and David Webb, and Tina Carvalho for their technical assistance, use of lab space, and equipment. The Army Natural Resources, Environmental Division, Conservation and Restoration Branch also provided logistical support for preliminary studies. 1 express my gratitude to Mr. Alvin Yoshinaga for all his help and hospitality. 1 thank my family and friends for all the love and support they provided, whether local or long-distance. Most importantly, 1 extend my deepest appreciation to my wife Kelly Perez. The work presented in this dissertation was successful due to her love, strength, perseverance, and encouragement. IV ABSTRACT Pritchardia remota (Kuntze) Becc. is an endemic Hawaiian palm. This species is listed as endangered due to threats such as invasive plants and animals, stochastic events, and/or reduced reproductive vigor. However, P. remota seeds are not presently stored in genebanks because it is presumed that they do not remain viable in storage. Yet, there are no studies that report the tolerance of Pritchardia species to storage conditions. Assessing the desiccation tolerance of Pritchardia is hampered by their difficult physiology. Germination is delayed in seeds of Pritchardia species and the mechanisms that control these delays are not understood. Clarifying these mechanisms can assist in forming propagation protocols for the creation of genetically diverse populations useful for recovery actions. Moreover, introduced vertebrate predators are known to damage seeds o f Pritchardia. It is not known if Pritchardia seeds can tolerate damage and remain viable. The studies reported here show that developmental patterns in P. remota are intermediate to those observed for species that can and can not tolerate storage in genebanks. Embryos tolerate relatively high levels of drying and remain viable. However, they do not tolerate the extreme drying (< 0.10 g H2 O/ g dry weight) necessary to maintain viability in conventional genebanks conditions. Therefore, it may be necessary to employ other techniques, such as cryo-preservation, to extend the longevity of this species in storage. Pritchardia remota seed are dormant at shedding. Emhryo growth is restricted by the operculum and endocarp. Emhryos are also underdeveloped and must reach a critical length, moisture content, and water potential for operculum displacement and subsequent radicle protrusion. Germination is hastened at warm constant temperatures {i.e., > 25°C) and if seeds are removed from the fruit coats. The combination of mechanisms suggests that P. remota seeds are morpho-physiologically dormant. Finally, moderate levels of seed damage may not be limiting in terms of germination for Pritchardia species. Damage to the endosperm caused seeds to germinate more rapidly compared to non-damaged seeds. Nevertheless, damaged seeds may succumb to fungal attack. Therefore, it is critical therefore to prevent predators from damaging Pritchardia seeds. VI TABLE OF CONTENTS D edication.................................................................................................................................. iii Acknowledgements.................................................................................................................. iv A bstract....................................................................................................................................... v List of Tables............................................................................................................................ x List of Figures........................................................................................................................... xi Chapter 1: Introduction........................................................................................................... 1 Chapter 2: The Protracted Developmental Program in Embryos of Pritchardia Remota (Kuntze) Becc. and its Deviation From Orthodox and Recalcitrant Developmental Patterns.......................................................................................................... 4 Introduction.................................................................................................................. 4 Seed Development in Palms is Prolonged.............................................. 5 The Assumption of Recalcitrance in Palm Seeds................................. 6 Materials and M ethods............................................................................................. 7 Plant Material............................................................................................... 7 W ater Content D eterm inations.................................................................. 8 Water Potential Determinations................................................................ 8 Ultrastructure Studies................................................................................. 9 Sugar Analyses............................................................................................. 9 R esu lts........................................................................................................................... 10 P. remota Embryos Undergo Substantial Maturation Drying 10 Embryonic Water Potential Drops Below -15 MPa In P la n ta 11 Vacuolar Volume is Reduced and Organelles De-Differentiated During Maturation......................................................................................... 12 Sucrose is the Predominant Sugar and Small Concentrations of Trehalose were Detected During Development..................................... 12 D iscussion.................................................................................................................... 13 Chapter 3: Germination of Maturing Embryos of Pritchardia remota (Kuntze) Becc.: Indications from Developmental and Desiccation Stress Experiments 30 Introduction.................................................................................................................. 30 Materials and M ethods.............................................................................................. 32 Plant M aterial............................................................................................... 32 Embryo Germination in Tissue Culture................................................... 32 Desiccation Tolerance of Developing Embryos................................... 32 Oxygen Consumption by Developing E m bryos.................................... 33 Trolox Equivalent Antioxidant Capacity (TEAC) of Developing E m bryos.......................................................................................................... 34 R esu lts........................................................................................................................... 36 Excised Embryos Germinate Early in the Developmental Program .. 36 Critical Water Contents Decrease Throughout Development but Embryos are not Tolerant Extreme Drying or Prolonged Storage at High Water Contents.................................................................................... 36 Metabolic Rates in P. remota Slow Considerably During Development.................................................................................................. 38 Vll TEAC Activity Peaks Before Mass Maturity Then Declines as Metabolic Rates Decline............................................................................. 39 D iscussion.................................................................................................................... 39 Chapter 4: Dormancy Mechanisms in Seeds of Pritchardia remota (Kuntze) Becc., An Endangered Palm Endemic to Hawaii........................................................................... 61 Introduction.................................................................................................................. 61 How Long Does it Take for Palm Seeds to Germinate?..................... 61 Environmental Factors That Influence Dormancy and Germination in P a lm s........................................................................................................... 62 Dormancy Mechanisms in Palm s.............................................................