ABSTRACT A MORPHOLOGICAL AND ANATOMICAL INVESTIGATION OF SHOOT APICAL MERISTEMS EXPRESSING RING FASCIATION IN CLARKIA TEMBLORIENSIS by Kilian TysonMayer Fasciation is a growth abnormality in shoot and root meristems of many vascular plants which leads to the development of enlarged, supernumerary, and misshapen stems, leaves, floral organs, and fruits. Artificial selection for modified phenotypes has occurred since the dawn of agriculture and is responsible for many commercially available fruits and vegetables today. Clarkia tembloriensis is a California wildflower expressing fasciation in certain populations when grown under abnormal environmental conditions in a laboratory setting. This makes it an excellent model organism to study the effect of abnormal environmental conditions on the expression of fasciation. In this investigation, shoot apex morphology and anatomy were observed throughout the development of wild- type plants and those expressing fasciation via scanning electron microscopy (SEM) and light microscopy. These observations revealed morphological and anatomical abnormalities of shoot apical meristem (SAM) development including an enlarged ring- shaped meristem, abnormal organs, and callus tissue. Comparing these observations with current literature for genetic and hormonal interactions in the SAM of plants, it is proposed that ring fasciation and callus formation in Clarkia tembloriensis occur as a result of abnormal environmental conditions via abnormal signaling of phytohormones and other developmental regulators which function in maintenance of meristem size and organ initiation. A MORPHOLOGICAL AND ANATOMICAL INVESTIGATION OF SHOOT APICAL MERISTEMS EXPRESSING RING FASCIATION IN CLARKIA TEMBLORIENSIS A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science by Kilian TysonMayer Miami University Oxford, Ohio 2019 Advisor: Nancy L. Smith-Huerta Reader: Alfredo J. Huerta Reader: Carolyn H. Keiffer ©2019 Kilian TysonMayer This Thesis titled A MORPHOLOGICAL AND ANATOMICAL INVESTIGATION OF SHOOT APICAL MERISTEMS EXPRESSING RING FASCIATION IN CLARKIA TEMBLORIENSIS by Kilian TysonMayer has been approved for publication by The College of Arts and Science and Department of Biology ____________________________________________________ Nancy L. Smith-Huerta ______________________________________________________ Alfredo J. Huerta _______________________________________________________ Carolyn H. Keiffer Table of Contents Introduction ........................................................................................................................1 Materials and Methods ......................................................................................................7 Plant Culture .....................................................................................................................7 Scanning Electron Microscopy, Light Microscopy, and Photography ............................8 Observations of Growth in the Field ................................................................................8 Results .................................................................................................................................9 Preliminary Observations of Abnormal Growth ..............................................................9 SEM Observations of SAM Development .......................................................................9 LM Observations of SAM Development .......................................................................11 Abnormal Floral Features ...............................................................................................12 Observations of Growth in the Field ..............................................................................13 Discussion..........................................................................................................................13 Preliminary Observations of Abnormal Growth ............................................................13 SEM Observations of SAM Development .....................................................................14 LM Observations of SAM Development .......................................................................18 Abnormal Floral Features ...............................................................................................20 Observations of Growth in the Field ..............................................................................21 Conclusion ........................................................................................................................22 Figures ...............................................................................................................................23 References .........................................................................................................................35 Appendices ........................................................................................................................44 Appendix 1 – Conceptual Diagram ................................................................................44 Appendix 2 – Apex Removal Experiment .....................................................................45 iii List of Tables Table 1. GPS Coordinates of Populations Visited...........................................................9 Supplemental Table 1. Results of Statistical Analyses for Apex Removal .................48 Supplemental Table 2. Summary of Apex Removal Experiment Data.......................48 iv List of Figures Figure 1. Digital Photographs of Morphological Abnormalities .................................23 Figure 2. SEM Images of Early SAM Development .....................................................24 Figure 3. SEM Images of Mid-Stage SAM Development .............................................25 Figure 4. SEM Images of Late SAM Development .......................................................26 Figure 5. SEM Images of Apical Callus Development..................................................27 Figure 6. LM Images of Early SAM Development .......................................................28 Figure 7. High-Magnification LM Images of Mid-Stage SAM Development ............29 Figure 8. Low-Magnification LM Images of Mid-Stage SAM Development .............30 Figure 9. LM Images of Callus Tissue Development ....................................................31 Figure 10. SEM Images of Abnormal Floral Features .................................................32 Figure 11. LM Image of Abnormal Floral Features .....................................................33 Figure 12. LM Images of Ovule and Abnormal Stigma ...............................................34 v Acknowledgements Gratitude is extended to the Willard Sherman Turrell Herbarium at Miami University and its curator, Dr. Michael Vincent, for facilitating the grant which funded my field collections trip. I would also like to thank Ryan O’Dell from the Bureau of Land Management’s Hollister field office for granting access to their land and offering expert field identification and navigation to populations of Clarkia tembloriensis. Much appreciation is owed to Dr. Richard Edelmann and Matthew Duley of Miami University’s Center for Advanced Microscopy and Imaging for their consistent help to perfect sample preparation and imaging techniques. Without the CAMI facility, the SEM course, and their expert input, this project would not have been possible. I would also like to thank Chelsea Obrebski for informative discussions during image analyses and additional assistance in troubleshooting sample preparation and imaging issues. My lab work would likely have taken much longer if Chelsea had not begun to perfect her light microscopy sample preparation procedure a few months prior to mine. I am grateful to my parents, Karen Tyson and Timothy Mayer, for constant encouragement, intermittent financial support, and for setting me on the path to higher education. In addition, I would like to thank Dr. Robert Verb for initially sparking my interest in botany and Dr. Stephen Kolomyjec for consistently going out of his way to help others understand science by promoting a positive, open-discussion approach to educating the general public. Lastly, I would like to thank my advisor Dr. Nancy Smith-Huerta for guidance in all aspects of my project as well as for continued correspondence and academic support despite retiring before the completion of my project. Likewise, I am grateful to my committee members Dr. Alfredo Huerta and Dr. Carolyn Keiffer as well as my former committee member Dr. Daniel Gladish for guidance on experimental design and for helping me to better understand the nuances of experimental botany. vi Introduction Variations in plant form have been the subject of scientific study for centuries to elucidate the details of plant organ development, and one such variation called fasciation has been artificially selected in agriculture for millennia (Moquin-Tandon 1841; Compton 1911; Johnson 1926; Bausor 1937; White 1948; Clark et al. 1993; Taguchi-Shiobara et al. 2001; Taiz 2013). Fasciation is an abnormal growth condition in plants which involves a change in shoot or root axis production from the normally round or polygonal form to one which is enlarged and irregular in shape (White 1948; Taguchi-Shiobara et al. 2001; Iliev and Kitin 2011; Reed et al. 2013). Fasciation can affect the morphology of any organ originating from the growing