UNDERSTANDING THE FLORAL TRANSITION IN AQUILEGIA COERULEA AND DEVELOPMENT OF A TISSUE CULTURE PROTOCOL A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Plant Science By Timothy A. Batz 2018 SIGNATURE PAGE THESIS: UNDERSTANDING THE FLORAL TRANSITION IN AQUILEGIA COERULEA AND DEVELOPMENT OF A TISSUE CULTURE PROTOCOL AUTHOR: Timothy A. Batz DATE SUBMITTED: Summer 2018 College of Agriculture Dr. Bharti Sharma Thesis Committee Co-Chair Department of Biological Sciences Dr. Valerie Mellano Thesis Committee Co-Chair Plant Science Department Dr. Kristin Bozak Department of Biological Sciences ii ACKNOWLEDGEMENTS I would like to thank the many faculty, family, and friends who helped me enormously throughout my master’s program. The endless support, mentorship, and motivation was crucial to my success now and in the future. Thank you! Dr. Mellano, as my academic advisor and mentor since my freshman year at Cal Poly Pomona, I greatly appreciate your time and dedication to my success. Thank you for guiding me towards my career in science. Dr. Sharma, thank you for taking me into your lab and taking the role of research mentor. Your letters of support allowed me the opportunities to grow as a scientist. Dr. Bozak, I always had a pleasure meeting with you for advice and constructive critiques. Thank you for the time spent reading my statements and the opportunities to gain presentation skills by lecturing in your classes. Dr. Still, thank you for introducing me into the world of research. Thank you for helping me understand the work and input required for scientific success. To lab mates Summer Blanco, Jesus Preciado, and Michael Speck, thank you for helping make research enjoyable. It was a pleasure to learn and grow as scientists with you all! To Dr. Adler, Dr. Buckley, Dr. Valdez, and Airan Jansen of the Cal Poly RISE Program, thank you for instilling values of hard work, determination, and cooperation in us budding scientists. It was pleasure learning from your experiences in science. To Dr. Washburn, Dr. Columbus, and Dylan Cohen of the Rancho Santa Ana Botanic Garden, my work relied heavily on your diligent training and generous allowance of the facilities and instruments. Thank you all for your support and collaboration. A mi familia amorosa y apoyoso, gracias por su amor y sacrificio. Eso me motivó a perseguir mis objetivos. Les dedico este trabajo a todos ustedes. iii ABSTRACT Vernalization, or exposure to prolonged cold, is a trigger which initiates the transition to flowering in many plants including crop and ornamental species. This environmental change in temperature is prevalent at high altitude environments and has become a strong selection pressure influencing the reproductive biology of plants. Species in the genus Aquilegia are primarily found in mountainous areas of Europe, Asia, and North America. In this study, we are using A. coerulea as a model system to understand the morphological and developmental changes that the shoot apical meristem undergoes during the transitioning from a vegetative to reproductive meristem. Plants were grown to maturity and then vernalized at 6°C for 4 weeks, then subsequently transferred to greenhouse conditions at 24°C. Meristems were dissected at each week throughout the treatments and prepared for histology and scanning electron microscopy. Results indicate that the formation of the reproductive inflorescence begins at the third week of vernalization. Floral meristems are established by the fourth week of greenhouse conditions. All vernalized plants flowered around the fourth week of greenhouse conditions while nonvernalized plants failed to do so. We conclude that vernalization is essential for flowering in A. coeruela. The ability to insert genes into plant models in vitro is a powerful tool for genetic investigations into morphological traits and developmental processes. A tissue culture protocol to establish transgenic plants is required. Seeds of A. coerulea were germinated in vitro and used as sterile explant tissues. Cotyledons grown in medium containing a [1.0/1.0mgL-1] ratio of auxin and cytokinin plant hormones, BAP/2,4-D (6- iv Benzylaminopurine/2,4-Dichlorophenoxyacetic acid) produced reliable callus growth. Shoot proliferation from callus was promoted by a BAP/2,4-D ratio of [0.5/0.25mgL-1]. A liquid medium containing [1.0mgL-1] of the auxin IAA (Indole-3-acetic acid) was found to promote root organogenesis from shooted callus in combination with 7 days of dark treatment. With further optimization of media, a tissue culture protocol can be established to genetically transform and regenerate A. coerulea plantlets in vitro, a technique useful in forward and reverse genetics experiments performed in the fields of plant evolution and developmental biology. v TABLE OF CONTENTS Signature Page…………..….……………………………………………………………ii Acknowledgements .......................................................................................................... iii Abstract ............................................................................................................................. iv List Of Tables ................................................................................................................. viii List Of Figures................................................................................................................... x Abbreviations………………………………………..………………………………….xii Chapter 1 Introduction..................................................................................................... 1 Background………………………………………………………………………..1 Literature Review………………………………………………………………….3 Chapter 2 Morphological & Developmental Studies To Understand The Floral Transition In Aquilegia coerulea.................................................................................... 11 Background………………………………………………………………………11 Objectives & Hypotheses…………………………………………………….......14 Materials & Methods…………………………………………………………….15 Results……………………………………………………………………………30 Conclusions...…………………………………………………………………….43 Chapter 3 Tissue culture of Aquilegia coerulea ............................................................ 44 Background………………………………………………………………………44 Objectives & Hypotheses………………………………………………………...45 Materials & Methods…………………………………………………………….46 vi Results…………………………………………………………………………....58 Conclusions...…………………………………………………………………….79 References……………………………………………………………………………….82 vii LIST OF TABLES CHAPTER 2: MORPHOLOGY OF THE FLORAL TRANSITION Table 1. Treatment of plants and number of meristems collected for histological analysis. ........................................................................................................................................... 17 Table 2. Tissue preparation protocol for histology .......................................................... 19 Table 3. Staining procedure using Sharman series ..................................................... 20-21 Table 4. Timepoints and conditions of plant collection and meristem dissection for SEM analysis before, during, and after vernalization treatment ................................. 23 Table 5. Dehydration timetable for dissected meristems in preparation for SEM ........... 26 CHAPTER 3: IN VITRO REGENERATION OF AQUILEGIA COERULEA Table 6. Solutions for sterilization of A. coerulea seeds .................................................. 47 Table 7. A. coerulea germination media: Ingredients and their respective amounts ....... 48 Table 8. Ratios and concentrations of BAP and 2,4-D used in callogenesis of A. coeruela cotyledons. ......................................................................................................... 51 Table 9. Concentration of AgNO3 used in callus media .................................................. 52 Table 10. Combinations of BAP, 2,4-D, and AgNO3 for callus induction of A. coerulea cotyledons ......................................................................................................................... 53 Table 11. Combinations of BAP, 2,4-D, used for shoot and root induction in A. coerulea callus tissues....................................................................................................... 55 Table 12. Treatments of BAP, 2,4-D, and darkness (aluminum foil) used for root induction in A. coerulea shooted callus ............................................................................ 56 viii Table 13. Ratios of BAP/2,4-D medias A-I and their resulting percentage of induction and organogenesis in A. coerulea cotyledons after 6 weeks of culture ............................ 63 Table 14. Media treatments with their respective ratios of BAP, 2,4-D, and AgNO3 ..... 67 Table 15. Ratios of BAP/2,4-D in callus medias and their resulting percentage of shoot and root induction in A. coerulea cotyledons after 9 weeks of culture ................... 75 Table 16. Number of roots produced from A. coerulea shooted calluses in BAP/2,4-D and IAA liquid media ± 7 days of darkness ................................................... 77 ix LIST OF FIGURES CHAPTER 2: MORPHOLOGY OF THE FLORAL TRANSITION Figure 1. Approximate weekly growth rate of A. coerulea seedlings at 24°C. ............... 16 Figure 2. Sharman stain series solutions .......................................................................... 22 Figure 3. Stereoscopic dissection of A. coerulea meristems. ........................................... 24 Figure 4. Meristems