MICROPROPAGATION OF THE RELICT GENUS CERCIDIPHYLLUM (CERCIDIPHYLLACEAE) A Thesis Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Craig Henry Carlson In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Major Department: Plant Sciences August 2013 Fargo, North Dakota North Dakota State University Graduate School Title Micropropagation of the Relict Genus Cercidiphyllum (Cercidiphyllaceae) By Craig Henry Carlson The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of MASTER OF SCIENCE SUPERVISORY COMMITTEE: Dr. Todd P. West Chair Dr. Harlene Hatterman-Valenti Dr. Wenhao Dai Dr. Joseph Zeleznik Approved: 11/21/14 Dr. Richard Horsley Date Department Chair ABSTRACT Focusing on various aspects of micropropagation with respect to well represented genotypes within Cercidiphyllum , this study is an attempt to broaden the experimental knowledge of the genus. The Tertiary relict Cercidiphyllum (Cercidiphyllaceae) is endemic to Japan and China and consists of two deciduous tree species, common katsura, C. japonicum Sieb. & Zucc. and the hiro-ha-katsura, C. magnificum Nakai. To date, there exists no literature on the in vitro requirements of C. magnificum and limited information on C. japonicum . Prized as specimen trees, the importance of Cercidiphyllum extends beyond its ornamental merit and regarded a multifaceted genetic resource. Subsequent protocols and analysis will not only be useful for nursery production and cultivar improvement but aid in ongoing conservation efforts of this rare genus. iii ACKNOWLEDGEMENTS This work would not have been possible without the support and patience of my advisor, Dr. Todd P. West, who has pushed me to becoming a more thoughtful and detailed scientist. I would like to thank the curators and staffs of the following institutions for access to their extensive and invaluable living collections: the Arnold Arboretum of Harvard University, Jamaica Plain, Massachusetts, the Morton Arboretum, Lisle, Illinois, The Hoyt Arboretum, Portland, Oregon, the United States National Arboretum, Washington, District of Columbia, and the North Dakota State University Dale E. Herman Research Arboretum. I would especially like to thank Dr. Dil Thavarajah in the School of Food Systems at North Dakota State University for her generosity and knowledge of secondary plant metabolites, as well as Philip Knutson for providing expert technical assistance, and my graduate committee: Drs. Harlene Hatterman- Valenti, Joseph Zeleznik, and Wenhao Dai. iv DEDICATION I especially thank my dad for always being a positive force in my life as well as my brother for all of the “brother projects” that have made us so very close. My propensity to dig deeper into the science of horticulture can be attributed to my long-time mentor, Neal Holland. Many interesting topics within this thesis have been hashed-out with my best friend, Adam Ogden (over a decade!). Arête! Axios! Alethes! To Ken Parker for the companionship, Josh Silbernagel for the danger, the Echevaria clan for loyalty, Ian Johnson for the Beam, Shane Ocshner for the riffs, Michael Dosmann for the wit, Hannah Passolt for the goofiness, Grady Reed for the shenanigans, and to Ana and Baird Heilman, Whitney Harchencko, Veronica Brotons, and all the other NDSU PLSc Latinos for your passion. Without my lab mate, Juan Ramon Franco-Coronado, I would have never heard Illmatic. You kept my head to the grindstone and taught me about the good things in life. You are a good man and I hope to be your friend and colleague for many years to come. Next time I see you, I hope we’re Stillmatic. Adios, mi amigo. To you, Lo. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………...iii ACKNOWLEDGMENTS………….…………………………………………………...……......iv DEDICATION…………..………………………………………………………………………...v LIST OF TABLES.………………………………………….……………………………………ix LIST OF FIGURES………………………………………………………...…………………….xi LIST OF DEFINITIONS…………………………………………………..………………….....xii LIST OF APPENDIX TABLES………………………………………………………………...xiv CHAPTER 1. INTRODUCTION………………………………………………………………….1 Taxonomy …………………………………………………………………………………1 Habitat and Genetics………………………………………………………………………3 Cultivation…………………………………………………………………………………7 Micropropagation …………………………………………………………………………9 Carbon Source ……………………………………………………………………………11 Secondary Metabolites…………………………………………………………………...13 Phenolics …………………………………………………………………………………14 Somaclonal Variation….…………………………………………………………………16 Alginate Encapsulation ………………………………………………………………….16 References ……………………………………………………………………………….18 CHAPTER 2. DEVELOPMENT OF THE MICROPROPAGATION PROTOCOL…………...25 Abstract…………………………………………………………………………………..25 Introduction………………………………………………………………….…............... 26 Materials and Methods………………………………………………………................... 29 vi Source of Explant Material ………………………………………………………29 Nutrient Media and Hormones …………………………………………………..30 Procedures ……………………………………………………………………….32 Data Collection and Statistical Analysis …………………………………………33 Results and Discussion…………………………………………………………………..34 Ex Vitro Initiation………………………………………………………………..34 In Vitro Initiation………………………………………………………………...36 Proliferation……………………………………………………………………...44 In Vitro Rooting………………………………………………………………….44 Conclusion……………………………………………………………………………….45 References………………………………………………………………………………..47 CHAPTER 3. REDUCING PHENOLIC COMPOUNDS IN CERCIDIPHYLLUM JAPONICUM CULTURES………………………………….…………………………………..50 Abstract……………………………………………………………………….………….50 Introduction………………………………………………………………………………50 Materials and Methods…………………………………………………………………...52 Source of Explant Material ………………………………………………………52 Disinfestation and Preparation of Plant Material………………………………...52 Data Collection and Statistical Analysis…………………………………………54 Results and Discussion ………………………………………………………………….54 Conclusion....…………………………………………………………………………….62 References ……………………………………………………………………………….63 CHAPTER 4. IDENTIFICATION AND QUANTIFICATION OF MAJOR PHENOLIC ACIDS IN CERCIDIPHYLLUM USING HPLC-DAD..………………………………………..65 vii Abstract…………………………………………………………………………………..65 Introduction…...………………………………………………………….………………65 Materials and Methods……...…………………………………………….……………...68 Source of Explant Material ………………………………………………………68 Standards…………………………………………………………………………68 Phenolic Extraction and Quantification …………………………………………69 Data Collection and Statistical Analysis …………………………………………71 Results and Discussion…………………………………………………………………...71 Conclusion……………………………………………………………………………….79 References ………………………………………………………………………………..80 CHAPTER 5. ALGINATE ENCAPSILATION OF CERCIDIPHYLLUM (CERCIDIPHYLLACEAE)………………………………………………………………...…....83 Abstract…………………………………………………………………………………..83 Introduction ………………………………………………………………………………83 Materials and Methods…………………………………………..………….……………86 Source of Explant Material ………………………………………………………86 Aseptic Nutrient Media ………………………………………………………….86 Nutrient Media and Hormones……………….…………………………………..87 Alginate Encapsulation …………………………………………………………..87 Data Collection and Statistical Analysis ………………………………………88 Results and Discussion …………………………………………………………………..88 Conclusion ……………………………………………………………………………….93 References ……………………………………………………………………………….94 APPENDIX. ANOVA OUTPUT ……………………………………………………………….97 viii LIST OF TABLES Table Page 2-1. Effects of BA and presence of 3% (w/v) sucrose on percent ex vitro initiation of dormant buds of three C. japonicum accessions after 2 w incubation (23±1 ºC in light)…………………………………………………………………………..…………36 2-2. Effects of basal salt and nutrient formulation, cytokinin concentration, and presence of IBA on mean shoot length, leaf length and width of C. japonicum Acc. No. TS9821-6 after 4w of incubation (23±1ºC in light)…………………………………….. 38 2-3. Effects of cytokinin type and concentration, and presence of auxin on mean shoot length, shoot number, and leaf length, for elongation of C. japonicum Acc. No. TS9821-6 from budbreak after 4w of incubation (23±1ºC in light).……….….……..….39 2-4. Effects of basal salt on mean shoot length, shoot number, and bud number of Acc. No. TS9821-6 from budbreak after 4w of incubation (23±1ºC in light)……………….. 40 2-5. Effects of cytokinin type concentration and auxin on mean shoot length, shoot number, and bud number of three Cercidiphyllum accessions after 4w of incubation (23±1ºC in light).……………………………………………...……………..41 2-6. Effects of basal salt on mean shoot length, shoot number, and bud number of three Cercidiphyllum accessions after 4w of incubation (23±1ºC in light)……………………42 2-7. Treatment effects of auxin type and concentration on root growth of C. japonicum Acc. No. TS9821-6 after 4w incubation (23±1ºC in light)..……………………...……...45 3-1. Effects of 1% (w/v) presoaking treatment solutions and presoak time on shoot length, shoot number, and bud number on C. japonicum Acc. No. TS9821-6 after 4w incubation (23±1ºC in light)………..……………………………………….………..55 3-2. Effects of incorporated treatment concentrations on shoot length, shoot number, and bud number on C. japonicum Acc. No. TS9821-6 after 4w of incubation (23±1ºC in light)…………………………………………………………………………………..….56 3-3. Effects of carbon source and concentration on leaf and shoot growth of C. japonicum Acc. No. TS9821-6 after 4w of incubation (23±1ºC in light)…………….58 4-1. Genotype accession number, species, origin, and tissue type of 10 Cercidiphyllum genotypes for HPLC-DAD analysis.………………………………………………….…70 4-2 The identification of phenolics in dried leaf and stem tissue samples