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Storage and Propagation Characteristics of Miscanthus X Giganteus by Cassandra Doll Downey

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Storage and Propagation Characteristics of Miscanthus x giganteus by Cassandra Doll Downey A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Plant Agriculture Guelph, Ontario, Canada © Cassandra Downey, September, 2018 ABSTRACT STORAGE AND PROPAGATION CHARACTERISTICS OF MISCANTHUS X GIGANTEUS Cassandra Doll Downey Advisor: University of Guelph 2018 Dr. Andrew Maxwell Phineas Jones Miscanthus x giganteus Anderss. exhibits favourable characteristics as a dedicated second- generation biomass feedstock. Propagation of this sterile species occurs mainly through micropropagation or rhizome cuttings, resulting in high establishment costs. This thesis sought to investigate the improvement of storage duration for field-harvested rhizomes, prolongment of callus regeneration potential through impairment of the phenylpropanoid biosynthetic pathway, and the induction of microrhizomes through the modification of media compositions. It was found that plant growth from rhizomes yielded similar results after spring and autumn harvests, plantlet regeneration was achieved from calli after 12 months in culture when media was supplemented with 2-aminoindan-2-phosphonic acid, and microrhizomes were successfully induced over a 10- week incubation period in liquid media supplemented with 8% sucrose, 6-benzylaminopurine, and 1-naphthaleneacetic acid. These findings may be used to improve conservation, reduce establishment costs, and allow for a greater supply of Miscanthus biomass in the marketplace. Acknowledgements I would like to begin by acknowledging my advisor, Dr. Andrew Maxwell Phineas Jones, for providing me with the opportunity to pursue my MSc. His guidance, understanding, and encouragement for me to engage in activities and projects both within and outside the scope of my thesis was valuable and greatly appreciated. I would also like to acknowledge my co-advisor, Dr. Praveen Kumar Saxena, for allowing me with the chance to gain valuable experience working with the Gosling Research Institute for Plant Preservation before beginning my MSc; Dr. Bill Deen, who helped with revising this thesis by incorporating his expertise in biomass crop production; Dr. Mahendra Thimmanagari of OMAFRA for supporting this research, providing valuable insight into biomass cultivation, and agreeing to be a member on my defence committee; and Dr. Ralph Martin for agreeing to chair my defence exam. I also would like to show my appreciation to our funding partner, BioFuelNet, for their considerate financial support. I am thankful for David Smith and the team at All Weather Farming Inc. for their generosity, depth of knowledge, and enthusiasm for this project. I would also like to thank Henk Williams and the Elora Research Station for providing us with additional planting material and support; Dr. Xin Hu at NSF International for helping us process samples in a timely manner; Dr. Michelle Edwards for her guidance in statistical analyses; Dr. Gale Bozzo for permitting us to use his cold storage and freezer space whenever we were in a bind; and Tara Israel for helping me coordinate my defence exam and complete necessary administrative tasks. I would like to express my gratitude for the Gosling Research Institute for Plant Preservation and the fantastic students and staff I’ve had the pleasure of working alongside. You are all extremely talented scientists who have inspired me to continually pursue truth. I am especially grateful for Bob Nichols, who was always willing to answer my many questions; Dr. Mukund Shukla, who took the time to teach me various tissue culture techniques and experimental design; Dr. Elena Popova, who familiarized me with cryopreservation technologies; Dr. Sherif Sherif, who encouraged me to take part in worthwhile teaching assistantships; and Lauren Erland, who offered her metabolite quantification services to us without grievance, even when she was overwhelmed with other requests and commitments. The Department of Plant Agriculture has a great sense of community, and I would like to thank the Plant Agriculture Social Committee for allowing me to be more involved with this body. Also, iii an abundance of thanks to Dr. Jay Subramanian for allowing me to TA his tissue culture course during my MSc so I could extend my passion for plant tissue culture to others. I am forever grateful for my parents, Glen and Karen Downey, who modelled sacrifice and exceptional work ethic to provide me with the opportunities to fulfill my dreams; for my many friends and family who continue to love and support me; and for my significant other who perfectly balances me out. And above all else, I acknowledge and praise God. 1 Thessalonians 5:16-18. iv Table of Contents 1: Introduction……………………………………………………………………………………..1 1.1 Summary………………………………………………………………………………1 1.2 Hypotheses and objectives……………………………………………………………..5 2: Literature Review……………………………………………………………………………….6 2.1 Miscanthus spp………………………………………………………………………...6 2.1.1 Miscanthus ancestry and taxonomy………………………………………….6 2.1.2 Aboveground physiology and morphology………………………………….6 2.1.3 Rhizome physiology and morphology……………………………………….7 2.2 Seedless triploid propagation………………………………………………………......9 2.2.1 Storage and establishment properties………………………………...………9 2.2.2 Storage and carbohydrate dynamics………………………………………..11 2.3 Micropropagation…………………………………………………………………….13 2.3.1 Fundamentals of micropropagation………………………………………...13 2.3.2 Miscanthus micropropagation………………………………...……………13 2.3.3 Miscanthus micropropagation challenges………………………...………..14 2.3.4 Phenylpropanoid biosynthetic pathway and in vitro culture…………...…...14 2.4 Microrhizomes……………………………………………………………………….16 2.4.1 Introduction……………………………………………………………...…16 2.4.2 Carbohydrate requirements………………………………………………...17 2.4.3 Carbohydrate and plant growth regulator interactions………………...........18 2.4.4 Synthetic seeds…………………………………………………………..…21 3: Storage and propagation of rhizomes from five Miscanthus x giganteus genotypes grown in southwestern Ontario…………………………………………………………………………….24 Abstract…………………………………………………………………………………..24 3.1 Introduction…………………………………………………………………………..24 3.2 Materials and methods ……………………………………………………………28 3.2.1 Harvest – Port Ryerse……………………………………………………....28 3.2.2 Harvest – Elora…………………………………………………...………...32 3.2.3 Sample preparation…………………………………………………...…….34 3.2.4 Cold storage conditions…………………………………………………….36 3.2.5 Greenhouse conditions……………………………………………………..37 3.2.6 Planting conditions…………………………………………………………38 3.2.7 Growth parameters…………………………………………………………38 3.2.8 Total moisture content……………………………………………………...39 3.2.9 Sample preparation………………………………………………………....40 3.2.10 Total starch quantification………………………………………………...40 3.2.11 Reducing and non-reducing sugar quantification………………………....41 3.2.12 Experimental design and statistical analysis………………………………42 3.3 Results………………………………………………………………………………..43 3.3.1 Rhizome viability……………………………………………………….….43 3.3.2 Culm emergence…………………………………………………………....44 3.3.3 Tiller height………………………………………………………………...47 3.3.4 Tiller number…………………………………………………………….....50 3.3.5 Leaf number…………………………………………………………….….52 3.3.6 Stem node number……………………………………………………….....55 v 3.3.7 Stem diameter……………………………………………………...……….58 3.3.8 Chlorophyll content………………………………………………………...61 3.3.9 Moisture content………………………………………………………...….61 3.3.10 Total starch content……………………………………………………….63 3.3.11 Total soluble carbohydrate content………………………………………..66 3.3.12 Total soluble carbohydrates consisting of D-glucose………………….......68 3.3.13 Total soluble carbohydrates consisting of sucrose………………………...71 3.3.14 Total soluble carbohydrates consisting of D-fructose………………..…....74 3.4 Discussion…………………………………………………………………………....76 3.5 Conclusions…………………………………………………………………………..81 3.6 Acknowledgements………………………………………………………………..…81 4: Improving regeneration capacity of Miscanthus x giganteus ‘M161’ calli through inhibition of the phenylpropanoid biosynthetic pathway…………………………………………..…………..82 Abstract……………………………………………………………………………..……82 4.1 Introduction…………………………………………………………………………..82 4.2 Materials and methods…………………………………………………………….….85 4.2.1 Plant material……………………………………………………………….85 4.2.2 Callus induction and multiplication………………………………………...85 4.2.3 Callus morphology assessment……………………………………………..86 4.2.4 Development of regenerants and embryo-like structures…………………...86 4.2.5 Soluble phenolic content…………………………………………………...87 4.2.6. Regeneration and plantlet formation……………………………………….88 4.2.7. Experimental design and statistical analysis…………………………..…...88 4.3 Results………………………………………………………………………………..88 4.3.1 Callus morphology frequencies…………………………………………….88 4.3.2 Regenerant number…………………………………………………….…...95 4.3.3 Soluble phenolic content…………………………………………………...98 4.3.4 Plantlet development……………………………………………….………98 4.4 Discussion……………………………………………………………………….…...99 4.5 Conclusions…………………………………………………………………………102 4.6 Acknowledgements…………………………………………………………………102 5: In Vitro Induction and Encapsulation of Miscanthus x giganteus Anderss. Microrhizomes….........................................................................................................................103 Abstract…………………………………………………………………………………103 5.1 Introduction…………………………………………………………………………103 5.2 Materials and methods……………………………………………………………....105 5.2.1 Culture initiation…………………………………………………………..105 5.2.2 Microrhizome induction………………………………………………..…106 5.2.3 Physiological assessment………………………………………………....106 5.2.4 Growth capacity…………………………………………………………..107
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