MODIFYING TUBER SIZE DISTRIBUTION, LOW TEMPERATURE SWEETENING AND TOLERANCE TO HEAT STRESS IN PROCESSING POTATOES (Solanum tuberosum L.) By DEREK JAMES HERMAN A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Department of Horticulture DECEMBER 2016 © Copyright by DEREK JAMES HERMAN, 2016 All Rights Reserved © Copyright by DEREK JAMES HERMAN, 2016 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of DEREK JAMES HERMAN find it satisfactory and recommend that it be accepted. ___________________________________ N. Richard Knowles, Ph.D. ___________________________________ Mark Pavek, Ph.D. ___________________________________ John Fellman, Ph.D. ___________________________________ Nora Olsen, Ph.D. ii ACKNOWLEDGEMENTS I would like to begin by thanking Dr. Rick Knowles for allowing me to further my education by attaining my Horticulture Doctorate of Philosophy degree in his prestigious laboratory. Your guidance, mentorship and generosity have allowed me to develop into the scientist, student and young man I am today. A special thanks to Dr. Lisa Knowles and Dr. Mohan Kumar, you are both world-class scientists and with your leadership and training I developed the skills necessary to thrive in the world of research. A special thank you to my undergraduate advisor and committee member Dr. John Fellman, your unique perspective and advice drove me to continue my education and for that I am so grateful. To the remainder of my committee, Dr. Mark Pavek and Dr. Nora Olsen, thank you for your advice on anything and everything potato. I would also like to thank all of those who helped with my research at Washington State University, but not limited to: Dr. Daniel Zommick, Zachary Holden, Chandler Dolezal, Dr. Rhett Spear, Dr. Jacob Blauer, Nora Fuller, Josh Rodriguez and Rudy Garza, without your assistance none of this would have been possible. Finally, thank you to my entire family, but most importantly my parents Drs. Doug and Wendy Herman. You instilled in me the hard work ethic and passion needed to be successful. I am forever grateful for the life you have given me, thank you and I love you. I appreciate you all so very much, without you I would not be where I am today. Thank you and Go Cougs! iii MODIFYING TUBER SIZE DISTRIBUTION, LOW TEMPERATURE SWEETENING AND TOLERANCE TO HEAT STRESS IN PROCESSING POTATOES (Solanum tuberosum L.) Abstract by Derek James Herman, Ph.D. Washington State University December 2016 Chair: N. Richard Knowles Controlling tuber size distribution and the propensity for low temperature sweetening (LTS) of processing potatoes are key to optimizing quality and maximizing crop value. My research focused on (1) developing methods to increase tuber set and shift tuber size distribution in cv. Bondi, (2) investigating how low O 2 storage modulates LTS of cvs. Innovator and Russet Burbank, (3) screening cultivars for tolerance to heat stress for retention of LTS-resistant phenotype and (4) determining the mechanism by which heat stress abolishes resistance to LTS. Bondi produces vigorous foliar growth, low tuber set and high yields of large tubers that frequently exceed optimum size for seed and processing markets. We evaluated several methods for altering apical dominance, tuber set and size distribution of Bondi and its maternal parent, Ranger Russet. Gibberellic acid (GA) applied to cut seed prior to planting reduced apical dominance, increased tuber set and decreased average tuber size; however, the optimal concentration to maximally shift tuber size distribution without decreasing marketable yield was iv 4-5-fold greater for Bondi than Ranger. The reduced sensitivity of Bondi to GA was likely inherited from its paternal parent Karaka, which displays similar morphological and developmental traits, indicative of high levels of endogenous GA. Russet Burbank and Innovator are frozen processing cultivars with inherently different susceptibilities to LTS. Here we show how low O 2 storage modulates cold-induced sweetening to reveal metabolic differences in LTS metabolism intrinsic to these cultivars. While storage of tubers in 2.5 kPa O 2 greatly attenuated the initial LTS responses for both cultivars, the effect was only temporary in Innovator. Low O 2 attenuated LTS by inhibiting invertase. Heat stress exacerbated cold-induced sweetening of Ranger and Russet Burbank tubers (LTS-susceptible cultivars), and abolished the inherent LTS-resistance of Sage Russet, GemStar Russet, POR06V12-3 and A02138-2 tubers. Heat stress rendered invertase cold-inducible in the LTS-resistant but non-heat tolerant cultivars/clones. Payette and its maternal parent EGA09702- 2, however, demonstrated considerable tolerance to heat stress for retention of LTS-resistance. Payette’s heat tolerance was conferred by the lack of cold-induction of invertase, similar to Innate ® Russet Burbank (W8) tubers, where silenced invertase activity conferred robust tolerance to heat stress. v Table of Contents Page Acknowledgements………………………………………………………………………… iii Abstract…………………………………………………………………………………….. iv List of Tables……………………………………………………………………………….. x List of Figures………………………………………………………………………………. xi Abbreviations……………………………………………………………………………….. xiii Introduction…………………………………………………………………………………. 1 References………………………………………………………………………………. 5 Chapter 1 : DIFFERENTIAL SENSITIVITIES OF GENETICALLY RELATED POTATO CULTIVARS TO TREATMENTS DESIGNED TO ALTER APICAL DOMINANCE , TUBER SET AND SIZE DISTRIBUTION Abstract…………………………………………………………………………………. 8 Introduction……………………………………………………………………………... 10 Materials and Methods………………………………………………………………….. 13 Synopsis of Trials…………………………………………………………………… 13 Seed Preparation and Treatments…………………………………………………… 14 Field Plot Design and Maintenance………………………………………………… 15 Plant Establishment, Stem Counts, Harvesting and Sorting………………………... 15 Data Analysis and Presentation……………………………………………………... 16 Results…………………………………………………………………………………... 16 GA x BA Dose Response…………………………………………………………… 16 CV x GA Dose Response…………………………………………………………… 18 GA x Seed Age Response…………………………………………………………... 22 vi Discussion………………………………………………………………………………. 24 Acknowledgements……………………………………………………………………... 29 References………………………………………………………………………………. 30 Figures and Tables……………………………………………………………………… 36 Chapter 2: LOW OXYGEN STORAGE MODULATES INVERTASE ACTIVITY TO ATTENUATE COLD -INDUCED SWEETENING AND LOSS OF PROCESS QUALITY IN POTATO (Solanum tuberosum L.) Abstract……………………………………………………………………………….… 53 Introduction……………………………………………………………………………... 55 Materials and methods………………………………………………………………….. 57 Plant Materials, Storage Temperature, and Controlled Atmosphere Regimes……... 57 Whole Tuber Respiration…………………………………………………………… 58 Sprouting, Fry Process Quality, and Tissue Sample Preparation…………………... 58 Carbohydrate Analysis……………………………………………………………… 59 Invertase and Starch Phosphorylase Activities……………………………………... 60 Data Analysis and Presentation……………………………………………………... 61 Results…………………………………………………………………………………... 61 Tuber Respiration…………………………………………………………………… 61 Tuber Dormancy and Sprouting…………………………………………………….. 63 Process Quality (Fry Color, Reducing Sugars, Sucrose)…………………………… 63 Invertase and Starch Phosphorylase Activities……………………………………... 66 Discussion………………………………………………………………………………. 68 Conclusions……………………………………………………………………………... 74 Acknowledgements……………………………………………………………………... 74 vii References………………………………………………………………………………. 75 Figures and Tables……………………………………………………………………… 84 Chapter 3: HEAT STRESS AFFECTS CARBOHYDRATE METABOLISM DURING COLD -INDUCED SWEETENING OF POTATO (S OLANUM TUBEROSUM L.) Abstract…………………………………………………………………………………. 95 Introduction……………………………………………………………………………... 96 Materials and methods………………………………………………………………….. 98 Plant Material……………………………………………………………………….. 98 In-season Heat Stress Studies………………………………………………………. 98 Postharvest Handling and Storage………………………………………………….. 101 Tissue Sampling and Process Quality Assessment…………………………………. 101 Sucrose and Reducing Sugar Analyses……………………………………………... 102 Dormancy Break……………………………………………………………………. 103 PHHS Studies………………………………………………………………………. 103 Enzyme Analysis…………………………………………………………………… 104 RNA Extraction and qPCR…………………………………………………………. 105 Data Analysis and Presentation…………………………………………………….. 106 Results…………………………………………………………………………………... 107 In-season Heat Stress Studies………………………………………………………. 107 Tuber Set, Size, Yield and Raw Quality……………………………………………. 108 At Harvest Process Quality and Reducing Sugar Accumulation During LTS……… 109 Dormancy Break and Sprout Growth………………………………………………. 111 PHHS Studies………………………………………………………………………. 112 viii Invertase and Starch Phosphorylase Activities……………………………………... 113 qPCR Analysis of Invertase and Invertase Inhibitors………………………………. 115 Discussion………………………………………………………………………………. 116 Acknowledgements……………………………………………………………………... 124 References………………………………………………………………………………. 125 Figures and Tables……………………………………………………………………… 136 Appendix………………………………………………………………………………... 154 ix List of Tables Chapter 1 1. Table 1. Seed Treatments and Experimental Design……………………………….. 36 2. Table 2. GA and BA affect Tuber Yield and Size Distribution…………………….. 37 3. Table 3. Yield Responses to GA Concentrations in 2013…………………………... 38 4. Table 4. Yield Responses to GA Concentrations in 2014………………………….. 39 5. Table 5. Effects of GA and Seed Age on Plant Emergence and Yield in 2014…….. 40 Chapter 2 6. Table 1. P-values for