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Influence of Ethylene Suppression and Ripening on Tomato Fruit Color, Firmness, and Volatile Emissions

HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Figures
 Abstract
 Introduction
 Literature review
 Materials and methods
 Results and discussion
 Appendices
 References
 Biographical sketch
 

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INFLUENCE OF ETHYLENE SUPPRESSION AND RIPENING ON TOMATO FRUIT COLOR, FIRMNESS, AND VOLATILE EMISSIONS By SONYA LEONORE STAHL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Sonya Leonore Stahl

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In memory of Dr. Isaac Asimov, whose popular sc ience essays inspired my interest in biochemistry, and in memory of Antonn Dvo k, whose music was my constant companion throughout my e xperiments and writing

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ACKNOWLEDGMENTS I would like to express my gratitude towards my committee chair, Dr. Don Huber, for his patience, encouragement, and wisdom during the construction of this document, during my research, and throughout my graduate program. I would also like thank the other members of my committee for their guidance: I thank Dr. Harry Klee, the Tomato God, for his inspiring course in phytohormones, for his advice, and for the use of his laboratory facilities. I thank Dr. Stephen Talcott for his advice as well, and for valuable assistance with statistics software. I would like to thank Dr. Jiwon Jeong and the West Coast and North Florida Tomato packinghouses for providing me with the fruit used in these experiments. I would also like to thank James Lee, who taught me how to use the laboratory equipment. I am very grateful for the assistance of the members of the Klee Lab who helped me with my research. I thank Dr. Denise Tieman for gas chromatography and Mark Taylor for providing information about laboratory procedure and equipment. I would especially like to thank Michelle Zeigler for instructing me in the techniques I used to sample volatiles and quantify the gas chromatograph results. I would like to thank those who provided physical assistance for me when there were simply too many tomatoes to dice in the time allotted or when I needed an escort to the cold rooms in the middle of the night, Caitlin Mirek, Anastasia Ducky Bower, Jeanmarie Mitchell, Swapna Mony, and Gzregorz Cieslewski. I also express my appreciation towards Mary Frances Folz Donahue and Sjn Myhr, not only for assisting iv

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me with my research but also for the support and understanding they both showed me throughout the writing process. It takes a village to raise a scientist, apparently, but most importantly, I thank my mother, Ellen Stahl, who is a staunch believer in the benefits of fresh fruits and vegetables, for always expecting the best of me, for opening my mind to science at an early age, and for buying me numerous books on exotic produce when I was a child. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF FIGURES .........................................................................................................viii ABSTRACT ...................................................................................................................xviii CHAPTER 1 INTRODUCTION........................................................................................................1 Tomato Ripening..........................................................................................................1 Volatile Profile of Tomato............................................................................................1 Objectives.....................................................................................................................2 2 LITERATURE REVIEW.............................................................................................3 General Information......................................................................................................3 Flavor Chemistry..........................................................................................................5 Ethylene........................................................................................................................8 Inhibitors of Ethylene Synthesis Action.....................................................................11 Effects of 1-MCP on Fruit Volatiles...........................................................................14 Factors Affecting Tomato Volatiles...........................................................................16 3 MATERIALS AND METHODS...............................................................................22 Plant Material..............................................................................................................22 Fruit Color Measurements..........................................................................................23 Analysis of Whole Fruit Firmness..............................................................................23 Evaluation of Volatile Profile of Ripe to Overripe Fruit............................................23 Fruit Selection and Cleaning...............................................................................24 Identifying Onset of Ripe Stage Using Color Measurements.............................24 Determination of Fruit Developmental Stage for 1-MCP Treatment.........................24 Exposure of Tomato Fruit to 1-MCP..........................................................................25 Analysis of the Effects of 1-MCP on Tomato Ripening and Volatile Profiles..........26 Analysis of Tomato Volatiles.....................................................................................27 Statistical Analysis......................................................................................................28 vi

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4 RESULTS AND DISCUSSION.................................................................................29 Evaluation of Volatile Profile of Ripe to Overripe Fruit............................................29 Determination of Fruit Developmental Stage for 1-MCP Treatment.........................32 Fatty-Acid Derived Volatiles..............................................................................35 Carotenoid Derived Volatiles..............................................................................37 Amino Acid Derived Volatiles............................................................................38 Volatiles of Other Derivation..............................................................................39 Response of Tomato Fruit Color and Firmness to 1-MCP.........................................40 Tomato Volatile Production in Ripe, Overripe, and 1-MCP-treated FL-91 Tomato Fruit...........................................................................................................41 Fatty-Acid Derived Volatiles..............................................................................42 Carotenoid Derived Volatiles..............................................................................44 Amino Acid Derived Volatiles............................................................................45 Volatiles of Other Derivation..............................................................................47 General Discussion.....................................................................................................47 APPENDIX A 'BNH586' HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS..................51 B FL-47 HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS.....................59 C FL-91 HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS.....................75 LIST OF REFERENCES.................................................................................................104 BIOGRAPHICAL SKETCH...........................................................................................110 vii

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LIST OF FIGURES Figure page A-1. Hue angle of 'BHN586' tomato fruit during storage at 20C.....................................51 A-2. Hue angle of 'BHN586' tomato fruit during storage at 20C after attaining a hue angle of 50..............................................................................................................52 A-3. Fruit firmness (N) of 'BHN586' tomato fruit during storage at 20C after attaining a hue angle of 50.....................................................................................................53 A-4. Cis-3-hexenal production in ripe BNH586' tomato fruit..........................................54 A-5. Hexanal production in ripe BNH586' tomato fruit...................................................55 A-6. Ethyl vinyl ketone production in ripe BNH586' tomato fruit...................................56 A-7. -ionone production in ripe BNH586' tomato fruit..................................................57 A-8. Isovaleraldehyde production in ripe BNH586' tomato fruit.....................................58 B-1. Firmness (N) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the pink stage and ripened at 20C...................................................59 B-2. Firmness (N) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the turning stage and ripened at 20C...............................................60 B-3. Hue angle () of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the pink stage and ripened at 20C...................................................61 B-4. Hue angle () of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the turning stage and ripened at 20C...............................................62 B-5. Hue angle () of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C..............................................63 B-6. Cis-3-hexenal production in 'FL-47 tomato fruit......................................................64 B-7. Hexanal production in 'FL-47 tomato fruit...............................................................65 B-8. Ethyl vinyl ketone production in 'FL-47 tomato fruit..............................................66 viii

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B-9. Cis-3-hexenol production in 'FL-47 tomato fruit.....................................................67 B-10. Trans-2-hexenal production in 'FL-47 tomato fruit................................................68 B-11. -ionone production in 'FL-47 tomato fruit............................................................68 B-12. 6-methyl-5-hepten-2-one production in 'FL-47 tomato fruit..................................69 B-13. Isovaleraldehyde production in 'FL-47 tomato fruit...............................................70 B-14. 2-methylbutyraldehyde production in 'FL-47 tomato fruit.....................................71 B-15. 3-methyl-1-butanol production in 'FL-47 tomato fruit...........................................72 B-16. 2-phenylethanol production in 'FL-47 tomato fruit................................................73 B-17. 1-nitro-2-phenylethane production in 'FL-47 tomato fruit.....................................74 B-18. 2-isobutylthiazole production in 'FL-47 tomato fruit.............................................74 C-1. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the average of five fruit, +/standard error.................75 C-2. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the average of five fruit, +/standard error.................76 C-3 Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the average of five fruit, +/standard error.................76 C-4. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the average of five fruit, +/standard error.................77 C-5. Hue angle () of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the average of five fruit, +/standard error. C-5-a) Experiment 1. C-5-b) Experiment 2. C-5-c) Experiment 3. C-5-d) Experiment 4...77 C-6. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................78 ix

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C-7. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................78 C-8. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................79 C-9. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................79 C-10. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................................80 C-11. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................80 C-12. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................................81 C-13. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which x

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time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................................81 C-14. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........82 C-15. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..........................................................................................................................82 C-16. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........83 C-17. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........83 C-18. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................84 C-19. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................84 xi

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C-20. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................85 C-21. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................85 C-22. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................86 C-23. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................86 C-24. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................87 C-25. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................87 C-26. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which xii

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time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................................88 C-27. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................88 C-28. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................................89 C-29. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................................89 C-30. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........90 C-31. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..........................................................................................................................90 C-32. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........91 xiii

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C-33. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........91 C-34. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................92 C-35. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................92 C-36. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................93 C-37. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................93 C-38. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........94 C-39. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) xiv

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sample was analyzed. Values represent the average of five fruit, +/standard error..........................................................................................................................94 C-40. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........95 C-41. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........95 C-42. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........96 C-43. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..........................................................................................................................96 C-44. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........97 C-45. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error...........97 xv

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C-46. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................98 C-47. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................98 C-48. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................99 C-49. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error..................99 C-50. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error.........100 C-51. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error........................................................................................................................100 C-52. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue xvi

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value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error.........101 C-53. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.........101 C-54. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error.........102 C-55. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error........................................................................................................................102 C-56. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error.........103 C-57. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.........103 xvii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INFLUENCE OF ETHYLENE SUPPRESSION AND RIPENING ON TOMATO FRUIT COLOR, FIRMNESS, AND VOLATILE EMISSIONS By Sonya Leonore Stahl December 2006 Chair: D. J. Huber Major Department: Horticultural Sciences Ripening parameters of tomato (Lycopersicon esculentum BHN586, FL-47, and FL-91) were evaluated to determine the effects of ethylene suppression on color, firmness, and volatile emissions. Tomato fruit BHN586 were obtained at turning stage and held at 20C until ripe. Fruit color, firmness, and volatile emissions were measured over a period of 16 d following ripening. Hue angle did not change significantly in the 16 d post ripening, but firmness decreased from 5 to 3 N. Production of cis-3-hexenal decreased and ethyl vinyl ketone increased as the fruit progressed from ripe to overripe, and hexenal production fell and rose again. -ionone production remained low during the first week after attainment of ripe stage, but then rose sharply through day 11 and remained high through day 16. Tomato fruit FL-47 were obtained at green, breaker, turning, and pink stages, stored at 20C, and treated with air or 1-methylcyclopropene (1-MCP) at 250 ppb for 12 h at 20C to determine the stage at which ethylene suppression would most delay color xviii

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change but not prevent ripening altogether. Breaker was determined to be the optimal stage for this treatment regimen, due to the difficulty of distinguishing between immature and mature green fruit, and the lack of response to 1-MCP exhibited by pink or turning fruit in this experiment. Production of C6 volatiles (cis-3-hexenal, cis-3-hexenol, trans-2-hexenal, and hexanal) was higher in ripe fruit derived from 1-MCP-treated breaker fruit than in ripe control fruit, but production of the carotenoid-derived volatiles (-ionone and 6-methyl-5-hepten-2-one) was unaffected by 1-MCP treatment at breaker stage. Amino acid derived volatile production was not affected consistently by 1-MCP: isovaleraldehyde and 3-methyl-1-butanol production decreased but 1-nitro-2-phenylethane production increased as a result of 1-MCP treatment. Tomato fruit FL-91 were treated at breaker stage with air or 500 ppb 1-MCP for 12 h at 20C and held until attainment of a hue angle below 50, signifying full ripe. Red color developed more slowly and with less uniformity in 1-MCP-treated fruit, and softening was also delayed by treatment with 1-MCP. Upon reaching full ripe, both ripe control and ripe fruit derived from 1-MCP treated breaker fruit were sampled for volatile production. A second group of control fruit was held until overripe so that volatile production of untreated fruit could be quantified at the same chronological age as treated fruit. Where differences were found between treatments, they were often inconsistent for individual volatiles between experimental replications. Cis-3-hexenal production was observed to decrease in 1-MCP-treated fruit in some instances, but the C6 volatiles were largely unaffected by 1-MCP treatment. In most cases, carotenoid-derived volatiles were also unaffected by 1-MCP treatment, and the effect of 1-MCP treatment on amino-acid derived volatiles was inconsistent and varied. xix

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CHAPTER 1 INTRODUCTION Tomato Ripening The tomato (Lycopersicon esculentum) is a solanaceous fruit that is important to the agricultural economy of the United States of America, and especially in Florida. Many developmental parameters are altered during tomato fruit ripening. Color change occurs as chlorophyll breaks down and lycopene production is initiated. Fruit firmness decreases to a softness acceptable for consumption. The development of red color and the softening of tomato fruit tissue can be delayed by 1-methylcyclopropene (1-MCP), a cyclic alkene that volatizes as a non-toxic, odorless gas. 1-MCP acts by suppressing the response to the hormone ethylene, thereby preventing or delaying processes dependent on ethylene. 1-MCP can therefore be used to determine which processes are directly regulated by that hormone. Volatile Profile of Tomato The flavor of tomatoes is derived from a combination of sugars, acids, and aroma volatiles. While sugars and acids are perceived by the gustatory receptors on the tongue, aroma volatiles are perceived by olfactory receptors and are therefore a smell rather than a taste contributor to tomato flavor. The volatile constituents most important to tomato flavor are the C6 compounds hexanal and cis-3-hexenal, which contribute a green, grassy odor that was also identified as sweet, ripe, and tomato flavor. These, and other related compounds, are derived from fatty acids; however, other 1

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2 important volatiles in tomato are derived from the metabolism of carotenoids and amino acids. Volatile emissions in tomato are affected by a number of parameters throughout tomato development. Fertilizer regime, maturity at time of harvest, and postharvest handling such as bruising or storage at low or high temperatures can have profound effects on the volatile profile. Choice of cultivar can also influence volatile emissions, as some varieties are low-quality and produce less aroma. Objectives The primary objective of this study was to determine whether the volatile profile of tomato fruit would be altered by treatment with an inhibitor of ethylene action. The first objective was to evaluate the volatile emissions in ripe tomato fruit, continuing through overripening. Another objective was to determine the ripening stage at which to treat tomatoes with 1-MCP which would have the most pronounced effect on delaying development of ripe color, but would still allow fruit to continue to attain a full ripe stage. Then tomato volatile emissions in ripe, overripe, and ripe fruit derived from breaker fruit treated with an ethylene action inhibitor were evaluated and compared within treatments to determine if volatile production in treated fruit was more similar to ripe or to overripe untreated fruit. It was also of interest to employ 1-MCP treatment as a method of discovering which volatile pathways were directly dependent on ethylene.

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CHAPTER 2 LITERATURE REVIEW General Information The tomato (Lycopersicon esculentum) is a member of the family Solanaceae. The genus name, Lycopersicon, is derived from a Greek word meaning "wolf peach" (Davies & Hobson, 1981). The fruit is a berry that is commonly eaten as a fresh or cooked vegetable and also has numerous applications in processed foods, such as ketchup, marinara sauce, and pre-prepared soups. It is a perennial plant, but it is grown as an annual. In the United States of America, it is second only to its relative the potato as the most abundantly produced of any vegetable. Nearly thirteen million metric tons of tomatoes were grown in the United States in 2005 (FAO 2006, http://faostat.fao.org/). The majority of the fresh-market cultivars are the familiar large round red varieties, but plums, clusters, round yellow, cherry, grape, and other specialty sizes are increasing in popularity (Sargent and Moretti, 2004). The average American eats over nineteen pounds of fresh tomato every year (http://www.floridatomatoes.org/facts.html). It is third in popularity among vegetables, under the potato and lettuce. Tomatoes are a plentiful source of vitamins C and A, certain minerals, and antioxidants, especially lycopene, the pigment responsible for the color of red tomatoes. The tomato was legally declared a vegetable in 1893 in a Supreme case centering on tariffs (Nix v. Hedden, 149 U.S. 304, 1893). Over four and a half million hectares of the world are devoted to tomato production (http://www.floridatomatoes.org/facts.html). Seventeen thousand hectares of this are in 3

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4 the state of Florida, which is half of the fresh tomatoes produced in the United States. This represents a 660 million dollar value to Florida farmers each year and jobs for 33 thousand workers (http://www.floridatomatoes.org/facts.html). The tomato fruit, a berry originating from a single, inferior, multi-locular ovary, undergoes experience a period of sigmoidal expansion and maturation prior to exhibiting the characteristic features of ripening. In this pre-ripening developmental period, the tomato fruit remain green. Upon completion of physical growth, the fruit attains a developmental state typically referred to as 'green' (Sargent and Moretti, 2004). For descriptive purposes, this green period is divided into four stages, M1 through M4. At the M1 stage, the locular tissue is firm and the seeds are immature (seeds are white and can be easily cut by passing a sharp blade through the fruit.) At the M2 stage, the seeds have matured and are tan, and gel is present in at least two locules. At the M3 stage, well developed gel is present in all of the locules, and the internal color is still green. The green maturation process concludes with 'mature green', which is the stage at which red color appears in the locule gel and internal, radial pericarp and columnella tissues (Sargent and Moretti, 2004, adapted from Kader and Morris 1976). Fruit harvested prior to attaining a physiologically mature, green stage will continue to ripen, but will not attain the maximum potential quality (Hurr, Huber, and Lee, 2005). The developmental period beyond the mature green stage through the full red-ripe stage has been divided for descriptive purposes into four stages based on the accumulation of surface lycopene distribution (USDA 1991: http://www.ams.usda.gov/standards/tomatfrh.pdf). Each stage indicates further breakdown of chlorophyll and accumulation of lycopene. Breaker fruit are mostly green

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5 but show a blush of red or pink around the blossom end that constitutes less than ten percent of the fruit surface. Turning fruit have a larger area of red development, between 10% and one third of the surface. Pink fruit are between one and two thirds red, and light red are between two thirds and 90% red. When the tomato reaches the full red stage, it is table-ripe and ready to eat. In commercial practice, tomatoes are usually hand-harvested unripe to prevent bruising and spoilage during storage and transportation. Care is taken to keep damaged, defective, immature, or fruit of an advanced stage of ripeningwhich will rot more quickly--out of postharvest channels to reduce the need for grading later on. Once out of the field and into the packinghouse, the fruit are washed in a chlorinated dump tank and then brushed, rinsed, and dried. Sometimes they are also waxed (Sargent, Brecht, and Olczyk, 2005 http://edis.ifas.ufl.edu/VH079). Tomato sorting occurs along a conveyor belt, as workers remove unsatisfactory fruit and classify the tomatoes by size and quality. In Florida, the quality grades are 'U.S. No. 1', '85% U.S. No. 1', 'U.S. Combination', and 'U.S. No. 2', and the sizes are '6x7' (between 2 9/32 and 2 19/32 inches in diameter), '6x6' (between 2 17/32 and 2 29/32 inches in diameter), and '5x6' (minimum diameter 2 25/32 inches) (http://www.floridatomatoes.org/domestic.html, USDA 1991: http://www.ams.usda.gov/standards/tomatfrh.pdf). The fruit are then packed into cartons and stacked into pallets. They are ripened by exposure to ethylene. This initiates ripening at a commercially desirable point in the supply chain and also assures uniform ripening of the harvest. Flavor Chemistry Flavor is an important component of a tomato's desirability, but with increasing focus from breeders on quality parameters including color, firmness, resistance to

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6 disease, yield, size, and physical uniformity, many of today's tomato cultivars do not meet consumer standards for good taste (Hobson, 1988). Poor flavor components include sourness, bite (the stinging present after imbibing carbonated beverages), a 'green' taste reminiscent of unripe fruit, low pH, and most relevant to this discussion, low levels of many aromatic volatiles (Tandon, Baldwin, Scott, and Shewfelt, 2003). Stevens, Kader, Albright-Holton, and Algazi (1977) reported that since it is more fluid than the other tomato tissue, qualities of the locule gel are more readily perceived by taste receptors on the tongue and it is therefore a more significant contributor to tomato flavor than either pericarp or placental tissue. The flavor of a ripe tomato is created by a complex combination of sugars, acids, and aroma volatiles (Krumbein and Auerswald, 1998). Tandon et al. (2003) report that fructose and glucose are the sugars most relevant to tomato flavor. These sugars comprise approximately three percent of the fresh weight of a ripe tomato, increasing as the fruit ripens (Tandon et al., 2003; Davies and Hobson, 1981). The sweetness of tomato fruit positively correlates to the amount of these sugars. Organic acids are another important constituent of tomato flavor. Citric acid is the most abundant, at a concentration of 135-675 micrograms per gram fresh weight (mg/gfw) (Davies and Hobson, 1981). Citric acid is most plentiful in the locular gel, where it is present in concentrations between 634-1560 mg/100 gfw. It is followed by malic acid, which is present in concentrations between 21 and 225 mg/100 gfw in the whole fruit and 30-412 mg/100 gfw in just the locular gel (Davies and Hobson, 1981). As also reported by Davies and Hobson (1981), amino acids are also present, the most numerous in tomato fruit being glutamic acid, arginine, aspartic acid, and asparagine.

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7 Aroma volatiles are present in very low concentrations--nanoliters per liter of blended fresh tomato fruit tissue--but are still very important to flavor. They are perceived by olfactory receptors in the nose, not by the taste receptors in the tongue (Acree, 1993). According to Buttery (1993), over four hundred volatiles have been identified in tomato fruit, but only around thirty of them comprise a significant portion of perceived flavor. These include various aldehydes, ketones, and alcohols. The volatiles that appear in the highest concentrations are cis-3-hexenal, hexanal, and 2-phenylethanol. Volatiles can be classified according to their synthetic pathways and the compounds from which they originate. Some, like hexanal, cis-3-hexenal, trans-2-hexenal, cis-3-hexenol, and ethyl vinyl ketone, are derived from fatty acids (Buttery & Ling, 1992) including linolenic and linoleic acids (Goff and Klee, 2006). These lipid-derived volatiles are present in both leaves and fruit tissue. The maceration and blending with CaCl 2 of tomato leaves by Buttery and Ling (1992) and the consequent sampling of the resultant volatiles revealed high levels of cis-3-hexenal in the leaf tissue. This indicates that cis-3-hexenal is the volatile responsible for the green, grassy scent of tomato leaves. Buttery and Ling (1992) also sampled the cis-3-hexenal produced by tomato fruit tissue and found that it increased as the fruit ripened. The compound was found at a concentration of 0.1 parts per million (ppm) in mature green tomato, 0.3 ppm at breaker stage, and 15 ppm in ripe fruit. Buttery and Ling (1992) also identify volatiles, including -ionone and 6-methyl-5-hepten-2-one, which are formed from carotenoids. -ionone is a cyclic compound, whereas 6-methyl-5-hepten-2-one is an open chain formed by oxidative cleavage of lycopene (Goff and Klee, 2006). Carotenoid-derived volatiles are found only in the fruit,

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8 and not in leaf, flower, or stem tissue. There are also volatiles that are derived from amino acids. These are found exclusively in the fruit and are formed between the breaker and full-ripe stage (Buttery & Ling, 1992). For example, 1-nitro-2-phenylethane and 2-phenylethanol are formed from phenylalanine, isovaleraldehyde (3-methylbutanal) is formed from leucine, and 2-methylbutanal is formed from isoleucine. The pathways of some, like 2-isobutylthiazole, are unknown (Goff and Klee, 2006) although Schutte (1974) suggests that 2-isobutylthiazole may also originate from leucine via isovaleraldehyde. Trained taste panelists have identified relationships between specific volatiles and certain facets of tomato flavor. Krumbein and Auerswald (1998) identified cis-3-hexenal as one of the most powerful components of flavor, producing a fresh green and sweet flavor. Hexanal was characterized as green and grassy by Krumbein and Auerswald (1998), and as sweet, ripe, and "tomato flavor" by Maul et al. (2000). 2-isobutylthiazole was described in Krumbein and Auerswald (1998) as producing both sweet and fruity flavors and causing a bite feeling in the mouth. It was also identified as musty. -ionone contributed to a sour taste, and trans-2-heptenal and 1-penten-3-one (ethyl vinyl ketone) produced a green aroma. 3-methylbutanal (isovaleraldehyde) was described as unpleasant (Krumbein and Auerswald, 1998; Baldwin, Scott, Shewmaker, and Schuch, 2000). Ethylene Ethylene (C 2 H 4 ) is the structurally simplest of the plant hormones, as well as being the simplest alkene. It is derived from methionine via a pathway elucidated in detail by Yang and Hoffman (1984). The regulatory effects of ethylene on plant development are

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9 numerous and varied. They include initiation of flower opening, seed germination, and leaf/flower senescence, and control of responses to both biotic and abiotic stresses (Yang and Hoffman, 1984). In climacteric fruit, those that experience an increase in respiration rate and ethylene production accompanying ripening, ethylene also regulates changes associated with fruit ripening (Yang and Hoffman, 1984). For thousands of years, humans have augmented the natural ethylene production of crops with their own artificially added doses in order to produce a uniform ripening response. For example, Theophrastus, who lived in the third century B.C.E., reports that agriculturists of his time damaged the skin of figs in order to initiate ripening through what is now understood to be a method of stimulating ethylene production via a wounding response (Galil, 1968). The process of wounding figs to promote ripening is still performed by present-day Egyptian farmers. In more modern times, citrus fruit such as oranges were placed in close proximity to burning kerosene in order to achieve degreening necessary for a brilliantly-colored orange or yellow product. Chace and Denny (1924) determined that the active factor in the burning kerosene was ethylene gas. In studies of apple fruit, Gane (1934) was the first to demonstrate that ethylene was a natural product of plant tissues. The biosynthesis of ethylene begins with the amino acid methionine, which is converted to S-adenosylmethionine (SAM) by SAM synthetase (E.C. 2.5.1.6). SAM is converted to 1-aminocyclopropane-1-carboxylic acid, or ACC, by ACC synthase (E.C. 4.4.1.14) also called ACS (Yang and Hoffman, 1984). It then is oxidized by ACC oxidase (E.C. 1.14.17.4), also called ACO and once referred to as the ethylene forming enzyme (EFE), in order to produce ethylene (Alexander and Grierson, 2002).

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10 Both the prevention of ethylene synthesis and the suppression of either ethylene perception or the response to ethylene interfere with ripening and other senescence processes. This was demonstrated by Brandt and Woodson (1992) by examining genotypic variation in carnation flowers. Cultivars with low amounts of ACC, indicating reduced activity of ACS, exhibited extended vase-life by an average of five days compared with a cultivar with normal levels of ethylene and its precursors. In addition, low activity of ACO, referred to in this study as EFE, the ethylene forming enzyme [as it was known before activity was obtained in vitro by Ververidis and John (1991)], resulted in even lower levels of ethylene in certain cultivars. One of these cultivars, 87-37G-2, responded to exogenously applied ethylene by finally producing its own ethylene and senescing earlier than untreated controls, indicating that its deficiency was in ethylene synthesis, not perception. Another synthesis-deficient cultivar, 799, failed to respond to external ethylene, indicating that is was both a perception and a synthesis mutant. It is clear that interfering with ethylene synthesis and/or perception will result in delayed, reduced, or halted senescence activities. Although carnation served as an early model for exploration of ethylene effects, ripening fruits have become increasingly the system of choice. Carnations are still investigated as a floral system, but the bulk of the molecular work dealing with ethylene perception and action has been performed with fruit tissue. For example, the Never-ripe tomato is a mutant that is unable to perceive ethylene because of a mutation to its ethylene receptor. The altered receptor is unable to bind to ethylene, leaving it unengaged and able to repress ethylene responses even in the presence of the hormone (Hackett et al., 2000). Hackett et al. (2000) further demonstrated that inhibiting the Nr gene using

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11 antisense constructs enabled the mutant tomatoes to recover ethylene perception and ripen normally. Inhibitors of Ethylene Synthesis Action One method of preventing the effects of ethylene on both cut flowers and edible crops is to employ an ethylene scrubber made of cement, silica gel, aluminum pellets, and more recently, potassium permanganate. The scrubbers absorb the ethylene from the surrounding atmosphere and prevent it from being absorbed by the product. Jayaraman and Raju (1992) found the potassium permanganate-based scrubber more effective than previously used materials in extending the shelf life of tomato, mango, and okra by a period of between three to eight days. Silver thiosulfate (STS) is a compound that interferes with ethylene perception (Veen & Geijn, 1978; Bleecker & Kende, 2000). Ethylene is believed to interact with a copper cofactor in the receptor. It has been speculated that if silver is present, it takes the place of copper and interacts with the ethylene instead. While it has the same ability as copper to interact with ethylene, it lacks the ability to complete the receptor's function as copper does, disabling the receptor (Rodriguez et al., 1999). However, Veen and Geijn (1978) observed brown, necrotic spots on the leaves resulting from the toxic effects of high concentrations of silver. Silver is a toxic heavy metal that can cause s injury even in small amounts because it binds to sulfhydryl groups non-discriminately. It may have effects of which science is still unaware. The toxicity of STS restricts it to use with flowers only, not for food crops, and in some countries its use or method of application have been restricted due to concerns about environmental pollution (Serek & Reid, 1993). Sisler, Reid, and Yang (1986), Serek, Sisler, and Reid (1994), and Sisler and Blankenship (1993) examined a wide range of cyclic olefins in search of compounds that

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12 might serve as antagonists or agonists of ethylene reception. 2,5-norbornadiene (NBD) and diazocyclopentadiene (DACP) were shown to have effects on the ethylene receptor, but they were deemed unsuitable for practical use. In the case of the former, the compound must be present continually in order to be effective (which is impractical for the supply chain of most produce), and in the case of the latter it was highly explosive. NBD also has a foul odor which further limits its desirability (Serek et al., 1994). 1-Methylcyclopropene, or 1-MCP, is a non-toxic, odorless gas that is effective in rendering ethylene receptor molecules inactive after short-term treatment. 1-MCP is a simple molecule, a cyclic alkene with a single methyl group attached to the olefinic carbons. 1-MCP apparently acts by non-competitively binding to the ethylene receptor because it has a lower binding constant, Kd=8 nlliter -1 (Serek et al., 1994). It is presumed that by blocking ethylene binding, 1-MCP causes the receptor to remain engaged, maintaining the downstream cascade that suppresses ethylene-induced genes (Sisler and Serek, 1997). The effectiveness of 1-MCP at extending the shelf life of many fruit and vegetable crops has been well documented. It has been approved for commercial use on horticultural crops since 2002, starting with apples (American Fruit Grower, 2002), and has now been approved for numerous other crops, including the tomato. Effects such as a delay in softening or in the development of ripe coloration have also been demonstrated on banana, avocado, melon, papaya, persimmon, and other crops, both climacteric and nonclimacteric (reviewed in detail in Blankenship & Dole, 2003). Commercially, it is available as EthylBloc for ornamentals and SmartFresh for edible crops. It is presently used as a gas that is volatized when a powder formulation is added to water, but a liquid

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13 application is currently being investigated (Todd Eddington, Agrofresh, personal communication). 1-MCP inhibits many parameters of ripening, including ethylene synthesis, ethylene perception, loss in firmness, and changes in pigmentation. The effects can either be permanent or dissipate after a period of 1 to 2 weeks, depending on the concentration used and the period of exposure. The fact that 1-MCP interferes with perception of ethylene makes it a useful tool to study which ripening processes are ethylene dependent and which occur independent of ethylene. Most research with the effects of 1-MCP has focused on firmness and color. For example, tomato fruit were treated with 1 ppm 1-MCP for 24 h at 20C. It was determined that the stage at which a tomato fruit is treated can determine the effectiveness of the 1-MCP treatment in delaying pigmentation changes associated with ripening. Tomatoes treated at mature green or breaker stage delay development of external red color for a week, and then ripen normally (Hurr et at., 2005). 1-MCP produces a delay in the loss of firmness associated with ripening. The amount of force required to compress the external tissue of a tomato a distance of 2 mm ranges from 20 Newtons (N) at the mature green stage to four or five N at full red ripe. 1-MCP causes this softening to be delayed from five to ten days later than usual (depending on the developmental stage at which the 1-MCP was applied). One possible explanation is that the enzymes polygalacturonase and cellulase, which are responsible for the breakdown of the pectin and cellulose that provide structural support for plant cells, are affected by ethylene suppression. The blocking of ethylene perception in avocado interferes with the activity of polygalacturonase and cellulase, but softening is inhibited only during the later stages of ripening with little effect during early to mid ripening

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14 (Jeong, Huber, and Sargent, 2002; Jeong and Huber, 2004). This seems to indicate that the softening that occurs during early ripening may be largely ethylene independent, whereas the softening during overripening may be ethylene dependent since it can be stopped by the inhibition of ethylene recognition. A tomato transgenically altered with an antisense gene for polygalacturonase exhibited a definite decrease in postharvest softening compared with wild-type fruit (Kramer et al., 1992). There is a point at which ripening can be delayed excessively, however, where fruits do not soften sufficiently and become shriveled and decayed prior to ripening. Effects of 1-MCP on Fruit Volatiles Documented effects of 1-MCP on fruit volatile profile are variable and dependent on the species of fruit and specifics of treatment (concentration, duration of exposure, and temperature). Golding, Shearer, McGlasson, and Wyllie (1999) reported a decrease in total volatile production of bananas ripened at 20C after treatment with 1-MCP (45 ppm, 1 h) at a mature green stage compared with ripe untreated bananas. This decrease was attributed mainly to a decrease in ester production. An increase in alcohol production in the treated fruit, compared with control fruit, was also observed. Golding et al. (1999) suggested that this increase in alcohols was due to a disruption of the final step in the formation of esters, in which alcohols are esterified by the enzyme alcohol acyl transferase, also known as AAT (E.C. 2.3.1.84) (Yahyaoui et al., 2002). Two esters, 3-methylbutyl isovalerate and pent-2-yl butanoate, increased in treated fruit compared with control fruit, implying that the enzyme remained functional and that availability of the substrate acyl CoA was the limiting factor in ester production (Golding et al., 1999). Pelayo, Vilas-Boas, Benichou, and Kader (2003) reported that ester production was delayed, but not reduced, in bananas treated with 1-MCP (1 ppm, 24 h), and stored at

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15 20C, compared with untreated bananas. Aldehyde loss was also delayed in treated fruit compared with control fruit. The concentration of 1-MCP applied is critical. Bagnato, Barrett, Sedgley, and Klieber (2003) found that when bananas were treated at 300 ppb, ripening was delayed and the flavor was unaffected. Too low of a concentration (3 ppb) did not extend shelf life and too high of a concentration (30 ppm) prevented ripening entirely. Apples have also been studied for the response of aroma volatiles to 1-MCP. During ripening of untreated apples, acetates and some esters increase and alcohols and aldehydes decrease (Lurie, Pre-Aymard, Ravid, Larkov, and Fallik, 2002). When 'Anna' apples were treated with 1-MCP (1 ppm, 20 h) at 20C, the volatile profile associated with harvest time was retained. Taste panelists preferred the treated apples, which tasted freshly harvested, to the control apples, which displayed the characteristic aroma of very ripe apples. In another study of apples using the cultivar 'Borkh', Defilippi, Dandekar, and Kader (2004) reported delayed and reduced esters when fruit were treated with 1-MCP (1 ppm, 20 h) at 20C just after harvest, but aldehydes were unaffected. Data from adding ethylene back to transgenic fruit with the inability to synthesize it on their own implied that a continuous supply of ethylene is necessary for volatile synthesis (Defilippi et al., 2004). Abdi, McGlasson, Holford, Williams, and Mizrahi (1998) used 1-MCP to determine whether parameters of plum ripening, such as the development of skin color and production of aroma volatiles, were ethylene dependent or independent. The cultivar 'Beauty' was treated with 1-MCP (26 and 39 ppm, 24 h) at 20C and developed aroma

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16 volatiles one week later than untreated plums, according to a taste panel. When the cultivar 'Shiro' received the same treatment, only the untreated fruit developed aroma volatiles at all. Mitcham, Mattheis, Bower, Biasi, and Clayon (2001) found that European pears responded to various concentrations of 1-MCP with suppressed production of aroma volatiles, assessed subjectively but no quantities were recorded. Once the effects of the treatment wore off, volatiles began to form normally. The processes regulated by ethylene are complex, and the response of a particular species or cultivar to 1-MCP, a powerful growth regulator, is not always predictable. This limits its commercial application and indicates that it is not suitable for every circumstance. Factors Affecting Tomato Volatiles The quantities and composition of volatiles in tomato fruit are highly dependent on genetic and environmental factors active throughout fruit development. Volatiles can vary between cultivars. For example, the variety 'FL-47' is known to be low-volatile, and consequently, low-quality (Tandon et al., 2003). Preharvest conditions such as fertilizer regime can also affect the tomato's volatile production. The amount of aroma volatiles including hexenal, phenylacetaldehyde, benzaldehyde, -ionone, and 6-methyl-5-hepten-2-one was increased by elevated levels of nitrogen and potassium (Wright and Harris, 1985). This increase had a negative impact on tomato flavor (Wright and Harris, 1985). Fruit maturity at harvest can influence the volatile profile. Maul et al. (1998) determined that tomato fruit that were harvested immature green, as indicated by the fruit taking 5 days to reach breaker stage in ethylene treatment, displayed lower levels of

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17 many aroma volatiles, including cis-3-hexenal, 6-methyl-5-hepten-2-one, 2-isobutylthiazole, and ethyl vinyl ketone, compared with tomato fruit that were harvested mature green and only took one day to break in ethylene treatment. After the tomato is harvested, ideally, care should be taken by the handlers and packers to ensure that an ideal volatile composition is preserved. Appropriate storage temperature is an important component of postharvest handling. The temperature during the final stages of ripening has the greatest effect on the fruit volatile profile once ripe. Maul et al. (2000) found that too low of a storage temperature will cause an increase in off-flavors and a decrease in aroma. Storage at a temperature below 12.5C for even two days in the case of cv. 'BHN-189' or four days in the case of cv. 'Solimar' resulted in a loss of flavor quality, as evaluated by a trained taste panel. The quality was further reduced if the storage temperature was 5C. The main loss in aroma volatiles consisted of a reduction in the lipid-derived volatiles (hexanal, cis-3-hexenal, ethyl vinyl ketone, trans-2-hexenal, trans-2-heptenal, and cis-3-hexenol) that accompanied the lower storage temperatures. The carotenoid-derived volatiles (-ionone and 6-methyl-5-hepten-2-one) were also reduced but only slightly. Tomatoes held at 20C achieved higher ratings from the taste panel in aroma, flavor, and intensity of sweetness, even after twelve days of storage. In another study, Boukobza and Taylor (2002) compared low temperature (6C), moderate temperature (21C and 35C), and high temperature (45C) storage environments. The tomatoes were held at the trial temperature for three days (in the low temperature vs moderate temperature experiment) or six hours (in the high temperature vs moderate temperature experiment) before being transferred to 21C for 4-6 hours to

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18 mimic retail conditions. Low-temperature storage caused an eventual decrease in the amounts of isobutylthiazole, methylbutanal, and methylbutanol, possibly due to a reduction in enzyme activity or a decreased level of available amino acid precursors. High-temperature storage reduced the amounts of hexanal, hexenal, hexenol, isobutylthiazole, methylbutanal, methylbutanol, and 6-methyl-5-hepten-2-one, an effect believed to be due to the denaturing effect of the heat on the enzymes of the lipid oxidation pathway from which the volatiles would have formed. The reduction in aroma volatiles was aggravated by increasing the temperature further or increasing the duration of holding at 21C. Bruising can cause changes in a tomato's volatile profile, most notably a reduction in the amounts of cis-3-hexenal, 6-methyl-5-hepten-2-one, ethyl vinyl ketone, cis-3-hexenol, and 2-isobutylthiazole. These changes were identified by comparing bruised locular tissue to unbruised locular tissue. Placental tissue responded to bruising with increased levels of ethyl vinyl ketone and -ionone compared with unbruised placental tissue, and the amount of trans-2-hexenal in bruised pericarp tissue was higher than in unbruised pericarp tissue. These alterations are due possibly to the disruption of the enzymatic schedule by the damage resulting from the physical impacts. (Moretti, Baldwin, Sargent, and Huber, 2002). A relationship between ethylene and the synthesis of many tomato volatiles is evident from a number of studies. Oeller, Wong, Taylor, and Pike (1991) investigated tomato plants which expressed an antisense construct for ACC synthase, suppressing the ethylene biosynthesis pathway. Ethylene production in the transformant was reduced nearly 200-fold, and the fruit failed to ripen beyond an orange color. Their aroma was

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19 never developed. Antisense fruits ripened upon exposure to exogenous ethylene, and ripening was accompanied by the development of aroma volatiles. Baldwin et al. (2000) reported that ethylene is required for carotenoid synthesis, possibly increasing ethylene's effects on at least those volatiles that are derived from carotenoids. Kausch and Handa (1997) found that the ethylene-insensitive Never-ripe (Nr) mutant accumulated the mRNA for lipoxygenase, an enzyme important in the synthesis of volatiles from linolenic and linoleic acids, but did not accumulate the lipoxygenase protein. This implies that ethylene is not necessary for the accumulation of the mRNA for lipoxygenase, but that it is necessary for the translation or stability of the lipoxygenase protein and, consequently, for the formation of lipid-derived volatiles, which is supported by Ealings (1994) work showing lipoxygenase activity increasing and then decreasing along with ethylene production in the early stages of tomato ripening (Ealing, 1994). Another important enzyme, alcohol dehydrogenase (ADH), catalyzes the conversion of hexanal to hexenol. ADH activity was reported to be ethylene-independent, which is supported by its elevated activity, possibly due to decreasing oxygen concentration, in ripening grape fruit, which are nonclimacteric (Zhu et al., 2005). During normal tomato ripening, Baldwin, Nisperos-Carreido, and Moshonas (1991) found that the amounts of most aroma volatiles increased, including cis-3-hexenol, cis-3-hexenal, trans-2-hexenal, 6-methyl-5-hepten-2-one, and 2-isobutylthiazole. The increase in many of the volatiles followed the same trend as the climacteric increase in respiration and production of ethylene and lycopene during ripening. Ethyl vinyl ketone decreased during ripening, but in only one of the cultivars investigated, 'Sunny'. In the cultivar 'Solimar', a small increase in ethylene production during the breaker stage corresponded

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20 with the appearance or significant increase in the amounts of cis-3-hexenol, cis-3-hexenal, trans-2-hexenal, 6-methyl-5-hepten-2-one, and 2-isobutylthiazole. The production of 1-hexenal, trans-2-hexenal, and 6-methyl-5-hepten-2-one was reduced in table ripe tomato fruit ( and ) treated at mature green stages with 1-MCP (2 ppm, 15 h) and stored at 21C, compared with untreated fruit. Production of ethyl vinyl ketone and cis-3-hexenal was enhanced in 1-MCP-treated fruit compared with the controls (Mir, Canoles, and Beaudry, 2004). The amount of 1-hexanal and 2-isobutylthiazole was higher in turning-harvested fruit than in green-harvested fruit, but the amount of ethyl vinyl ketone and cis-3-hexenal was greater in green-harvested fruit than in turning-harvested fruit. The volatiles altered by treating tomatoes with 1-MCP were the same as the volatile altered by harvesting fruit at an earlier developmental stage. This implies that 1-MCP treatment may encourage flavors associated with harvesting tomatoes at an earlier stage of maturity (Mir et al., 2004), which often has a deleterious effect on flavor (Maul et al., 1998). However, the magnitude of the changes in aroma caused by 1-MCP in this study is small enough to indicate that it may not be a concern to the perception of consumers. Tieman et al. (2006) monitored the production of M82 tomato volatiles as the fruit progressed from immature green to light red stage. Emissions of cis-3-hexenal, trans-2-hexenal, -ionone, and 6-methyl-5-hepten-2-one remained low during early ripening and increased dramatically between turning stage and light red stage. The largest increase in 2-phenylethanol was at breaker stage. Maximum emission of 3-methyl-1-butanol was at turning stage, and 2-isobutylthiazole decreased from immature green onwards. Mir et al. (2004) examined the volatile profile of and tomatoes

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21 during the mature green, turning, and full ripe stages. However, neither work quantitatively addressed the changes occurring in tomato volatile emissions during the period following the attainment of full ripe stage, nor has the volatile profile of 1-MCP treated fruit been compared with fruit past this initial attainment of full ripe. The objective of the present study was to examine the development of overripe tomatoes and compare them with tomatoes of the same age which had undergone 1-MCP treatment to determine if 1-MCP continues to affect tomatoes past the initial point of ripening, or if changes in volatile emissions between 1-MCP ripened and untreated ripe tomatoes were due to the 1-MCP treatment directly or to the age of the fruit. It was also of interest to examine untreated tomatoes past full ripe stage because tomatoes are often purchased when full ripe or nearly full ripe, and subsequently held for a period of days prior to consumption, and it was of interest to discover for how many days is the volatile profile associated with the first day of full ripe (as defined by attaining a hue angle of 50) maintained before being altered by overripening.

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CHAPTER 3 MATERIALS AND METHODS Plant Material Tomato (Lycopersicon esculentum) fruit, cvs. Florida 47, Florida 91, and BHN586, employed for the studies performed were obtained from West Coast Tomato, Inc., in Palmetto, FL, North Florida Tomatoes in Quincy, FL, and University of Florida North Florida Research & Education Center Suwannee Valley extension stations in Live Oak, FL. Depending on the specific experiment, fruit were obtained at several developmental stages, including mature-green, breaker, turning, pink and full red, as defined on the basis of surface lycopene distribution (USDA 1991: http://www.ams.usda.gov/standards/tomatfrh.pdf). Each stage reflects further breakdown of chlorophyll and accumulation of lycopene. Breaker fruit are mostly green but show a blush of red or pink around the blossom end that constitutes less than ten percent of the fruit surface. Turning fruit have a larger area of red development, between 10% and one third of the surface. Pink fruit are between one and two thirds red, and light red are between two thirds and 90% red. When the tomato reaches the full red stage, it is generally considered by consumers to be ripe and ready to eat. Fruit used in all experiments were obtained on the day of harvest, and transported immediately to Postharvest facilities at the University of Florida. Upon arrival, fruit were washed in 1.34 millimolar Na-hypochlorite prepared from household bleach, selected for uniformity in color and freedom from blemishes, and placed in storage facilities at 20C until treated. No fruit used in any of these studies were treated with exogenous ethylene. 22

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23 Specific conditions of 1-MCP treatment and storage duration are described in separate sections below. Fruit Color Measurements Fruit external color was measured daily using a Minolta Chroma Meter CR-400 (Minolta Camera Co Ltd, Japan) calibrated using a white tile as the standard. The chroma meter records lightness (on a scale of black to white, where black is numerically zero), chroma (color saturation), and hue angle, a spectral scale in which 45 represents red and 180 represents green. The measurements were taken at three equatorial locations and the three hue angle values were averaged. Analysis of Whole Fruit Firmness Fruit firmness was measured every other day nondestructively using an Instron Universal Testing Instrument (model 4411; Canton, Mass). The Instron was fitted with a 5 kg load cell and a blunt-ended, convex 7.5 mm probe. After establishing zero force contact with the surface of the tomato, the probe was driven with a cross-head speed of 50 mm/min, targeting an area over locular tissue and not internal walls. The force of compression was recorded in Newtons (N) at the point at which the tomato skin had been compressed a distance of 2 mm. The same fruit were measured for the duration of the experiment. Firmness was measured at three equatorial points for each tomato, and the values averaged. Evaluation of Volatile Profile of Ripe to Overripe Fruit This experiment was performed to determine the changes in volatile profile of tomatoes between the onset of full ripeness (as designated by an average hue angle of below 50) and over the subsequent 16 d as the tomato becomes overripe.

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24 Fruit Selection and Cleaning Tomato (cv. BHN586) fruit ranging from breaker to pink stage were obtained from a packinghouse in February 2006. From these, 110 turning fruit were selected, dipped in 1.34 millimolar hypochlorite, rinsed, and arranged on paper towels to dry. Each fruit was individually marked with a ballpoint pen on a white adhesive label. Turning fruit were selected for this experiment so that the precise day of ripening (as designated by an average hue angle below 50) could be identified through daily monitoring. Identifying Onset of Ripe Stage Using Color Measurements Fruit external color was measured daily using a Minolta Chroma Meter CR-400 (Minolta Camera Co Ltd, Japan) as described in the section 'Fruit Color Measurements'. The measurements were taken at three equatorial locations designated by the points 90, 180, and 270 in reference to the white label sticker. The three hue angle values were averaged, and those fruit that reached an average hue angle of below 50 were selected and divided into two identical samples. The day on which the hue angle reached this starting point was designated Day 0. On Day 1, 2, 6, 11, and 16 during storage at 20C, five fruit were selected from each sample and removed for volatile analysis, with the Day 1 and 2 fruit representing ripe fruit and the Day 16 fruit representing significantly overripe fruit. Volatiles were analyzed using the method described above in the section 'Analysis of tomato volatiles'. Determination of Fruit Developmental Stage for 1-MCP Treatment This experiment was designed to determine the developmental stage at which 1-methylcyclopropene (1-MCP) would be most effective at delaying, but not preventing, the attainment of a full-ripe condition, as defined on the basis of fruit reaching a hue angle of 45, representing full-red external pigmentation. Fruit (cv. FL-47) were obtained

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25 from the University of Florida North Florida Research & Education Center in Quincy, FL. Upon return to Gainesville, fruit were cleaned and washed as described under 'Plant Material'. One experiment was performed using fruit that was obtained at the 'green' stage. Fruit for other experiments were obtained after color break. Fruit at green, breaker, and pink stages were subsequently treated with 250 ppb 1-MCP for 12 h at 20C in 174-L containers as described in detail under 'Exposure of Tomato Fruit to 1-MCP'. Following treatment, fruit were transferred to 20C storage facilities for up to 14 d and evaluated daily for color using a Minolta Chroma Meter CR-400 (Minolta Camera Co Ltd, Japan) as described in detail under 'Fruit Color Measurements'. Fruit were also measured every other day for firmness using an Instron Universal Testing Instrument) model 4411; Canton, Mass) as described in detail under the section 'Analysis of whole fruit firmness'. Volatile analysis was performed on the breaker-stage treated fruit, once they had reached full ripe, using the method described in the section 'Analysis of tomato volatiles'. Exposure of Tomato Fruit to 1-MCP For all 1-MCP experiments in this study, 1-MCP gas was generated by weighing the appropriate quantity of SmartFresh powder (1-Methylcyclopropene, 0.14% A.I.) (AgroFresh, a division of Rohm & Haas) to produce the desired atmosphere inside 174-L and 20-L treatment chambers. The chamber selected was based on the number of tomatoes being treated. The powder (26 mg for the 20-L chambers and 112 mg for the 174-L chambers) necessary to produce an atmosphere of 250 ppb (for the 174-L chambers) or 500 ppb (for the 20-L chambers) 1-MCP in the treatment chambers was added to a 125-mL Erlenmeyer flask containing 40 mL of tap water. After swirling to dissolve the 1-MCP powder, the flask was placed in the treatment chambers that were

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26 immediately sealed. The chambers were sealed by clamping a clear Plexiglas cover and foam gasket to the open end of each treatment chamber (174-L chambers) or by inserting a threaded Gamma-Seal (B&A Products Ltd. Co., Oklahoma) (20-L containers). Control fruit were placed in a treatment chamber with a flask containing 40 mL of water alone. Treatment was performed at 20C for twelve hours, after which time the containers were opened and the fruit transferred to 20C normoxic storage facilities. At intervals during storage at 20C, the fruit were removed and evaluated for color, firmness, and volatile production. Analysis of the Effects of 1-MCP on Tomato Ripening and Volatile Profiles Tomato fruit (cv. 'FL-91') were obtained in October 2005 from a packinghouse at the green stage and transported to Gainesville the same day. Fruit were dipped in 1.34 millimolar Na-hypochlorite, rinsed, and dried at room temperature. Fruit were held at 20C until color break and then selected for color uniformity at breaker stage. Two experiments were performed. For each experiment, fifteen identical breaker-stage fruit were selected and these fifteen fruit were subdivided into three groups of five fruit each. One group was treated with 500 ppb 1-MCP for 12 h at 20C in 20-L treatment chambers as described earlier in the section 'Exposure of Tomato Fruit to 1-MCP'. The other two groups were designated as untreated controls. The extra control group was needed as described below. Following treatment with 1-MCP, the fruit were transferred to 20C storage facilities for up to 14 d and evaluated daily for color using a Minolta Chroma Meter CR-400 (Minolta Camera Co Ltd, Japan) as described in detail under 'Fruit Color Measurements'. Fruit were also measured every other day for firmness using an Instron

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27 Universal Testing Instrument) model 4411; Canton, Mass) as described in detail under 'Analysis of Whole Fruit Firmness' above. The untreated tomatoes reached a full red condition, as designated by the hue angle of each fruit averaging below 50, after 7 to 10 d. Within 1 day of this, one of the two groups of untreated fruit was sampled to determine the aroma volatile constituency as described below. At this point, the 1-MCP treated fruit were still at a pink stage. 1-MCP treated fruit were held until they reached full red (average hue angle < 50), approximately 4 to 11 d later than the control fruit, and then sampled to determine their volatile profile as described below. The other untreated control group, which had been full red (average hue angle < 50) for 4 to 11 d, was analyzed at the same time. Thus for this experiment, the three treatments consisted of: 1. Fruit treated with 1-MCP at the breaker stage and ripened at 20C, 2. Control fruit ripened at 20C, and 3. Control fruit held at 20C until the 1-MCP-treated fruit reached a full-ripe condition. The experiment was repeated using fruit from another harvest from the same source three weeks later. Analysis of Tomato Volatiles In both the ripening evaluation and 1-MCP experiment described in these studies, fruit with an average hue angle of below 50 were used to determine the aroma volatile profile. To compose a single 100 g sample, a longitudinal wedge of approximately 60 degrees was removed from each of the five fruit and the tissue diced on a plastic tray using a sharp knife into cubes of approximately 1 cm 3 All tissues, including the pericarp, locular gel, juice, and seeds were retained in order to obtain a representative whole-fruit volatile profile. Each sample was held between 5 min to 1 h in a hexagonal

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28 weigh-boat (Fisher, Canada) and then loaded into glass tubes measuring 2.54 cm x 61 cm (Volume: 309.1 mL). Both ends of the tubes were sealed with a rubber septum. Volatiles were collected on Super-Q resin columns for 1 h using the method described by Schmelz, Alborn, and Tumlinson (2001). Filtered air was passed through the glass tubes and across the tomato samples at a flow rate of 618 mL/min. After 1 hour, a 5 l nonyl acetate internal standard was injected into each resin column using a glass syringe. The contents of the columns were eluted with 150 l methylene chloride, and stored in 300 L glass inserts inside 1.8 mL glass jars at -80C to preserve volatile integrity. The volatiles within the glass jars were analyzed using an Agilent Technologies 6890N gas chromatograph with a DB-5 column (Agilent Technologies) and a flame ionization detector, using helium as the carrier gas. The injector temperature was 220C, the detector temperature was 280C, and the oven temperature was between 40 and 250C. The carrier gas flow rate was 1.2 mL/min. The software package ChemStation (Agilent Technologies) was used to analyze the peaks produced by the volatiles, where retention times were compared with known standards and used to identify peaks. The peak areas were then exported into Microsoft Excel. Statistical Analysis Volatile production data was analyzed using the least squares method with a confidence interval of 95% in JMPIN, the student version of the JMP program made by SAS. Firmness and color data was analyzed using standard errors calculated by Microsoft Excel.

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CHAPTER 4 RESULTS AND DISCUSSION Evaluation of Volatile Profile of Ripe to Overripe Fruit The changes in volatile emissions as tomato fruit (M82) progress from immature green, through mature green, breaker, turning, and pink, to light red stage, have been observed and recorded in Tieman et al. (2006). The experiments in the present study were designed to determine the changes in tomato volatile emissions in the successive stages, starting at the day on which fruit reach a full ripe stage (as defined by an average hue angle below 50) and continuing until 16 d following ripening. Tomato fruit ('BHN586') were obtained at turning stage and ripened at 20C. Color was monitored daily until individual fruit attained an individual average hue angle below 50. Figure A-1 shows the hue angles of the fruit at the start of the experiment and each consecutive day during storage. The hue angle quickly decreased as the tomato ripened in 20C storage. The tomatoes upon reaching hue angle values below 50 (Figure A-1, day 4) were held for up to 17 days at 20C (Figure A-2), during which time fruit were periodically measured for volatile emissions. According to these results, a tomato, once reaching red, does not experience further significant color change even as many as seventeen days after ripening. Figure A-3 shows the average firmness of the tomatoes sampled. A ripe tomato is usually around 5 N, and for the first three days after ripening (using hue angle < 50 as an indicator of ripening) the tomatoes maintained a firmness comparable to the normal 29

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30 firmness of full ripe tomatoes. However, firmness continued to decrease as the tomatoes progressed from ripe to overripe, declining to between 3 and 4 N by 11 d following ripening. Changes in volatile emissions of the 'BHN586' tomato fruit during ripening and over-ripening are shown in Figures A-4 to A-8. Volatiles are discussed in order of their importance to tomato aroma, as noted by other researchers (Buttery and Ling, 1992; Buttery, 1993; Krumbein and Auerswald, 1998). The C6 compounds hexanal and cis-3-hexenal greatly impact the aroma of tomato leaves and fruit, producing a green, grassy scent (Buttery and Ling, 1992) or a sweet or fresh green flavor (Krumbein and Auerswald, 1998). The flavor of hexanal alone has also been referred to as sweet, ripe, and tomato flavor (Maul et al., 2000), identifying it as a key component of tomato aroma and taste. Cis-3-hexenal and hexanal are derived from the fatty acids linoleic and linolenic acids and increase in concentration as the tomato ripens (Buttery and Ling, 1992). Tieman et al. (2006) observed low levels of cis-3-hexenal in M82 tomato fruit up until the turning stage, at which point a dramatic increase in production occurred through the light red stage. According to Buttery and Ling (1992), cis-3-hexenal emission in tomato fruit increases slightly at color break, but experiences a fifty-fold increase at the full-ripe stage. In the present study, BNH586 tomato fruit experienced a decrease in cis-3-hexenal production as fruit progressed from full ripe to 16 d overripe (Figure A-4). Since cis-3-hexenal is a key component of ripe tomato aroma, it is not surprising that its production rises to the highest point 1 or 2 d after attaining a hue angle below 50 and then drops again. Interestingly, Mir et al. (2004) found that cis-3-hexenal production decreased in

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31 and tomatoes as they progressed from mature green to turning and then to full red, but this contrasting data may be explained by the difference in cultivar. Hexanal production in BNH586 tomatoes fruit fell between 1 and 2 d after attaining full red, but thereafter increased and remained high during the 16 d of observation, as shown in Figure A-5. Ethyl vinyl ketone, or 1-penten-3-one, is another C6 volatile derived from fatty acids and found in both vegetative and reproductive tissues of the tomato plant. As with the other C6 volatiles, it imparts a green aroma (Krumbein and Auerswald, 1998). The and tomatoes investigated in Mir et al. (2004) were found to produce less ethyl vinyl ketone at the turning stage than when mature green, after which production remained constant through the full ripe stage. As shown in Figure A-6, production of ethyl vinyl ketone in BNH586 tomato fruit remained constant within the first three days after attaining a full ripe stage, after which it rose over the course of the next week. Ethyl vinyl ketone emissions peaked on day 11 and remained statistically constant for the next 5 d. This pattern is consistent with the trend noted by Mir et al. (2004). -ionone is a cyclic compound synthesized from the metabolism of carotenoids (Buttery and Ling, 1992). Carotenoid-derived volatiles are formed in the fruit but not in the vegetative tissues of tomato plants,. -ionone has been characterized as imparting a sour taste to taste panelists (Tandon et al., 2003). Production of ionone increased dramatically as the 'BHN586' tomato fruit progressed from ripe to overripe (Figure A-7). ionone production was relatively constant within the first three days after fully ripening, and increased sharply at 11 d. At 11 and 16 d after ripening, -ionone production had increased approximately six-fold compared with values observed at the

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32 full-ripe stage. Tieman et al. (2006) reported a dramatic increase in -ionone production between the turning and light red stages, at which time their observations ceased. The data in Figure A-7 illustrate that this volatile continues to increase through and after full ripening. There was no significant difference in -ionone production between the two identical groups sampled on any of the days measured. Other tomato flavor volatiles are synthesized from amino acids. Isovaleraldehyde, also called 3-methylbutanal, is formed from the amino acid leucine. It has been associated with an unpleasant flavor (Krumbein and Auerswald, 1998). Tieman et al. (2006) observed a steady increase in isovaleraldehyde production in M82 tomato fruit in the time from immature green to turning stage, after which point emissions of isovaleraldehyde decreased again as the tomato progressed to light red. The BNH586 tomatoes used in the present experiment experienced steady isovaleraldehyde production for the first three days after attaining full ripe stage, after which production increased slightly. By day 11, however, it had fallen to well below the full-ripe rate of production, and emissions of isovaleraldehyde remained low over the next 5 d (Figure A-8). Determination of Fruit Developmental Stage for 1-MCP Treatment The purpose of these experiments was to determine the optimal stage for treating the tomato fruit with 1-MCP in order to be certain the 1-MCP would sufficiently delay ripening. Tomato fruit (Lycopersicon esculentum 'FL-47') at green, breaker, turning, and pink stages of development were treated with 1-MCP (250 ppb, 12 h, 20C) or air and ripened at 20C. Fruit surface color was recorded daily and firmness every other day. Fruit at the green stage were found to be unsuitable for 1-MCP experiments, due to the unpredictable numbers of immature-green fruit in the samples. As noted by Hurr et al., (2005), green tomato fruit treated with 1-MCP eventually diverge into 2 distinct

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33 populations in terms of the degree of suppression of softening and surface red color development. Softening and color development of immature green fruit (an assignment made retrospectively based on the number of days to color break of the untreated controls) were acutely and irreversibly inhibited by the ethylene antagonist. Mature-green fruit, on the other hand, eventually softened and colored to a degree exhibited by control fruit, though both ripening parameters were significantly delayed. Aside from the difficulties in properly classifying green fruit, there is ample evidence that even mature-green fruit, once detached, never develop the characteristic volatiles associated with high quality. Definitive classification of green fruit as immature green or mature green requires either nuclear magnetic resonance technology (NMR) (Abbott, 1999) or X-ray technology (Brecht, Shewfelt, Garner, and Tollner, 1991) which are expensive and time-consuming, or cutting the fruit open. In view of the inability of distinguishing mature versus immature-green fruit, subsequent experiments were performed using fruit at the breaker stage and beyond. Treating tomato fruit with 1-MCP at the pink or turning stage was also problematic because in the first five days following treatment, no difference in color or firmness was observed between the ripening of control fruit and 1-MCP treated fruit. This parallel ripening pattern is shown in Figures B-1 through B-4. Color of fruit treated at the pink stage did not appear to have been affected by the 1-MCP treatment, and the difference in average hue angle between 1-MCP treated and control fruit was less than one degree throughout the six days following ripening, indicating statistically similar values. No difference was observed between the firmness of treated and untreated pink fruit over the course of the observation. Fruit treated with 1-MCP at either the pink or turning stages

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34 softened just as rapidly as air-treated control fruit. It is possible that differences may have emerged after the six days observed, but since at the time the experiment was terminated all fruit had already softened and reddened sufficiently to indicate full ripe within a similar period of time, there was nothing to indicate that 1-MCP had any effect on their development during ripening. These results are in contrast with those of Hurr et al. (2005), who found that pink and turning FL-47 tomato fruit did respond to 1-MCP treatment (1 ppm, 24 h, 20C), as did all other stages of tomato fruit development through full-ripe, with delayed softening and color development. This may be due to the fact that the concentration used was four times what was used in these experiments (1 ppm vs 250 ppb) and the duration of treatment was twice as long (24 h vs 12 h), and a treatment of that magnitude may be required to affect the development of pink or turning fruit. However, an initial concentration of only 150 ppb successfully produced a delay in ripening parameters such as softening and development of red color in Prisca tomato fruit after 20 h of treatment (Hoeberichts, Van der Plas, and Woltering, 2002). It is possible that the difference in cultivar may be responsible for the difference in 1-MCP effectiveness. Blankenship and Dole (2003) stated that there is evidence of cultivar differences in response to 1-MCP for apple and banana fruits. This might be due to cultivar differences in the magnitude of ethylene production or in inherent sensitivity to ethylene. Another possible explanation for the deviation of the present results from previous work is that the fruit used in this experiment were stressed due to being transported in the trunk of a car for 1 h just after harvest, where high temperatures may have had a negative effect. This is unlikely,

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35 however, since fruit color development, which is highly sensitive to high-temperature injury, was not impaired. Due to the problems with green, pink, and turning fruit enumerated above, breaker fruit were employed for all further experiments. Fruit (FL-47) treated with 1-MCP (250 ppb, 12 h, 20C) at the breaker stage were monitored for changes in color and firmness until they attained a full ripe stage, the stage at which 90% or more of the fruit is covered by red pigmentation and the hue angle is below 50. In this experiment, the length of time required for fruit to reach full red was significantly affected by 1-MCP. As shown in Figure B-5, control fruit reached an average hue angle below 45 eight days earlier than 1-MCP-treated fruit. Additionally, development of red pigmentation occurred with more uniformity over the surface in control fruit, although uniform red pigmentation did eventually develop in all fruit. Fruit treated at breaker stage with 1-MCP also exhibited a delay in loss of firmness compared with control fruit. 1-MCP-treated fruit experienced a delay in development of the ripening parameters of color and firmness compared with untreated fruit. It was of interest to discern whether this delay would affect volatile emissions. With that in mind, volatile analysis was performed as described earlier on ripe fruit derived from airor 1-MCP-treated breaker fruit, and on breaker fruit taken before 1-MCP treatment. Fatty-Acid Derived Volatiles The amounts of cis-3-hexenal and cis-3-hexenol produced in ripe control fruit were higher than the amounts produced in breaker fruit (Figures B-6 and B-9). This increase in cis-3-hexenal during ripening in tomato fruit corroborates the data from Tieman et al. (2006), which indicated an increase in cis-3-hexenal between the turning and light red stages, but not Mir et al. (2004), who reported a decrease in cis-3-hexenal between

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36 mature green, turning, and ripe fruit. The amount of both cis-3-hexenal and cis-3-hexenol produced in ripe fruit derived from 1-MCP-treated breaker fruit was higher than the amounts in ripe control fruit (Figs B-6 and B-9). In the case of cis-3-hexenal, this is consistent with the increase observed by Mir et al. (2004) in and tomatoes treated with 2 ppm 1-MCP for 15 h at 21C. However, Mir et al. (2004) did not observe any change in the amount of cis-3-hexenol throughout their experiment. This difference might be attributed to cultivar differences, or difference in 1-MCP treatment or volatile sampling procedure. Mir measured volatiles in fruit homogenates whereas in the present study the volatiles were measured from a heterogenous mixture of skin, pericarp, locule gel, juice, and seeds diced into cubes of approximately 1 cm 3 Hexanal and trans-2-hexenal were found in statistically similar amounts in breaker and ripe control fruit, and higher levels in ripe fruit derived from 1-MCP-treated breaker fruit (Figures B-7 and B-10). This contradicts the decrease observed in Mir et al. (2004) between ripe control and ripe fruit derived from 1-MCP-treated mature green fruit, and also the increase observed in that study in hexanal production between the mature green and turning stages. However, once the tomatoes in Mir et al. (2004) had reached turning stage, their hexanal emissions remained constant, and it is possible that the conclusion can be drawn from these combined results that the increase in hexanal associated with tomato ripening is initiated at the breaker stage, and not turning stage. Mir et al. (2004) did not measure tomatoes at breaker stage, and the tomatoes observed in that study retained constant hexanal emissions from the turning through full-ripe stage. An increase in the amount of trans-2-hexenal produced in ripe control fruit compared with breaker fruit does appear at the 90% confidence level, although not at a 95% confidence level.

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37 Tieman et al. (2006) had found an increase in the amount of trans-2-hexenal produced as M82 tomato fruit ripen, but this increase was not observed in FL-47. The increase in trans-2-hexenal may have occurred between the turning and light red stages, but remained undetected because the only stages at which volatiles were sampled were breaker and full red. A possible explanation is that the increase may have occurred transiently, only between the turning and light red stages, and this trend is corroborated by the data from Tieman et al. (2006). Additionally, the trans-2-hexenal production data presented in Figure B-10 is not consistent with the observations made by Mir et al. (2004), in which trans-2-hexenal was reported to have been produced at higher levels in untreated fruit, although in both experiments there was an increase in the amount produced in ripe fruit compared with fruit in the early stages of development. Mir et al. (2004) had observed a decrease in ethyl vinyl ketone production from mature green to turning, after which production levels remained constant. Treatment with 1-MCP (2 ppm, 15 h, 21C) reduced the levels of ethyl vinyl ketone in and tomatoes to the level they had exhibited when mature green. However, in this experiment, there was no difference in the amount of ethyl vinyl ketone produced by FL-47 tomatoes between any of the treatments/stages (Figure B-8). Carotenoid Derived Volatiles The amount of -ionone produced was statistically similar for all treatments (Figure B-11). The amount of 6-methyl-5-hepten-2-one produced was much higher in both ripe control and ripe fruit derived from 1-MCP-treated breaker fruit than in breaker fruit (Figure B-12). An increase in both of these volatiles was observed in Tieman et al. (2006) between the turning and light red stages and in Mir et al. (2004) between mature green, turning, and full ripe. In the case of 6-methyl-5-hepten-2-one, at least, this increase upon

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38 ripening seems not to have been compromised by exposure to 1-MCP at the breaker stage. In contrast to the results reported here, Mir et al. (2004) reported decreased levels of both volatiles in response to 1-MCP. In this case it may be more practical to attribute the differences in observations to the difference between the 1-MCP concentrations in each experiment [2 ppm in Mir et al (2004) vs 250 ppb in the present study] rather than cultivar differences because the untreated FL-47 fruit in Figure R behaved consistently with the M82 fruit in Tieman et al. (2006) and the and fruit in Mir et al. (2004). Amino Acid Derived Volatiles The amounts of isovaleraldehyde and 3-methyl-1-butanol produced in ripe control fruit were higher than the amounts in both breaker fruit and ripe fruit derived from 1-MCP-treated breaker fruit, which were statistically similar at the 95% confidence level (Figures B-13 and B-15) but different at the 90% confidence level, where ripe fruit derived from 1-MCP-treated breaker fruit were found to produce higher emissions of 3-methyl-1-butanol than breaker fruit. Tieman et al. (2006) observed maximum emissions of 3-methyl-1-butanol at the turning stage, after which it decreased, which may seem to contradict these results. However, comparing only the breaker and light red stage data from Tieman et al. (2006), there was a small overall increase in 3-methyl-1-butanol. Whether or not the 1-MCP-treated fruit observed here experienced this transient increase is unknown because turning fruit were not measured for volatile content. In contrast with the results presented in Figure P, untreated and fruit observed in Mir et al. (2004) maintained a constant level of 3-methyl-1-butanol production throughout the mature green, turning, and full ripe stages, while 1-MCP treated fruit produced higher levels of 3-methyl-1-butanol.

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39 2-methylbutyraldehyde was produced in higher amounts in ripe control fruit than in breaker fruit. Neither was statistically different from the amount produced in ripe fruit derived from 1-MCP-treated breaker fruit at the 95% confidence level (Figure B-14) but at the 90% confidence level ripe control fruit was observed to produce higher levels of 2-methylbutyraldehyde than ripe fruit derived from 1-MCP-treated breaker fruit. 1-nitro-2-phenylethane was produced in higher amounts in ripe fruit derived from 1-MCP-treated breaker fruit than in breaker or ripe control fruit (Figure B-17). There was no statistical difference observed in the amounts of 2-phenylethanol produced between any of the treatments/stages (Figure B-16). Since 1-nitro-2-phenylethane and 2-phenylethanol are both derived from phenylalanine, it is reasonable to assume that they would respond similarly to 1-MCP if their production trends were dependent on ethylene. This expectation was not fulfilled by the results in this study. However, as noted in Figures V and W, the volatiles exhibited different patterns of response to 1-MCP. Buttery and Ling (1992) stated that amino acid-derived volatiles such as 1-nitro-2-phenylethane and 2-phenylethanol are formed between the breaker and full ripe stages, indicating that an increase should have been detected at least between breaker and ripe control fruit. That increase was not observed in this study for either volatile. Volatiles of Other Derivation The derivation of 2-isobutylthiazole is unknown (Goff and Klee, 2006), although Schutte (1974) has suggested that 2-isobutylthiazole may originate from leucine using isovaleraldehyde as an intermediate. The flavor attributed to 2-isobutylthiazole is sweet and fruity and it was observed to cause a bite feeling in the mouth (Krumbein and Auerswald, 1998). Both ripe control and ripe fruit derived from 1-MCP-treated breaker fruit produced higher levels of 2-isobutylthiazole than those produced by breaker fruits,

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40 and there was no difference observed in the amounts produced between ripe control and ripe fruit derived from 1-MCP-treated breaker fruit (Figure B-18). In Tieman et al. (2006), the maximum emission of 2-isobutylthiazole was during the immature green stage, thereafter declining as tomato development proceeded. However, Tieman et al. (2006) only measured fruit volatiles up until light red stage, so it is possible that the increase observed in the present study, which appears not to have been impacted by exposure to 1-MCP, occurred entirely after the fruit attained a full red color. The increase observed by Tieman et al. (2006) is contradicted by the data in Mir et al. (2004), which show s an increase in 2-isobutylthiazole production between mature green and turning, after which the amount produced remains constant in untreated and tomato fruit. Mir et al. (2004) also shows a decrease in the amount of 2-isobutylthiazole in 1-MCP fruit compared with ripe control fruit, but this was not observed in the present study. Response of Tomato Fruit Color and Firmness to 1-MCP Tomato fruit (Lycopersicon esculentum 'FL-91') at the breaker stage were treated with 1-MCP (12 h, 20C, 500 ppb) or air and ripened at 20C. Firmness decreased more rapidly in air-treated fruit, as shown in Figures C-1 through C-4. This is consistent with previous studies involving the effect of 1-MCP in delaying FL-47 tomato softening, such as Hurr et al. (2005), in which a week to 10 d was required for 1-MCP treated fruit to soften to the same firmness as control fruit. Hoeberichts et al. (2002) also demonstrated that 1-MCP treatment causes tomato fruit (Prisca) to attain a softness associated with ripening after a delay, compared with controls. Development of red coloration occurred more slowly in 1-MCP -treated fruit than in control fruit, as shown in Figure C-5, and with less uniformity. Since color was

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41 measured at three equatorial positions, it is not evident that for some 1-MCP treated fruit, patches of green or yellow remained at the stem end of the fruit for several days. These areas did eventually develop red color, resulting in a uniform full red fruit. Tomato Volatile Production in Ripe, Overripe, and 1-MCP-treated FL-91 Tomato Fruit These experiments were designed to determine whether or not emission of the thirteen volatiles considered most important to tomato aroma would be affected by ethylene suppression. In addition, these experiments sought to compare the volatile emissions of 1-MCP treated fruit not only with control fruit of a similar ripening stage, but with control fruit of similar age, and which were therefore overripe. The four trials performed within this experiment will be discussed together due to the fact that they involved tomatoes of the same cultivar (FL-91), obtained at the same stage (breaker) from the same packing house, treated at the same stage (breaker) with the same concentration of 1-MCP (500 ppb) for the same time period (12 h). Experiments 3 and 4 were performed on fruit obtained three weeks later than Experiments 1 and 2. Where no interaction is described, no significant differences were found between treatments at the 95% confidence level using the least squares method. Fruit designated as ripe control were sampled upon reaching a hue angle of 50 after treatment in air. Fruit designated as '1-MCP' were treated at breaker stage with 500 ppb 1-Methylcyclopropene (1-MCP), 12 h, 20C) and then sampled upon reaching a hue angle of 50. Since 1-MCP delays the change in pigmentation associated with tomato ripening by suppressing the ethylene response, this change in pigmentation occurred 4 to 11 d later than the 'ripe control' fruit. Fruit designated as 'overripe' were treated in air, but

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42 sampled concurrently with the 1-MCP-treated fruit, between 4 to 11 days after the ripe' reached a hue angle of 50. The rationale behind the sampling of overripe air-treated fruit as well as ripe air-treated fruit was to rule out the possibility that differences in the tomato volatile profile between air-treated and 1-MCP-treated ripe fruit were due to fruit age rather than a response to delayed ripening. The 1-MCP-treated fruit were 4 to 11 d older than the air-treated fruit when they reached the 50 hue angle, and sampling the overripe fruit was necessary to distinguish between volatile differences due to fruit age and not to any direct effects of 1-MCP on ethylene-mediated processes. Fatty-Acid Derived Volatiles Cis-3-hexenal was found in higher amounts in ripe control fruit than in overripe fruit in Experiments 2 and 3, and higher amounts in ripe control fruit than in ripe fruit derived from 1-MCP-treated breaker fruit in experiment 2, which contradicts the results observed earlier in this study (Figures C-7 and C-8) and also the observations of Mir et al. (2004). There were no differences in cis-3-hexenal production between treatments in Experiments 1 or 4 (Figures C-6 and C-9). Hexanal was found in higher amounts in overripe fruit than in ripe control fruit in Experiment 1, but no significant differences were found between ripe fruit derived from 1-MCP-treated breaker fruit and either ripe control or overripe fruit in this experiment (Figure C-10). There were no differences between treatments in Experiments 2 and 3 (Figures C-11 and C-12). In Experiment 4, higher amounts of hexanal were found in ripe fruit derived from 1-MCP-treated breaker fruit than in ripe control fruit, supporting the results observed earlier in this study (Figure C-13). There were no differences between ripe fruit derived from 1-MCP-treated breaker fruit and overripe fruit or between overripe fruit and ripe control fruit at the 95% confidence level, although at the 90% confidence

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43 level, ripe fruit derived from 1-MCP-treated breaker fruit produced more hexanal than ripe control fruit in experiment 1, and overripe fruit produced more hexanal than ripe control fruit in experiment 4. These indications that 1-MCP treatment may cause some FL-91 tomato fruit to produce higher levels of hexanal contradicts data presented in Mir et al. (2004), in which and tomato fruit treated with1-MCP showed a 50% reduction in hexanal production, resulting in emission rates comparable to those of mature green fruit. Ethyl vinyl ketone production was statistically similar for all treatments in Experiments 1, 2, and 4 (Figures C-14, C-15, and C-17). In Experiment 3, greater amounts of ethyl vinyl ketone were found in overripe fruit than in ripe fruit derived from 1-MCP-treated breaker fruit (Figure C-16). No differences were found between ripe control fruit and either overripe fruit or ripe fruit derived from 1-MCP-treated breaker fruit at the 95% confidence level, but overripe fruit produced more ethyl vinyl ketone than ripe control fruit at the 90% confidence level. This lack of statistical difference mirrors the results for the ethyl vinyl ketone measured earlier in this study. There were no statistical differences in the production of trans-2-hexenal between any of the treatments at the 95% confidence level in all four experiments (Figures C-18 through C-21). However, at the 90% confidence level, overripe fruit produced more trans-2-hexenal than ripe control fruit in Experiments 3 and 4. Cis-3-hexenol production was statistically similar for all treatments at the 95% confidence level in Experiments 1, 2, and 4 (Figures C-22, C-23, and C-25). However, at the 90% confidence level, overripe fruit produced higher levels of cis-3-hexenol than ripe control fruit in Experiments 1 and 4. Overripe fruit in experiment 3 produced higher

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44 levels of cis-3-hexenol than ripe fruit or ripe fruit derived from 1-MCP-treated breaker fruit (Figure C-24). No differences were found between cis-3-hexenol production in ripe control fruit and ripe fruit derived from 1-MCP-treated breaker fruit. This similarity between treatments is fairly consistent with the observations of Mir et al. (2004), in which no differences were found in the amounts of cis-3-hexenol produced by mature green, turning, ripe control, and ripe fruit derived from 1-MCP-treated mature-green fruit. Carotenoid Derived Volatiles -ionone was found in higher amounts in both overripe and ripe fruit derived from 1-MCP-treated breaker fruit than in ripe control fruit in Experiment 3 (Figure C-28). There were no differences in -ionone production between treatments in Experiments 1, 2, or 4 (Figures C-26, C-27, and C-29), corroborating results presented earlier in this study. Both findings contradict the observations of Mir et al. (2004), in which ripe fruit derived from 1-MCP-treated mature green fruit produced much lower levels of -ionone than ripe control fruit. 6-methyl-5-hepten-2-one production was statistically similar for all treatments in Experiments 1 and 2 at the 95% confidence level (Figures C-30 and C-31), although at the 90% confidence level, ripe fruit derived from 1-MCP-treated breaker fruit produced more 6-methyl-5-hepten-2-one than ripe control fruit in Experiment 1. Higher levels of 6-methyl-5-hepten-2-one were identified in overripe fruit than in both control ripe and ripe fruit derived from 1-MCP-treated breaker fruit in Experiment 3 (Figure C-32). In Experiment 4. higher levels of 6-methyl-5-hepten-2-one were found in overripe fruit than in ripe fruit derived from 1-MCP-treated breaker fruit, and at the 95% confidence level, production of 6-methyl-5-hepten-2-one in ripe control fruit was statistically similar to both overripe fruit and ripe fruit derived from 1-MCP-treated breaker fruit. (Figure C-33).

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45 At the 90% confidence level, however, overripe fruit produced more 6-methyl-5-hepten-2-one than ripe control fruit in Experiment 4. The statistical similarity between ripe fruit derived from 1-MCP-treated breaker fruit and ripe control fruit echoes the similarity observed earlier in this study and contradicts the findings of Mir et al. (2004), in which 1-MCP was observed to produce a decrease in 6-methyl-5-hepten-2-one emissions in and tomato fruit. Amino Acid Derived Volatiles Higher levels of isovaleraldehyde were observed for ripe control fruit than in both overripe fruit and from ripe fruit derived from 1-MCP-treated breaker fruit in Experiment 2 (Figure C-35). In Experiment 3, isovaleraldehyde was found in higher amounts in ripe fruit derived from 1-MCP-treated breaker fruit than in both overripe and ripe control fruit (Figure C-36). In Experiment 4, isovaleraldehyde was found in higher amounts in the overripe fruit compared with ripe fruit derived from 1-MCP-treated breaker fruit, both of which were statistically similar to ripe control fruit (Figure C-37). There were no differences in isovaleraldehyde production between treatments in Experiment 1. (Figure C-34) 3-methyl-1-butanol was found in higher amounts in ripe control fruit than in overripe fruit, both of which were statistically similar to the amount in ripe fruit derived from 1-MCP-treated breaker fruit at the 95% confidence level, in Experiment 1 (Figure C-38). At the 90% confidence level, however, ripe control fruit produced higher levels of 3-methyl-1-butanol than ripe fruit derived from 1-MCP-treated breaker fruit in Experiment 1. In Experiment 2, higher amounts of 3-methyl-1-butanol were found in ripe control fruit than in both overripe fruit and in ripe fruit derived from 1-MCP-treated breaker fruit (Figure C-39). Higher levels of 3-methyl-1-butanol were identified in ripe

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46 fruit derived from 1-MCP-treated breaker fruit than in both overripe and ripe control fruit in experiment 3 (Figure C-40), corroborating results from Mir et al. (2004). There were no differences in 3-methyl-1-butanol production between treatments in Experiment 4 (Figure C-41). 1-nitro-2-phenylethane was found in higher amounts in both overripe fruit and in ripe fruit derived from 1-MCP-treated breaker fruit than in ripe control fruit in Experiments 1 and 3, corroborating observations described earlier (Figures C-42 and C-44). In Experiment 4, higher levels of 1-nitro-2-phenylethane were identified in overripe fruit than in both ripe fruit derived from 1-MCP-treated breaker fruit and in ripe control fruit (Figure C-45). There were no differences in production of 1-nitro-2-phenylethane between treatments in Experiment 2 (Figure C-43). Higher levels of both 2-phenylethanol and 2-methylbutyraldehyde were identified in ripe control fruit than in both overripe fruit and in ripe fruit derived from 1-MCP-treated breaker fruit in Experiment 2 (Figures C-47 and C-51). No differences were observed in the production of either 2-phenylethanol or 2-methylbutyraldehyde in overripe fruit and in ripe fruit derived from 1-MCP-treated breaker fruit in experiment 2 at the 95% confidence level, although at the 90% confidence level, higher levels of 2-methylbutyraldehyde were observed in ripe fruit derived from 1-MCP-treated breaker fruit than in overripe fruit. There were no differences in 2-phenylethanol or 2-methylbutyraldehyde production between treatments in Experiments 1, 3, or 4 (Figures C-46, C-48, C-49, C-50, C-52, and C-53).

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47 It is interesting to note that as with the earlier experiment in this study, the reaction of 1-nitro-2-phenylethane and 2-phenylethanol, both derived from phenylalanine, to ethylene suppression was not similar. Volatiles of Other Derivation 2-isobutylthiazole was found in higher amounts in ripe fruit derived from 1-MCP-treated breaker fruit than in both overripe and ripe control fruit in Experiment 3 (Figure C-56), consistent with the report of Mir et al. (2004). There were no differences in 2-isobutylthiazole production between treatments in Experiments 1, 2, or 4 (Figures C-54, C-55, and C-57). There were no differences observed between ripe control and ripe fruit derived from 1-MCP-treated breaker fruit in the study discussed earlier, and the decrease observed in Mir et al. (2004) was minor, indicating that ethylene action may not be an important factor in the formation of 2-isobutylthiazole. General Discussion It is evident from the results of these studies that factors other than ethylene recognition affect volatile emissions. Numerous preharvest factors can influence volatile production in tomato fruit, such as fertilizer regime (Wright and Harris, 1985), choice of cultivar (Tandon et al., 2003), and climatic conditions during the growing season (cite). Harvesting conditions, such as fruit maturity at harvest (Maul et al., 2000) and storage temperature (Boukobza and Taylor, 2002; Maul et al., 2000), can also play a role in altering the volatile profile of tomato fruit. Bruising is also a factor (Moretti et al., 2001), which means that postharvest handling practices are likely influential. These natural variations were minimized by obtaining the fruit for the experiments in this study from the same packing house during the same season, and from the same cultivar. However, in the FL-91 experiment, the tomato fruit for experiments 3 and 4 were

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48 obtained three weeks later than the fruit for experiments 1 and 2. A significant increase was found in the total volatile emissions (pooling data from all three treatments) from Experiments 1 and 2 compared with Experiments 3 and 4. Upon examination of the weather in October 2005 from Quincy, FL, the source of the tomatoes for this experiment, a possible explanation arises from the temperature data (Florida Automated Weather Network, http://fawn.ifas.ufl.edu ). In the first three weeks of October, during which the fruit from the first harvest were obtained, the temperature averaged around 20C. During the last week of October, approximately half a week before the start of experiments 3 and 4, temperatures dropped to between 1 and 4C and remained there for 9-12 h per night for three consecutive days. Johnson, Gould, Badenhop, and Johnson (1968) observed tomatoes of the same varieties differing in the amount of cis-3-hexenol emitted in their juice depending in which week the tomatoes were harvested, showing that there is precedent in the literature for such variation. Another source of volatile variation lies in the method of sample processing. In order to obtain a representative whole-fruit volatile profile, all tissues of the tomato fruitincluding the pericarp, locular gel, and seedswere retained after dicing and their volatile emissions collected and analyzed. Since each sample represents tissue taken from five fruit, precise amounts of locule gel, or any other fruit tissue, would not be consistent from sample to sample. In addition, sometimes as many as fourteen samples were prepared on one day before being loaded into the glass tubes and sealed with septa. The last sample diced was on the lab bench in an open weigh-boat for only five minutes, whereas the first sample diced might have remained in the open air for an hour, during

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49 which time some volatiles may have escaped, or experienced changes in synthetic/degradation pathways. There is evidence in the literature of volatile emissions being affected by the amount or method of tissue processing involved in the volatile sampling technique. Carbonell-Barrachina, Agust, and Ruiz (2006) compared hexanal production in blended tomatoes with hexanal production observed in tomato paste by Buttery et al. (1990) and hexanal production observed by Maul et al. (1998) using a gas chromatograph headspace analysis technique. Carbonell-Barrachina et al. (2006), who used a dynamic headspace technique, found that hexanal, as well as the other compounds analyzed, was higher in their study than in tomato paste and lower than in the tissues analyzed by gas chromatograph headspace analysis. In an effort to develop a non-destructive method of volatile sampling, they also compared aroma volatile emissions from blended tomatoes with emissions from whole fruit. Blended tomatoes exhibited much higher aroma production than whole tomatoes, which supports the statement that tissue disruption can lead to additional production of some volatiles. Since the present study involved tomatoes that were never blended, only finely chopped, some differences between these results and previous studies may be attributed to divergent techniques. Evidence in Zhu et al. (2005) would indicate that ethylene suppression would diminish production of fatty-acid derived volatiles and have no direct impact on production of volatiles derived from amino acids. While in one of the four FL-91 experiments cis-3-hexenal content was higher in untreated ripe fruit than in ripe fruit derived from 1-MCP-treated breaker fruit, the rest of the data from the fatty-acid derived volatiles in this study did not indicate that ethylene action inhibition had caused a

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50 decrease in fatty-acid derived volatile production. In fact, in some experiments, it produced an increase in hexanal emissions, contradicting previous studies (Mir et al., 2004). As for amino acid derived volatiles, whose production was not expected to be directly affected by ethylene suppression, there were isolated cases of ripe control fruit exhibiting higher production of amino acid derived volatiles than ripe fruit derived from 1-MCP-treated breaker fruit, but the reverse was also observed, as well as an observation of no difference between treatments. Since the results were inconsistent between samples and between experiments, it is possible that taste differences existed between individual samples, but it is difficult to formulate a broad declaration about what kind of difference in taste would be present. Although no taste panels were used in the present study, one worker in the laboratory could distinguish between treated and untreated ripe fruit by odor. In the future, it may be productive to augment quantitative volatile analysis, such as the gas chromatography employed within this study, with parallel taste tests performed by a trained taste panel. Such panels have already examined the volatile content of tomatoes, but have yet to explore potential differences in subjective taste due to ethylene inhibition.

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51 APPENDIX A 'BNH586' HUE ANGLE, FIRMNE SS, AND VOLATILE EMISSIONS Figure A-1. Hue angle of 'BHN 586' tomato fruit during stor age at 20C. Fruit were selected at the turning stage and placed directly in 20C storage facilities. Hue angle was determined daily at the equa torial region of twenty-five individual fruit. Values represent the means (n= 75. 3 measurements each on each of 25 individual fruit), +/standard error

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52 Figure A-2-a Figure A-2-b Figure A-2. Hue angle of 'BHN586 tomato fruit during storage at 20C after attaining a hue angle of 50. Fruit were obtained at the turning stage and hue angle determined daily at the equatorial region of five individual fruit. On the days indicated, 5 fruit were removed and vol atiles measured. Days after ripening refers to days following the date on which a 50 hue angle was observed on all

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53 five individual fruit measured. Figur es A-2-a and A-2-b depict the two identical groups of fruit from the same source, selected and stored at the same time and at the same stage, and belonging to the same cultivar. Values represent the average of those five fruit, +/standard error. Figure A-3. Fruit firmness (N) of 'BHN586' to mato fruit during storage at 20C after attaining a hue angle of 50. Fruit were obtained at the tu rning stage and hue angle determined daily at the equatorial region of five individual fruit. On the days indicated, 5 fruit were remove d and volatiles measured. Days after ripening refers to days following the time at which a 50 hue angle was observed on all five individua l fruit measured. Values represent the average of those five fruit, +/standard error.

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54 Figure A-4. Cis -3-hexenal production in ripe BNH586' tomato fruit. Fruit were ripened at 20C, and volatile production wa s measured on 1, 2, 4, 11, and 16 days after fruit reached an average hue angle of 50. Group 1 and Group 2 are identical samples taken from fruit obtai ned at the same time from the same source and stored for the same duration. Values represent the average of five fruit, +/standard error.

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55 Figure A-5. Hexanal production in ripe BNH586' to mato fruit. Fruit were ripened at 20C, and volatile production was measured on 1, 2, 4, 11, and 16 days after fruit reached an average hue angle of 50. Group 1 and Group 2 are identical samples taken from fruit obtained at the same time from the same source and stored for the same duration. Values repr esent the average of five fruit, +/standard error.

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56 Figure A-6. Ethyl vinyl ketone produc tion in ripe BNH586' tomato fruit. Fruit were ripened at 20C, and volatile produc tion was measured on 1, 2, 4, 11, and 16 days after fruit reached an average hue angle of 50. Group 1 and Group 2 are identical samples taken from fruit obtai ned at the same time from the same source and stored for the same duration. Values represent the average of five fruit, +/standard error.

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57 -ionone0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 1241116Days after ripening Group 1 Group 2 Figure A-7. -ionone production in ripe BNH586' tomato fruit. Fruit were ripened at 20C, and volatile production was measured on 1, 2, 4, 11, and 16 days after fruit reached an average hue angle of 50. Group 1 and Group 2 are identical samples taken from fruit obtained at the same time from the same source and stored for the same duration. Values repr esent the average of five fruit, +/standard error.

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58 Figure A-8. Isovaleraldehyde pr oduction in ripe BNH586' to mato fruit. Fruit were ripened at 20C, and volatile produc tion was measured on 1, 2, 4, 11, and 16 days after fruit reached an average hue angle of 50. Group 1 and Group 2 are identical samples taken from fruit obtai ned at the same time from the same source and stored for the same duration. Values represent the average of five fruit, +/standard error.

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59 APPENDIX B FL-47 HUE ANGLE, FIRMNE SS, AND VOLATILE EMISSIONS Figure B-1. Firmness (N) of 'FL-47' tomato fru it after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the pink stage and ripe ned at 20C. Days represent time since treatment. Values represent the av erage of fifteen fruit, +/standard error.

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60 Figure B-2. Firmness (N) of 'FL-47' tomato fru it after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the turning stage and ripened at 20C. Days represent time since treatment. Values represent the av erage of fifteen fruit, +/standard error.

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61 Figure B-3. Hue angle () of 'FL-47' tomato fr uit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the pink stage and ripe ned at 20C. Days represent time since treatment. Values represent the av erage of fifteen fruit, +/standard error.

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62 Figure B-4. Hue angle () of 'FL-47' tomato fr uit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the turning stage and ripened at 20C. Days represent time since treatment. Values represent the av erage of fifteen fruit, +/standard error.

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63 Figure B-5. Hue angle () of 'FL-47' tomato fr uit after treatment with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the av erage of fifteen fruit, +/standard error.

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64 Figure B-6. Cis -3-hexenal production in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit requi red an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error. Figure B-7. Hexanal production in 'FL-47 tomato fruit. Fruit were treated with air or 1MCP (250 ppb, 12 h, 20C) at the breaker st age and ripened at 20C. Volatile production was measured when fruit reac hed an average hue angle of 45. 1MCP treated fruit required an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error.

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65 Figure B-8. Ethyl vinyl ketone pr oduction in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit requi red an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error.

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66 Figure B-9. Cis -3-hexenol production in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit requi red an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error.

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67 Figure B-10. Trans -2-hexenal production in 'FL-47 toma to fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit requi red an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error. 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09ng/gfw/hr Breaker Ripe Control Ripe 1-MCPa a a Figure B-11. -ionone production in 'FL-47 tomato fruit. Fruit were treated with air or 1MCP (250 ppb, 12 h, 20C) at the breaker st age and ripened at 20C. Volatile production was measured when fruit reac hed an average hue angle of 45. 1MCP treated fruit required an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error.

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68 Figure B-12. 6-methyl-5-hepten2-one production in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 45. 1-MCP treated fruit required an additional 8 days to reach this hue value. Breaker fruit we re sampled without treatment. Values represent the average of fifteen fruit, +/standard error.

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69 Figure B-13. Isovaleraldehyde pr oduction in 'FL-47 tomato fru it. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit requi red an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error.

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70 Figure B-14. 2-methylbutyralde hyde production in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 45. 1-MCP treated fruit required an additional 8 days to reach this hue value. Breaker fruit we re sampled without treatment. Values represent the average of fifteen fruit, +/standard error.

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71 Figure B-15. 3-methyl-1-butanol production in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit required an additional 8 days to reach this hue value. Breaker fruit were sample d without treatment. Values represent the average of fifteen fruit, +/standard error.

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72 Figure B-16. 2-phenylethanol produc tion in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit requi red an additional 8 days to reach this hue value. Breaker fruit were sampled without treat ment. Values represent the average of fifteen fruit, +/standard error.

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73 Figure B-17. 1-nitro-2-phenyl ethane production in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 45. 1-MCP treated fruit required an additional 8 days to reach this hue value. Breaker fruit we re sampled without treatment. Values represent the average of fifteen fruit, +/standard error.

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74 Figure B-18. 2-isobutylthiazole production in 'FL-47 tomato fruit. Fruit were treated with air or 1-MCP (250 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 45. 1-MCP treated fruit required an additional 8 days to reach this hue value. Breaker fruit were sample d without treatment. Values represent the average of fifteen fruit, +/standard error.

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75 APPENDIX C FL-91 HUE ANGLE, FIRMNE SS, AND VOLATILE EMISSIONS Figure C-1. Firmness (N) of 'FL-91' tomato fru it after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the aver age of five fruit, +/standard error.

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76 Figure C-2. Firmness (N) of 'FL-91' tomato fru it after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the aver age of five fruit, +/standard error. Figure C-3. Firmness (N) of 'FL-91' tomato fru it after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the aver age of five fruit, +/standard error.

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77 Figure C-4. Firmness (N) of 'FL-91' tomato fru it after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the aver age of five fruit, +/standard error. Figure C-5. Hue angle () of 'FL-91' tomato fr uit after treatment with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Days represent time since treatment. Values represent the aver age of five fruit, +/standard error. C-5-a) Experiment 1. C-5-b) Experime nt 2. C-5-c) Experiment 3. C-5-d) Experiment 4.

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78 Figure C-6. Cis -3-hexenal production in ripe 'FL-91' to mato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 5 days to reach this hue value, at which time an additional control ( hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-7. Cis -3-hexenal production in ripe 'FL-91' to mato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an a dditional 11 days to reach this hue value, at which time an additional cont rol (hue angle at 50 + 11 days) sample was analyzed. Values represent the averag e of five fruit, +/standard error.

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79 Figure C-8. Cis -3-hexenal production in ripe 'FL-91' to mato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 6 days to reach this hue value, at which time an additional control ( hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-9. Cis -3-hexenal production in ripe 'FL-91' to mato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 4 days to reach this hue value, at which time an additional control ( hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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80 Figure C-10. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 5 days to reach this hue value, at which time an additional control ( hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-11. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an a dditional 11 days to reach this hue value, at which time an additional cont rol (hue angle at 50 + 11 days) sample was analyzed. Values represent the averag e of five fruit, +/standard error.

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81 Figure C-12. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 6 days to reach this hue value, at which time an additional control ( hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-13. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 4 days to reach this hue value, at which time an additional control ( hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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82 Figure C-14. Ethyl vinyl ketone production in ripe 'FL-91' toma to fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-15. Ethyl vinyl ketone production in ripe 'FL-91' toma to fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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83 Figure C-16. Ethyl vinyl ketone production in ripe 'FL-91' toma to fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-17. Ethyl vinyl ketone production in ripe 'FL-91' toma to fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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84 Figure C-18. Trans -2-hexenal production in ripe 'FL-91 tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-19. Trans -2-hexenal production in ripe 'FL-91 tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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85 Figure C-20. Trans -2-hexenal production in ripe 'FL-91 tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-21. Trans -2-hexenal production in ripe 'FL-91 tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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86 Figure C-22. Cis -3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-23. Cis -3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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87 Figure C-24. Cis -3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-25. Cis -3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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88 Figure C-26. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 5 days to reach this hue value, at which time an additional control ( hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-27. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an a dditional 11 days to reach this hue value, at which time an additional cont rol (hue angle at 50 + 11 days) sample was analyzed. Values represent the averag e of five fruit, +/standard error.

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89 Figure C-28. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 6 days to reach this hue value, at which time an additional control ( hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-29. -ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the br eaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit requi red an additional 4 days to reach this hue value, at which time an additional control ( hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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90 Figure C-30. 6-methyl-5-hepten2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-31. 6-methyl-5-hepten2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values repr esent the average of five fruit, +/standard error.

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91 Figure C-32. 6-methyl-5-hepten2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-33. 6-methyl-5-hepten2-one production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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92 Figure C-34. Isovaleraldehyde produ ction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-35. Isovaleraldehyde produ ction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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93 Figure C-36. Isovaleraldehyde produ ction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-37. Isovaleraldehyde produ ction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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94 Figure C-38. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-39. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values repr esent the average of five fruit, +/standard error.

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95 Figure C-40. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-41. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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96 Figure C-42. 1-nitro-2-phenylet hane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-43. 1-nitro-2-phenylet hane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values repr esent the average of five fruit, +/standard error.

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97 Figure C-44. 1-nitro-2-phenylet hane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-45. 1-nitro-2-phenylet hane production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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98 Figure C-46. 2-phenylethanol produc tion in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-47. 2-phenylethanol produc tion in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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99 Figure C-48. 2-phenylethanol produc tion in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-49. 2-phenylethanol produc tion in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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100 Figure C-50. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-51. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values repr esent the average of five fruit, +/standard error.

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101 Figure C-52. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-53. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production wa s measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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102 Figure C-54. 2-isobutylthiazole pr oduction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at which time an additional control (hue angle at 50 + 5 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-55. 2-isobutylthiazole pr oduction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 11 days to reach this hue value, at which time an additional control (hue angle at 50 + 11 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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103 Figure C-56. 2-isobutylthiazole pr oduction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue value, at which time an additional control (hue angle at 50 + 6 days) sample was analyzed. Values represent the average of five fruit, +/standard error. Figure C-57. 2-isobutylthiazole pr oduction in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-MCP (500 ppb, 12 h, 20C) at the breaker stage and ripened at 20C. Volatile production was measured when fruit reached an average hue angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue value, at which time an additional control (hue angle at 50 + 4 days) sample was analyzed. Values represent the average of five fruit, +/standard error.

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104 LIST OF REFERENCES Abbott JA, 1999. Quality measurement of fruits and vegetables. Po stharvest Biol and Technol 15:207-225. Abdi N, McGlasson WB, Holford P, W illiams M, Mizrahi Y, 1998. Responses of climacteric and suppressed-climacteric pl ums to treatment with propylene and 1methylcyclopropene. Postharves t Biol and Technol 14:29-39 Acree, 1993 Bioassays for flavor. In: Acree T., Teranishi R. (ed.) Flavor Science: Sensible Principles and Techniques. Am Chem Soc, Washington D.C., 1-20 Alexander L, Grierson D, 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripeni ng. J of Exp Bot 53:2039-2055. Bagnato N, Barrett R, Sedgley M, Klieber A, 2003. The effects on the quality of Cavendish bananas, which have been tr eated with ethylene, of exposure to 1methylcyclopropene. Int J of Food Sci and Technol 38:745-750. Baldwin EA, Nisperos-Carriedo MO, Mo shonas MG, 1991. Quantitative analysis of flavor and other volatiles and for certain constituents of two tomato cultivars during ripening. J. Amer. Soc. Ho rt. Sci. 116(2):265-269. Baldwin EA, Scott JW, Shewmaker CK, Sc huch W, 2000. Flavor trivia and tomato aroma: Biochemistry and possible mechanisms for control of important aroma components. HortScience 35(6):1013-1022. Blankenship SM, Dole JM, 2003. 1-Methylcycl opropene: a review. Postharvest Biol. Technol. 28:1-25 Bleecker AB, Kende H, 2000. Ethylene: a gase ous signal molecule in plants. Annu. Rev. Cell Dev. Biol. 16:1-18. Boukobza F, Taylor AJ, 2002. Effect of posth arvest treatment on flavour volatiles of tomatoes. Postharvest Biol and Technol 25:321-331. Brandt AS, Woodson WR, 1992. Variation in flower senescence and ethylene biosynthesis among carnations. HortScience 27(10):1100-1102. Brecht JK, Shewfelt RL, Garner JC, Tollner, EW, 1991. Using x-ray computer tomography to nondestructively determine to mato maturity. HortScience 26(1):4547.

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106 Golding JB, Shearer D, McGlasson WB Wyllie SG, 1999. Relationships between respiration, ethylene, and aroma producti on in ripening banana. J. Agric. Food Chem 47:1646-1651. Hackett RM, Ho CW, Lin Z, Foote HCC, Fray RG, Grierson D, 2000. Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor -inhibition model. Plant Physiol 124:10791085. Hobson G, September 1988. How the tomato lost its taste. New Scientist 29:46-50. Hoeberichts, FA, Van der Plas LHW, Woltering EJ, 2002. Ethylene perception is required for the expression of tomato ripening-related genes and associated physiological changes evene at advanced st ages of ripening. Postharvest Biol. And Technol. 26:125-133. Hurr BM, Huber DJ, Lee JH, 2005. Differential re sponses in color changes and softening of Florida-47 tomato fruit treated at gr een and advanced ripening stages with the ethylene antagonist 1-Methylcyclopr opene. HortTechnol 15(3):617-622 Jayaraman KS, Raju PS, 1992. Development a nd evaluation of a permanganate-based ethylene scrubber for extending the shelf life of fresh fruits and vegetables. J. Fd. Sci. Technol. 29(2):77-83. Jeong J, Huber DJ, 2004. Suppression of avocado ( Persea americana Mill.) fruit softening and changes in cell wall matrix polysaccharides and enzyme activities: Differential responses to 1-MCP and delaye d ethylene application. J. Amer. Soc. Hort. Sci. 129(5):752-759. Jeong J, Huber DJ, Sargent SA, 2002. Delay of avocado ( Persea americana ) fruit ripening by 1-methylcyclopr opene and wax treatments. Postharvest Biol and Technol 28:247-257. Johnson JH, Gould WA, Badenhop AF, Johnson RM, 1968. Quantitative comparison of isoamylol, pentanol, and 3-hexenol-1 in tomato juice: Varietal and harvest differences and processing effects. J. Agric. Food Chem. 16:255-258. Kausch KD, Handa AK, 1997. Molecular cloning of a ripening-speci fic lipoxygenase and its expression during wild-type and muta nt tomato fruit development. Plant Physiol. 113:1041-1050. Kramer M, Sanders R, Bolkan H, Waters C, Sheehy RE, Hiatt WR, 1992. Postharvest evaluation of transgenic tomatoes with reduced levels of polygalacturonase: Processing, firmness, and disease resistan ce. Postharvest Biol. and Technol. 1:241. Krumbein A, Auerswald H, 1998. Characteri zation of aroma volat iles in tomatoes by sensory analysis. Nahrung/Food 42(6):395-399.

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107 Lurie S, Pre-Aymard C, Ravid U, La rkov O, Fallik E, 2002. Effect of 1methylcyclopropene on volatile emission and aroma in Cv. Anna apples. J. Agric. Food Chem. 50:4251-4256. Maul F, Sargent SA, Balaban MO, Bald win EA, Huber DJ, Sims CA. 1998. Aroma volatile profiles from ripe tomato fruit ar e influenced by physiological maturity at harvest: an application for electronic nose technology. J. Amer. Soc. Hort. Sci.123(6):1094-1101. Maul F, Sargent SA, Sims CA, Baldwi n EA, Balaban MO, Huber DJ 2000. Tomato flavor and aroma quality as affected by storage temperature. J. Food Sci. 65:12281237. Mir N, Canoles M, Beaudry R, 2004. Inhibiting tomato ripening with 1methylcyclopropene. J. Am. Soc. Hort. Sci. 129(1):112-120. Mitcham B, Mattheis J, Bower J, Biasi B, Clayon M, 2001. Response of european pears to 1-MCP. Perishables Handling Quarterly 108:16-19. Moretti CL, Baldwin EA, Sargent SA, Hube r DJ, 2002. Internal bruising alters aroma volatile profiles in tomato fruit tissues. HortScience 37(2):378-382. Nix v. Hedden, 149 U.S. 304, 1893 Oeller PW, Wong LM, Taylor LP, Pike DA, Theologis A, 1991. Reversible inhibition of tomato fruit senescence by an tisense RNA. Science 254:437-439 Pelayo C, Vilas-Boas EVdeB, Benichou M, Kader AA 2003. Variability in responses of partially ripe bananas to 1-methylcycl opropene. Postharvest Biol and Technol 28:75-85. Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB, 1999. A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science 283: 996-998 Sargent and Moretti, 2004. Tomato. In: USDA Handbook 66. http://usna.usda.gov/hb66/138tomato.pdf accessed: July 2006. Sargent SA, Brecht JK, Olczyk T, 2005. Handli ng Florida Vegetables SeriesRound and Roma Tomato Types. SS-VEC-928 Gainesville FL: University of Florida IFAS, http://edis.ifas.ufl.edu/VH079 accessed: July 2006. Schmelz EA, Alborn HT, Tumlinson JH, 2001. The influence of intact-plant and excisedleaf bioassay designs on volicitinand ja smonic acid-induced sesquiterpene volatile release in Zea mays Planta 214:171-179. Schutte L, March 1974. Precursors of sulfu r-containing flavor compounds. Crit Rev in Food Technol 4:457-505.

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108 Serek M, Reid MS 1993. Anti-ethylene treatmen ts for potted Christmas cactus--Efficacy of inhibitors of ethylen e action and biosynthesis. HortScience 28(12):1180-1181. Sisler EC, Serek M, 1997. Inhibitors of ethylen e responses in plants at the receptor level: Recent developments. Physiologia Plantarum 100:577-582. Sisler EC, Reid MS, Yang SF, 1986. Effect of antagonists of ethylene action on binding of ethylene in cut carnations. Plant Growth Reg 4(3):213-218. Sisler EC, Blankenship SM, 1993. Effect of diazocyclopentadiene on tomato ripening. Plant Growth Reg. 12:155-160. Serek M, Sisler EC, Reid MS, 1994. Novel ga seous ethylene binding inhibitor prevents ethylene effects in potted flowering pl ants. J. Am. Soc. Hortic. Sci. 119, 1230 1233. Stevens MA, Kader AA, Albright-Holton M, Algazi, M. Genotypic variation for flavor and composition in fresh-market tomatoes. J. Am. Soc. Hortic. Sci. 102:680-689. Tandon KS, Baldwin EA, Scott JW, Shewfelt RL, 2003. Linking sensor y descriptors to volatile and nonvolatile components of fr esh tomato flavor. J of Food Sci 68(7):2366-2371. Tieman D, Zeigler M, Schmelz M, Taylor M, Bliss P, Kirst M, Klee H, 2006. Identification of loci affecting flavor volat ile emissions in tomato fruits. J Exp Bot 57(4):887-896. USDA Agricultural Marketing Service, Fr uit and Vegetable Division, Fresh Products Branch. 1991. United States standards for grades of fresh tomatoes. http://www.ams.usda.gov/standards/tomatfrh.pdf accessed: July 2006. Veen H, van de Geijn SC 1978. Mobility and ioni c form of silver as related to longevity of cut carnations. Planta 140:93-96. Ververidis P, John P, 1991. Complete recovery in vitro of ethylene-forming enzyme activity. Phytochemistry 30:725-727. Wright DH, Harris ND, 1985. Effect of nitr ogen and potassium fertilization on tomato flavor. J. Agric. Food Chem. 33:355-358. Yahyaoui FEL, Chalermchai WA, Latch A, Hackett R, Grierson D, Pech JC, 2002. Molecular and biochemical characteristic s of a gene encoding an alcohol acyltransferase involved in th e generation of aroma volat ile esters during melon ripening. Eur. J. Biochem 269:2359-2366.

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109 Yang SF, Hoffman NE 1984. Ethylene biosynthesi s and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155. Zhu HL, Zhu BZ, Fu DQ, Xie YH, Hao YL Luo YB 2005. Role of ethylene in the biosynthetic pathways of aroma volatiles in ripening fruit. Russian J of Plant Phys 52(5):691-695.

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110 BIOGRAPHICAL SKETCH Sonya Leonore Stahl was born on October 22, 1981, in New York City, NY, and grew up in Coral Springs, FL. She gradua ted from J. P. Taravella High School and entered college in 1999. In 2004, she graduated from the University of Florida with a Bachelor of Science degree in Natural Res ources and Conservation and a Bachelor of Arts degree in Violin Performance. That su mmer, she returned to the same university to pursue a Master of Science degree in Hor ticultural Sciences, which she obtained in December 2006. Ms. Stahl works at East Coast Fruit, c ontinuing her lifelong ambition of working with fruits and vegetables--an interest th at was born in the hydroponic greenhouse of Disney's EPCOT Center wh en she was six years old.


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Title: Influence of Ethylene Suppression and Ripening on Tomato Fruit Color, Firmness, and Volatile Emissions
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Title: Influence of Ethylene Suppression and Ripening on Tomato Fruit Color, Firmness, and Volatile Emissions
Physical Description: Mixed Material
Copyright Date: 2008

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Table of Contents
    Title Page
        Page i
        Page ii
    Dedication
        Page iii
    Acknowledgement
        Page iv
        Page v
    Table of Contents
        Page vi
        Page vii
    List of Figures
        Page viii
        Page ix
        Page x
        Page xi
        Page xii
        Page xiii
        Page xiv
        Page xv
        Page xvi
        Page xvii
    Abstract
        Page xviii
        Page xix
    Introduction
        Page 1
        Page 2
    Literature review
        Page 3
        Page 4
        Page 5
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    Materials and methods
        Page 22
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    Results and discussion
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    Appendices
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    References
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    Biographical sketch
        Page 110
Full Text












INFLUENCE OF ETHYLENE SUPPRESSION AND RIPENING ON TOMATO
FRUIT COLOR, FIRMNESS, AND VOLATILE EMISSIONS












By

SONYA LEONORE STAHL


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

Sonya Leonore Stahl

































In memory of Dr. Isaac Asimov, whose popular science essays inspired my interest in
biochemistry, and in memory of Antonin Dvofik, whose music was my constant
companion throughout my experiments and writing















ACKNOWLEDGMENTS

I would like to express my gratitude towards my committee chair, Dr. Don Huber,

for his patience, encouragement, and wisdom during the construction of this document,

during my research, and throughout my graduate program. I would also like thank the

other members of my committee for their guidance: I thank Dr. Harry Klee, the Tomato

God, for his inspiring course in phytohormones, for his advice, and for the use of his

laboratory facilities. I thank Dr. Stephen Talcott for his advice as well, and for valuable

assistance with statistics software.

I would like to thank Dr. Jiwon Jeong and the West Coast and North Florida

Tomato packinghouses for providing me with the fruit used in these experiments. I would

also like to thank James Lee, who taught me how to use the laboratory equipment.

I am very grateful for the assistance of the members of the Klee Lab who helped

me with my research. I thank Dr. Denise Tieman for gas chromatography and Mark

Taylor for providing information about laboratory procedure and equipment. I would

especially like to thank Michelle Zeigler for instructing me in the techniques I used to

sample volatiles and quantify the gas chromatograph results.

I would like to thank those who provided physical assistance for me when there

were simply too many tomatoes to dice in the time allotted or when I needed an escort to

the cold rooms in the middle of the night, Caitlin Mirek, Anastasia "Ducky" Bower,

Jeanmarie Mitchell, Swapna Mony, and Gzregorz Cieslewski. I also express my

appreciation towards Mary Frances Folz Donahue and Sjon Myhr, not only for assisting









me with my research but also for the support and understanding they both showed me

throughout the writing process.

It takes a village to raise a scientist, apparently, but most importantly, I thank my

mother, Ellen Stahl, who is a staunch believer in the benefits of fresh fruits and

vegetables, for always expecting the best of me, for opening my mind to science at an

early age, and for buying me numerous books on exotic produce when I was a child.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF FIGURES ................................................ viii

ABSTRACT .................................................. ................. xviii

CHAPTER

1 IN T R O D U C T IO N .................................................................. .. ... .... ............... 1

T o m ato R ip en in g .......................................................................................................... 1
V olatile P rofi le of T om ato ........................................... .......... ............................ .. 1...
O bjectiv e s ........................................................................ ................................. . .2

2 LITER A TU R E R EV IEW .................................................................... ...............3...

G general Inform action .......................................................................................3.........
Flavor Chem istry .............. ...................... ................ ..... ..5....
E thylene .................................................... .............. .......................8
Inhibitors of Ethylene Synthesis A ction.................................................................. 11
Effects of 1-M CP on Fruit Volatiles..................................................... 14
Factors A affecting Tom ato V olatiles ...................................................... ................ 16

3 M A TERIALS AND M ETH OD S .......................................................... ................ 22

P la n t M ate ria l..............................................................................................................2 2
Fruit Color M easurem ents .................. ............................................................. 23
A analysis of W hole Fruit Firm ness........................................................ ................ 23
Evaluation of Volatile Profile of Ripe to Overripe Fruit.......................................23
Fruit Selection and C leaning ........................................ ............... ... ................ 24
Identifying Onset of Ripe Stage Using Color Measurements ..........................24
Determination of Fruit Developmental Stage for 1 -MCP Treatment......................24
Exposure of Tom ato Fruit to 1-M CP ............................................. ........................ 25
Analysis of the Effects of 1-MCP on Tomato Ripening and Volatile Profiles ..........26
A analysis of T om ato V olatiles ....................................... ...................... ................ 27
Statistical A n aly sis...................................................................................................... 2 8









4 RE SU LTS AN D D ISCU SSION ............................................................ ................ 29

Evaluation of Volatile Profile of Ripe to Overripe Fruit.......................................29
Determination of Fruit Developmental Stage for 1-MCP Treatment......................32
F atty-A cid D erived V olatiles ......................................................... ................ 35
C arotenoid D erived V olatiles ......................................................... ............... 37
A m ino A cid D erived V olatiles....................................................... ................ 38
V olatiles of O their D erivation ............................................................................. 39
Response of Tomato Fruit Color and Firmness to 1-MCP .........................................40
Tomato Volatile Production in Ripe, Overripe, and 1-MCP-treated 'FL-91'
T o m ato F ru it ........................................................................................................... 4 1
F atty-A cid D erived V olatiles ......................................................... ................ 42
C arotenoid D erived V olatiles ......................................................... ................ 44
A m ino A cid D erived V olatiles....................................................... ................ 45
V olatiles of O their D erivation ......................................................... ................ 47
General Discussion .................... .. ........... .....................................47

APPENDIX

A 'BNH586' HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS...............51

B 'FL-47' HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS..................59

C 'FL-91' HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS.................. 75

L IST O F R E F E R E N C E S ................................................................................................. 104

BIO GRAPH ICAL SK ETCH .................. .............................................................1...... 10















LIST OF FIGURES


Figure page

A-1. Hue angle of 'BHN586' tomato fruit during storage at 20C................................51

A-2. Hue angle of 'BHN586' tomato fruit during storage at 200C after attaining a hue
an g le o f 5 00 ............................................................................................................. 5 2

A-3. Fruit firmness (N) of 'BHN586' tomato fruit during storage at 200C after attaining
a h u e an g le o f 5 0 ..................................................................................................... 5 3

A-4. Cis-3-hexenal production in ripe 'BNH586' tomato fruit....................................54

A-5. Hexanal production in ripe 'BNH586' tomato fruit................................................ 55

A-6. Ethyl vinyl ketone production in ripe 'BNH586' tomato fruit............... ............... 56

A-7. B-ionone production in ripe 'BNH586' tomato fruit.............................................57

A-8. Isovaleraldehyde production in ripe 'BNH586' tomato fruit................................58

B-1. Firmness (N) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb,
12 h, 200C) at the pink stage and ripened at 200C. ............................................59

B-2. Firmness (N) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb,
12 h, 200C) at the turning stage and ripened at 20C..........................................60

B-3. Hue angle (0) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb,
12 h, 200C) at the pink stage and ripened at 200C. ............................................61

B-4. Hue angle (0) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb,
12 h, 200C) at the turning stage and ripened at 20C..........................................62

B-5. Hue angle (0) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250 ppb,
12 h, 20C) at the breaker stage and ripened at 20C. .......................................63

B-6. Cis-3-hexenal production in 'FL-47' tomato fruit.................................................64

B-7. Hexanal production in 'FL-47' tom ato fruit.......................................... ................ 65

B-8. Ethyl vinyl ketone production in 'FL-47' tomato fruit. .......................................66









B-9. Cis-3-hexenol production in 'FL-47' tomato fruit. ............................... ................ 67

B-10. Trans-2-hexenal production in 'FL-47' tomato fruit...........................................68

B-11. 1-ionone production in 'FL-47' tom ato fruit....................................... ................ 68

B-12. 6-methyl-5-hepten-2-one production in 'FL-47' tomato fruit...............................69

B-13. Isovaleraldehyde production in 'FL-47' tomato fruit..........................................70

B-14. 2-methylbutyraldehyde production in 'FL-47' tomato fruit................................71

B-15. 3-methyl- -butanol production in 'FL-47' tomato fruit..................................72

B-16. 2-phenylethanol production in 'FL-47' tomato fruit ........................... ................ 73

B-17. 1-nitro-2-phenylethane production in 'FL-47' tomato fruit. ...............................74

B-18. 2-isobutylthiazole production in 'FL-47' tomato fruit .......................... 74

C-1. Firmness (N) of'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb,
12 h, 200C) at the breaker stage and ripened at 200C. Days represent time since
treatment. Values represent the average of five fruit, +/- standard error ..............75

C-2. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb,
12 h, 200C) at the breaker stage and ripened at 200C. Days represent time since
treatment. Values represent the average of five fruit, +/- standard error ..............76

C-3 Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb,
12 h, 200C) at the breaker stage and ripened at 200C. Days represent time since
treatment. Values represent the average of five fruit, +/- standard error ..............76

C-4. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb,
12 h, 200C) at the breaker stage and ripened at 200C. Days represent time since
treatment. Values represent the average of five fruit, +/- standard error ..............77

C-5. Hue angle (0) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500 ppb,
12 h, 200C) at the breaker stage and ripened at 200C. Days represent time since
treatment. Values represent the average of five fruit, +/- standard error. C-5-a)
Experiment 1. C-5-b) Experiment 2. C-5-c) Experiment 3. C-5-d) Experiment 4...77

C-6. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at
which time an additional control (hue angle at 50 + 5 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............78









C-7. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 11 days to reach this hue value,
at which time an additional control (hue angle at 500 + 11 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............78

C-8. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 6 days to reach this hue value, at
which time an additional control (hue angle at 500 + 6 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............79

C-9. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 4 days to reach this hue value, at
which time an additional control (hue angle at 500 + 4 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............79

C-10. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 5 days to reach this hue value, at which
time an additional control (hue angle at 500 + 5 days) sample was analyzed.
Values represent the average of five fruit, +/- standard error. ............................... 80

C-11. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 11 days to reach this hue value, at
which time an additional control (hue angle at 500 + 11 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............80

C-12. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 6 days to reach this hue value, at which
time an additional control (hue angle at 500 + 6 days) sample was analyzed.
Values represent the average of five fruit, +/- standard error. ................................ 81

C-13. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 4 days to reach this hue value, at which









time an additional control (hue angle at 500 + 4 days) sample was analyzed.
Values represent the average of five fruit, +/- standard error. ................................81

C-14. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 5 days to reach this hue
value, at which time an additional control (hue angle at 500 + 5 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........82

C-15. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 50. 1-MCP treated fruit required an additional 11 days to reach this
hue value, at which time an additional control (hue angle at 500 + 11 days)
sample was analyzed. Values represent the average of five fruit, +/- standard
erro r. ...................................................................................................... ........ .. 8 2

C-16. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue
value, at which time an additional control (hue angle at 500 + 6 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........83

C-17. Ethyl vinyl ketone production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 50. 1-MCP treated fruit required an additional 4 days to reach this hue
value, at which time an additional control (hue angle at 500 + 4 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........83

C-18. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
50. 1-MCP treated fruit required an additional 5 days to reach this hue value, at
which time an additional control (hue angle at 500 + 5 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............84

C-19. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
50. 1-MCP treated fruit required an additional 11 days to reach this hue value,
at which time an additional control (hue angle at 500 + 11 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............84









C-20. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 6 days to reach this hue value, at
which time an additional control (hue angle at 500 + 6 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ..............85

C-21. Trans-2-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 4 days to reach this hue value, at
which time an additional control (hue angle at 500 + 4 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ..............85

C-22. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 5 days to reach this hue value, at
which time an additional control (hue angle at 500 + 5 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ..............86

C-23. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 11 days to reach this hue value,
at which time an additional control (hue angle at 500 + 11 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error .............86

C-24. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 6 days to reach this hue value, at
which time an additional control (hue angle at 500 + 6 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ..............87

C-25. Cis-3-hexenol production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 4 days to reach this hue value, at
which time an additional control (hue angle at 500 + 4 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ..............87

C-26. B-ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 5 days to reach this hue value, at which









time an additional control (hue angle at 500 + 5 days) sample was analyzed.
Values represent the average of five fruit, +/- standard error. ................................88

C-27. B-ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 11 days to reach this hue value, at
which time an additional control (hue angle at 500 + 11 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............88

C-28. B-ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 6 days to reach this hue value, at which
time an additional control (hue angle at 500 + 6 days) sample was analyzed.
Values represent the average of five fruit, +/- standard error. ............................... 89

C-29. B-ionone production in ripe 'FL-91' tomato fruit. Fruit were treated with air or 1-
MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 500. 1-
MCP treated fruit required an additional 4 days to reach this hue value, at which
time an additional control (hue angle at 500 + 4 days) sample was analyzed.
Values represent the average of five fruit, +/- standard error. ............................... 89

C-30. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 5 days to reach this hue
value, at which time an additional control (hue angle at 500 + 5 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........90

C-31. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 11 days to reach this
hue value, at which time an additional control (hue angle at 500 + 11 days)
sample was analyzed. Values represent the average of five fruit, +/- standard
erro r ....................................................................................................... ........ .. 9 0

C-32. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 6 days to reach this hue
value, at which time an additional control (hue angle at 500 + 6 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........91









C-33. 6-methyl-5-hepten-2-one production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 4 days to reach this hue
value, at which time an additional control (hue angle at 500 + 4 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........91

C-34. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 5 days to reach this hue value, at
which time an additional control (hue angle at 500 + 5 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............92

C-35. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 11 days to reach this hue value,
at which time an additional control (hue angle at 500 + 11 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............92

C-36. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 6 days to reach this hue value, at
which time an additional control (hue angle at 500 + 6 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............93

C-37. Isovaleraldehyde production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 4 days to reach this hue value, at
which time an additional control (hue angle at 500 + 4 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............93

C-38. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 5 days to reach this hue
value, at which time an additional control (hue angle at 500 + 5 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........94

C-39. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 11 days to reach this
hue value, at which time an additional control (hue angle at 500 + 11 days)









sample was analyzed. Values represent the average of five fruit, +/- standard
erro r ....................................................................................................... ........ .. 9 4

C-40. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 6 days to reach this hue
value, at which time an additional control (hue angle at 500 + 6 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........95

C-41. 3-methyl-1-butanol production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 4 days to reach this hue
value, at which time an additional control (hue angle at 500 + 4 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........95

C-42. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 5 days to reach this hue
value, at which time an additional control (hue angle at 500 + 5 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........96

C-43. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 11 days to reach this
hue value, at which time an additional control (hue angle at 500 + 11 days)
sample was analyzed. Values represent the average of five fruit, +/- standard
erro r ....................................................................................................... ........ .. 9 6

C-44. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 6 days to reach this hue
value, at which time an additional control (hue angle at 500 + 6 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........97

C-45. 1-nitro-2-phenylethane production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 4 days to reach this hue
value, at which time an additional control (hue angle at 500 + 4 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error...........97









C-46. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 5 days to reach this hue value, at
which time an additional control (hue angle at 500 + 5 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............98

C-47. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 11 days to reach this hue value,
at which time an additional control (hue angle at 500 + 11 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............98

C-48. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 6 days to reach this hue value, at
which time an additional control (hue angle at 500 + 6 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............99

C-49. 2-phenylethanol production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 4 days to reach this hue value, at
which time an additional control (hue angle at 500 + 4 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error ...............99

C-50. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 5 days to reach this hue
value, at which time an additional control (hue angle at 500 + 5 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.........100

C-51. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 11 days to reach this
hue value, at which time an additional control (hue angle at 500 + 11 days)
sample was analyzed. Values represent the average of five fruit, +/- standard
erro r .................................................................................................... ........... 10 0

C-52. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 50. 1-MCP treated fruit required an additional 6 days to reach this hue









value, at which time an additional control (hue angle at 500 + 6 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.........101

C-53. 2-methylbutyraldehyde production in ripe 'FL-91' tomato fruit. Fruit were
treated with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened
at 200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 4 days to reach this hue
value, at which time an additional control (hue angle at 500 + 4 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.........101

C-54. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 5 days to reach this hue
value, at which time an additional control (hue angle at 500 + 5 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.........102

C-55. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 11 days to reach this
hue value, at which time an additional control (hue angle at 500 + 11 days)
sample was analyzed. Values represent the average of five fruit, +/- standard
erro r .................................................................................................... ............ 10 2

C-56. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 6 days to reach this hue
value, at which time an additional control (hue angle at 500 + 6 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.........103

C-57. 2-isobutylthiazole production in ripe 'FL-91' tomato fruit. Fruit were treated
with air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 500. 1-MCP treated fruit required an additional 4 days to reach this hue
value, at which time an additional control (hue angle at 500 + 4 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.........103















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

INFLUENCE OF ETHYLENE SUPPRESSION AND RIPENING ON TOMATO
FRUIT COLOR, FIRMNESS, AND VOLATILE EMISSIONS

By

Sonya Leonore Stahl

December 2006

Chair: D. J. Huber
Major Department: Horticultural Sciences

Ripening parameters of tomato (Lycopersicon esculentum 'BHN586', 'FL-47', and

'FL-91') were evaluated to determine the effects of ethylene suppression on color,

firmness, and volatile emissions.

Tomato fruit 'BHN586' were obtained at turning stage and held at 200C until ripe.

Fruit color, firmness, and volatile emissions were measured over a period of 16 d

following ripening. Hue angle did not change significantly in the 16 d post ripening, but

firmness decreased from 5 to 3 N. Production of cis-3-hexenal decreased and ethyl vinyl

ketone increased as the fruit progressed from ripe to overripe, and hexenal production fell

and rose again. 8-ionone production remained low during the first week after attainment

of ripe stage, but then rose sharply through day 11 and remained high through day 16.

Tomato fruit 'FL-47' were obtained at green, breaker, turning, and pink stages,

stored at 200C, and treated with air or 1-methylcyclopropene (1-MCP) at 250 ppb for 12

h at 200C to determine the stage at which ethylene suppression would most delay color


xviii









change but not prevent ripening altogether. Breaker was determined to be the optimal

stage for this treatment regimen, due to the difficulty of distinguishing between immature

and mature green fruit, and the lack of response to 1-MCP exhibited by pink or turning

fruit in this experiment. Production of C6 volatiles (cis-3-hexenal, cis-3-hexenol, trans-2-

hexenal, and hexanal) was higher in ripe fruit derived from 1-MCP-treated breaker fruit

than in ripe control fruit, but production of the carotenoid-derived volatiles (8-ionone and

6-methyl-5-hepten-2-one) was unaffected by 1-MCP treatment at breaker stage. Amino

acid derived volatile production was not affected consistently by 1-MCP:

isovaleraldehyde and 3-methyl-l-butanol production decreased but 1-nitro-2-

phenylethane production increased as a result of 1-MCP treatment.

Tomato fruit 'FL-91' were treated at breaker stage with air or 500 ppb 1-MCP for

12 h at 200C and held until attainment of a hue angle below 500, signifying full ripe. Red

color developed more slowly and with less uniformity in 1-MCP-treated fruit, and

softening was also delayed by treatment with 1-MCP. Upon reaching full ripe, both ripe

control and ripe fruit derived from 1-MCP treated breaker fruit were sampled for volatile

production. A second group of control fruit was held until overripe so that volatile

production of untreated fruit could be quantified at the same chronological age as treated

fruit. Where differences were found between treatments, they were often inconsistent for

individual volatiles between experimental replications. Cis-3-hexenal production was

observed to decrease in 1-MCP-treated fruit in some instances, but the C6 volatiles were

largely unaffected by 1-MCP treatment. In most cases, carotenoid-derived volatiles were

also unaffected by 1-MCP treatment, and the effect of 1-MCP treatment on amino-acid

derived volatiles was inconsistent and varied.














CHAPTER 1
INTRODUCTION

Tomato Ripening

The tomato (Lycopersicon esculentum) is a solanaceous fruit that is important to

the agricultural economy of the United States of America, and especially in Florida.

Many developmental parameters are altered during tomato fruit ripening. Color change

occurs as chlorophyll breaks down and lycopene production is initiated. Fruit firmness

decreases to a softness acceptable for consumption. The development of red color and the

softening of tomato fruit tissue can be delayed by 1-methylcyclopropene (1-MCP), a

cyclic alkene that volatizes as a non-toxic, odorless gas. 1-MCP acts by suppressing the

response to the hormone ethylene, thereby preventing or delaying processes dependent on

ethylene. 1-MCP can therefore be used to determine which processes are directly

regulated by that hormone.

Volatile Profile of Tomato

The flavor of tomatoes is derived from a combination of sugars, acids, and aroma

volatiles. While sugars and acids are perceived by the gustatory receptors on the tongue,

aroma volatiles are perceived by olfactory receptors and are therefore a 'smell' rather

than a 'taste' contributor to tomato flavor. The volatile constituents most important to

tomato flavor are the C6 compounds hexanal and cis-3-hexenal, which contribute a

"green, grassy" odor that was also identified as "sweet", "ripe", and "tomato flavor".

These, and other related compounds, are derived from fatty acids; however, other









important volatiles in tomato are derived from the metabolism of carotenoids and amino

acids.

Volatile emissions in tomato are affected by a number of parameters throughout

tomato development. Fertilizer regime, maturity at time of harvest, and postharvest

handling such as bruising or storage at low or high temperatures can have profound

effects on the volatile profile. Choice of cultivar can also influence volatile emissions, as

some varieties are low-quality and produce less aroma.

Objectives

The primary objective of this study was to determine whether the volatile profile of

tomato fruit would be altered by treatment with an inhibitor of ethylene action. The first

objective was to evaluate the volatile emissions in ripe tomato fruit, continuing through

overripening. Another objective was to determine the ripening stage at which to treat

tomatoes with 1-MCP which would have the most pronounced effect on delaying

development of ripe color, but would still allow fruit to continue to attain a full ripe stage.

Then tomato volatile emissions in ripe, overripe, and ripe fruit derived from breaker fruit

treated with an ethylene action inhibitor were evaluated and compared within treatments

to determine if volatile production in treated fruit was more similar to ripe or to overripe

untreated fruit. It was also of interest to employ 1-MCP treatment as a method of

discovering which volatile pathways were directly dependent on ethylene.














CHAPTER 2
LITERATURE REVIEW

General Information

The tomato (Lycopersicon esculentum) is a member of the family Solanaceae. The

genus name, Lycopersicon, is derived from a Greek word meaning "wolf peach" (Davies

& Hobson, 1981). The fruit is a berry that is commonly eaten as a fresh or cooked

vegetable and also has numerous applications in processed foods, such as ketchup,

marinara sauce, and pre-prepared soups. It is a perennial plant, but it is grown as an

annual. In the United States of America, it is second only to its relative the potato as the

most abundantly produced of any vegetable. Nearly thirteen million metric tons of

tomatoes were grown in the United States in 2005 (FAO 2006, http://faostat.fao.org/).

The majority of the fresh-market cultivars are the familiar large round red varieties, but

plums, clusters, round yellow, cherry, grape, and other specialty sizes are increasing in

popularity (Sargent and Moretti, 2004).

The average American eats over nineteen pounds of fresh tomato every year

(http://www.floridatomatoes.org/facts.html). It is third in popularity among vegetables,

under the potato and lettuce. Tomatoes are a plentiful source of vitamins C and A, certain

minerals, and antioxidants, especially lycopene, the pigment responsible for the color of

red tomatoes. The tomato was legally declared a vegetable in 1893 in a Supreme case

centering on tariffs (Nix v. Hedden, 149 U.S. 304, 1893).

Over four and a half million hectares of the world are devoted to tomato production

(http://www.floridatomatoes.org/facts.html). Seventeen thousand hectares of this are in









the state of Florida, which is half of the fresh tomatoes produced in the United States.

This represents a 660 million dollar value to Florida farmers each year and jobs for 33

thousand workers (http://www.floridatomatoes.org/facts.html).

The tomato fruit, a berry originating from a single, inferior, multi-locular ovary,

undergoes experience a period of sigmoidal expansion and maturation prior to exhibiting

the characteristic features of ripening. In this pre-ripening developmental period, the

tomato fruit remain green. Upon completion of physical growth, the fruit attains a

developmental state typically referred to as 'green' (Sargent and Moretti, 2004). For

descriptive purposes, this green period is divided into four stages, Ml through M4. At the

Ml stage, the locular tissue is firm and the seeds are immature (seeds are white and can

be easily cut by passing a sharp blade through the fruit.) At the M2 stage, the seeds have

matured and are tan, and gel is present in at least two locules. At the M3 stage, well

developed gel is present in all of the locules, and the internal color is still green. The

green maturation process concludes with 'mature green', which is the stage at which red

color appears in the locule gel and internal, radial pericarp and columnella tissues

(Sargent and Moretti, 2004, adapted from Kader and Morris 1976). Fruit harvested prior

to attaining a physiologically mature, 'green' stage will continue to ripen, but will not

attain the maximum potential quality (Hurr, Huber, and Lee, 2005).

The developmental period beyond the mature green stage through the full red-ripe

stage has been divided for descriptive purposes into four stages based on the

accumulation of surface lycopene distribution (USDA 1991:

http://www.ams.usda.gov/standards/tomatfrh.pdf). Each stage indicates further

breakdown of chlorophyll and accumulation of lycopene. Breaker fruit are mostly green









but show a blush of red or pink around the blossom end that constitutes less than ten

percent of the fruit surface. Turning fruit have a larger area of red development, between

10% and one third of the surface. Pink fruit are between one and two thirds red, and light

red are between two thirds and 90% red. When the tomato reaches the full red stage, it is

table-ripe and ready to eat.

In commercial practice, tomatoes are usually hand-harvested unripe to prevent

bruising and spoilage during storage and transportation. Care is taken to keep damaged,

defective, immature, or fruit of an advanced stage of ripening-which will rot more

quickly--out of postharvest channels to reduce the need for grading later on. Once out of

the field and into the packinghouse, the fruit are washed in a chlorinated dump tank and

then brushed, rinsed, and dried. Sometimes they are also waxed (Sargent, Brecht, and

Olczyk, 2005 http://edis.ifas.ufl.edu/VH079). Tomato sorting occurs along a conveyor

belt, as workers remove unsatisfactory fruit and classify the tomatoes by size and quality.

In Florida, the quality grades are 'U.S. No. 1', '85% U.S. No. 1', 'U.S. Combination', and

'U.S. No. 2', and the sizes are '6x7' (between 2 9/32 and 2 19/32 inches in diameter), '6x6'

(between 2 17/32 and 2 29/32 inches in diameter), and '5x6' (minimum diameter 2 25/32

inches) (http://www.floridatomatoes.org/domestic.html, USDA 1991:

http://www.ams.usda.gov/standards/tomatfrh.pdf). The fruit are then packed into cartons

and stacked into pallets. They are ripened by exposure to ethylene. This initiates ripening

at a commercially desirable point in the supply chain and also assures uniform ripening of

the harvest.

Flavor Chemistry

Flavor is an important component of a tomato's desirability, but with increasing

focus from breeders on quality parameters including color, firmness, resistance to









disease, yield, size, and physical uniformity, many of today's tomato cultivars do not

meet consumer standards for good taste (Hobson, 1988). Poor flavor components include

sourness, bite (the stinging present after imbibing carbonated beverages), a 'green' taste

reminiscent of unripe fruit, low pH, and most relevant to this discussion, low levels of

many aromatic volatiles (Tandon, Baldwin, Scott, and Shewfelt, 2003). Stevens, Kader,

Albright-Holton, and Algazi (1977) reported that since it is more fluid than the other

tomato tissue, qualities of the locule gel are more readily perceived by taste receptors on

the tongue and it is therefore a more significant contributor to tomato flavor than either

pericarp or placental tissue.

The flavor of a ripe tomato is created by a complex combination of sugars, acids,

and aroma volatiles (Krumbein and Auerswald, 1998). Tandon et al. (2003) report that

fructose and glucose are the sugars most relevant to tomato flavor. These sugars comprise

approximately three percent of the fresh weight of a ripe tomato, increasing as the fruit

ripens (Tandon et al., 2003; Davies and Hobson, 1981). The sweetness of tomato fruit

positively correlates to the amount of these sugars.

Organic acids are another important constituent of tomato flavor. Citric acid is the

most abundant, at a concentration of 135-675 micrograms per gram fresh weight

(mg/gfw) (Davies and Hobson, 1981). Citric acid is most plentiful in the locular gel,

where it is present in concentrations between 634-1560 mg/100 gfw. It is followed by

malic acid, which is present in concentrations between 21 and 225 mg/100 gfw in the

whole fruit and 30-412 mg/100 gfw in just the locular gel (Davies and Hobson, 1981). As

also reported by Davies and Hobson (1981), amino acids are also present, the most

numerous in tomato fruit being glutamic acid, arginine, aspartic acid, and asparagine.









Aroma volatiles are present in very low concentrations--nanoliters per liter of

blended fresh tomato fruit tissue--but are still very important to flavor. They are

perceived by olfactory receptors in the nose, not by the taste receptors in the tongue

(Acree, 1993). According to Buttery (1993), over four hundred volatiles have been

identified in tomato fruit, but only around thirty of them comprise a significant portion of

perceived flavor. These include various aldehydes, ketones, and alcohols. The volatiles

that appear in the highest concentrations are cis-3-hexenal, hexanal, and 2-phenylethanol.

Volatiles can be classified according to their synthetic pathways and the

compounds from which they originate. Some, like hexanal, cis-3-hexenal, trans-2-

hexenal, cis-3-hexenol, and ethyl vinyl ketone, are derived from fatty acids (Buttery &

Ling, 1992) including linolenic and linoleic acids (Goff and Klee, 2006). These lipid-

derived volatiles are present in both leaves and fruit tissue. The maceration and blending

with CaCl2 of tomato leaves by Buttery and Ling (1992) and the consequent sampling of

the resultant volatiles revealed high levels of cis-3-hexenal in the leaf tissue. This

indicates that cis-3-hexenal is the volatile responsible for the green, grassy scent of

tomato leaves. Buttery and Ling (1992) also sampled the cis-3-hexenal produced by

tomato fruit tissue and found that it increased as the fruit ripened. The compound was

found at a concentration of 0.1 parts per million (ppm) in mature green tomato, 0.3 ppm

at breaker stage, and 15 ppm in ripe fruit.

Buttery and Ling (1992) also identify volatiles, including 8-ionone and 6-methyl-5-

hepten-2-one, which are formed from carotenoids. 8-ionone is a cyclic compound,

whereas 6-methyl-5-hepten-2-one is an open chain formed by oxidative cleavage of

lycopene (Goff and Klee, 2006). Carotenoid-derived volatiles are found only in the fruit,









and not in leaf, flower, or stem tissue. There are also volatiles that are derived from

amino acids. These are found exclusively in the fruit and are formed between the breaker

and full-ripe stage (Buttery & Ling, 1992). For example, 1-nitro-2-phenylethane and 2-

phenylethanol are formed from phenylalanine, isovaleraldehyde (3-methylbutanal) is

formed from leucine, and 2-methylbutanal is formed from isoleucine. The pathways of

some, like 2-isobutylthiazole, are unknown (Goff and Klee, 2006) although Schutte

(1974) suggests that 2-isobutylthiazole may also originate from leucine via

isovaleraldehyde.

Trained taste panelists have identified relationships between specific volatiles and

certain facets of tomato flavor. Krumbein and Auerswald (1998) identified cis-3-hexenal

as one of the most powerful components of flavor, producing a "fresh green" and "sweet"

flavor. Hexanal was characterized as "green" and "grassy" by Krumbein and Auerswald

(1998), and as "sweet", "ripe", and "tomato flavor" by Maul et al. (2000). 2-

isobutylthiazole was described in Krumbein and Auerswald (1998) as producing both

sweet and fruity flavors and causing a "bite" feeling in the mouth. It was also identified

as "musty". B-ionone contributed to a sour taste, and trans-2-heptenal and 1-penten-3-one

(ethyl vinyl ketone) produced a "green" aroma. 3-methylbutanal (isovaleraldehyde) was

described as "unpleasant" (Krumbein and Auerswald, 1998; Baldwin, Scott, Shewmaker,

and Schuch, 2000).



Ethylene

Ethylene (C2H4) is the structurally simplest of the plant hormones, as well as being

the simplest alkene. It is derived from methionine via a pathway elucidated in detail by

Yang and Hoffman (1984). The regulatory effects of ethylene on plant development are









numerous and varied. They include initiation of flower opening, seed germination, and

leaf/flower senescence, and control of responses to both biotic and abiotic stresses (Yang

and Hoffman, 1984). In climacteric fruit, those that experience an increase in respiration

rate and ethylene production accompanying ripening, ethylene also regulates changes

associated with fruit ripening (Yang and Hoffman, 1984). For thousands of years,

humans have augmented the natural ethylene production of crops with their own

artificially added doses in order to produce a uniform ripening response. For example,

Theophrastus, who lived in the third century B.C.E., reports that agriculturists of his time

damaged the skin of figs in order to initiate ripening through what is now understood to

be a method of stimulating ethylene production via a wounding response (Galil, 1968).

The process of wounding figs to promote ripening is still performed by present-day

Egyptian farmers. In more modern times, citrus fruit such as oranges were placed in close

proximity to burning kerosene in order to achieve degreening necessary for a brilliantly-

colored orange or yellow product. Chace and Denny (1924) determined that the active

factor in the burning kerosene was ethylene gas. In studies of apple fruit, Gane (1934)

was the first to demonstrate that ethylene was a natural product of plant tissues.

The biosynthesis of ethylene begins with the amino acid methionine, which is

converted to S-adenosylmethionine (SAM) by SAM synthetase (E.C. 2.5.1.6). SAM is

converted to 1-aminocyclopropane-1-carboxylic acid, or ACC, by ACC synthase (E.C.

4.4.1.14) also called ACS (Yang and Hoffman, 1984). It then is oxidized by ACC oxidase

(E.C. 1.14.17.4), also called ACO and once referred to as the ethylene forming enzyme

(EFE), in order to produce ethylene (Alexander and Grierson, 2002).









Both the prevention of ethylene synthesis and the suppression of either ethylene

perception or the response to ethylene interfere with ripening and other senescence

processes. This was demonstrated by Brandt and Woodson (1992) by examining

genotypic variation in carnation flowers. Cultivars with low amounts of ACC, indicating

reduced activity of ACS, exhibited extended vase-life by an average of five days

compared with a cultivar with normal levels of ethylene and its precursors. In addition,

low activity of ACO, referred to in this study as EFE, the ethylene forming enzyme [as it

was known before activity was obtained in vitro by Ververidis and John (1991)], resulted

in even lower levels of ethylene in certain cultivars. One of these cultivars, 87-37G-2,

responded to exogenously applied ethylene by finally producing its own ethylene and

senescing earlier than untreated controls, indicating that its deficiency was in ethylene

synthesis, not perception. Another synthesis-deficient cultivar, 799, failed to respond to

external ethylene, indicating that is was both a perception and a synthesis mutant. It is

clear that interfering with ethylene synthesis and/or perception will result in delayed,

reduced, or halted senescence activities.

Although carnation served as an early model for exploration of ethylene effects,

ripening fruits have become increasingly the system of choice. Carnations are still

investigated as a floral system, but the bulk of the molecular work dealing with ethylene

perception and action has been performed with fruit tissue. For example, the Never-ripe

tomato is a mutant that is unable to perceive ethylene because of a mutation to its

ethylene receptor. The altered receptor is unable to bind to ethylene, leaving it unengaged

and able to repress ethylene responses even in the presence of the hormone (Hackett et

al., 2000). Hackett et al. (2000) further demonstrated that inhibiting the Nr gene using









antisense constructs enabled the mutant tomatoes to recover ethylene perception and

ripen normally.

Inhibitors of Ethylene Synthesis Action

One method of preventing the effects of ethylene on both cut flowers and edible

crops is to employ an ethylene scrubber made of cement, silica gel, aluminum pellets, and

more recently, potassium permanganate. The scrubbers absorb the ethylene from the

surrounding atmosphere and prevent it from being absorbed by the product. Jayaraman

and Raju (1992) found the potassium permanganate-based scrubber more effective than

previously used materials in extending the shelf life of tomato, mango, and okra by a

period of between three to eight days.

Silver thiosulfate (STS) is a compound that interferes with ethylene perception

(Veen & Geijn, 1978; Bleecker & Kende, 2000). Ethylene is believed to interact with a

copper cofactor in the receptor. It has been speculated that if silver is present, it takes the

place of copper and interacts with the ethylene instead. While it has the same ability as

copper to interact with ethylene, it lacks the ability to complete the receptor's function as

copper does, disabling the receptor (Rodriguez et al., 1999). However, Veen and Geijn

(1978) observed brown, necrotic spots on the leaves resulting from the toxic effects of

high concentrations of silver. Silver is a toxic heavy metal that can causes injury even in

small amounts because it binds to sulfhydryl groups non-discriminately. It may have

effects of which science is still unaware. The toxicity of STS restricts it to use with

flowers only, not for food crops, and in some countries its use or method of application

have been restricted due to concerns about environmental pollution (Serek & Reid, 1993).

Sisler, Reid, and Yang (1986), Serek, Sisler, and Reid (1994), and Sisler and

Blankenship (1993) examined a wide range of cyclic olefins in search of compounds that









might serve as antagonists or agonists of ethylene reception. 2,5-norbornadiene (NBD)

and diazocyclopentadiene (DACP) were shown to have effects on the ethylene receptor,

but they were deemed unsuitable for practical use. In the case of the former, the

compound must be present continually in order to be effective (which is impractical for

the supply chain of most produce), and in the case of the latter it was highly explosive.

NBD also has a foul odor which further limits its desirability (Serek et al., 1994).

1-Methylcyclopropene, or 1-MCP, is a non-toxic, odorless gas that is effective in

rendering ethylene receptor molecules inactive after short-term treatment. 1-MCP is a

simple molecule, a cyclic alkene with a single methyl group attached to the olefinic

carbons. 1-MCP apparently acts by non-competitively binding to the ethylene receptor

because it has a lower binding constant, Kd=8 nl*liter-1 (Serek et al., 1994). It is

presumed that by blocking ethylene binding, 1-MCP causes the receptor to remain

engaged, maintaining the downstream cascade that suppresses ethylene-induced genes

(Sisler and Serek, 1997).

The effectiveness of 1-MCP at extending the shelf life of many fruit and vegetable

crops has been well documented. It has been approved for commercial use on

horticultural crops since 2002, starting with apples (American Fruit Grower, 2002), and

has now been approved for numerous other crops, including the tomato. Effects such as a

delay in softening or in the development of ripe coloration have also been demonstrated

on banana, avocado, melon, papaya, persimmon, and other crops, both climacteric and

nonclimacteric (reviewed in detail in Blankenship & Dole, 2003). Commercially, it is

available as Etli)lBloc for ornamentals and SmartFresh for edible crops. It is presently

used as a gas that is volatized when a powder formulation is added to water, but a liquid









application is currently being investigated (Todd Eddington, Agrofresh, personal

communication).

1-MCP inhibits many parameters of ripening, including ethylene synthesis,

ethylene perception, loss in firmness, and changes in pigmentation. The effects can either

be permanent or dissipate after a period of 1 to 2 weeks, depending on the concentration

used and the period of exposure. The fact that 1-MCP interferes with perception of

ethylene makes it a useful tool to study which ripening processes are ethylene dependent

and which occur independent of ethylene. Most research with the effects of 1-MCP has

focused on firmness and color. For example, tomato fruit were treated with 1 ppm 1-MCP

for 24 h at 200C. It was determined that the stage at which a tomato fruit is treated can

determine the effectiveness of the 1-MCP treatment in delaying pigmentation changes

associated with ripening. Tomatoes treated at mature green or breaker stage delay

development of external red color for a week, and then ripen normally (Hurr et at., 2005).

1-MCP produces a delay in the loss of firmness associated with ripening. The

amount of force required to compress the external tissue of a tomato a distance of 2 mm

ranges from 20 Newtons (N) at the mature green stage to four or five N at full red ripe. 1-

MCP causes this softening to be delayed from five to ten days later than usual (depending

on the developmental stage at which the 1-MCP was applied). One possible explanation

is that the enzymes polygalacturonase and cellulase, which are responsible for the

breakdown of the pectin and cellulose that provide structural support for plant cells, are

affected by ethylene suppression. The blocking of ethylene perception in avocado

interferes with the activity of polygalacturonase and cellulase, but softening is inhibited

only during the later stages of ripening with little effect during early to mid ripening









(Jeong, Huber, and Sargent, 2002; Jeong and Huber, 2004). This seems to indicate that

the softening that occurs during early ripening may be largely ethylene independent,

whereas the softening during overripening may be ethylene dependent since it can be

stopped by the inhibition of ethylene recognition. A tomato transgenically altered with an

antisense gene for polygalacturonase exhibited a definite decrease in postharvest

softening compared with wild-type fruit (Kramer et al., 1992). There is a point at which

ripening can be delayed excessively, however, where fruits do not soften sufficiently and

become shriveled and decayed prior to ripening.

Effects of 1-MCP on Fruit Volatiles

Documented effects of 1-MCP on fruit volatile profile are variable and dependent

on the species of fruit and specifics of treatment (concentration, duration of exposure, and

temperature). Golding, Shearer, McGlasson, and Wyllie (1999) reported a decrease in

total volatile production of bananas ripened at 200C after treatment with 1-MCP (45 ppm,

1 h) at a mature green stage compared with ripe untreated bananas. This decrease was

attributed mainly to a decrease in ester production. An increase in alcohol production in

the treated fruit, compared with control fruit, was also observed. Golding et al. (1999)

suggested that this increase in alcohols was due to a disruption of the final step in the

formation of esters, in which alcohols are esterified by the enzyme alcohol acyl

transferase, also known as AAT (E.C. 2.3.1.84) (Yahyaoui et al., 2002). Two esters, 3-

methylbutyl isovalerate and pent-2-yl butanoate, increased in treated fruit compared with

control fruit, implying that the enzyme remained functional and that availability of the

substrate acyl CoA was the limiting factor in ester production (Golding et al., 1999).

Pelayo, Vilas-Boas, Benichou, and Kader (2003) reported that ester production was

delayed, but not reduced, in bananas treated with 1-MCP (1 ppm, 24 h), and stored at









200C, compared with untreated bananas. Aldehyde loss was also delayed in treated fruit

compared with control fruit. The concentration of 1-MCP applied is critical. Bagnato,

Barrett, Sedgley, and Klieber (2003) found that when bananas were treated at 300 ppb,

ripening was delayed and the flavor was unaffected. Too low of a concentration (3 ppb)

did not extend shelf life and too high of a concentration (30 ppm) prevented ripening

entirely.

Apples have also been studied for the response of aroma volatiles to 1-MCP.

During ripening of untreated apples, acetates and some esters increase and alcohols and

aldehydes decrease (Lurie, Pre-Aymard, Ravid, Larkov, and Fallik, 2002). When 'Anna'

apples were treated with 1-MCP (1 ppm, 20 h) at 200C, the volatile profile associated

with harvest time was retained. Taste panelists preferred the treated apples, which tasted

freshly harvested, to the control apples, which displayed the characteristic aroma of very

ripe apples.

In another study of apples using the cultivar 'Borkh', Defilippi, Dandekar, and

Kader (2004) reported delayed and reduced esters when fruit were treated with 1-MCP (1

ppm, 20 h) at 20C just after harvest, but aldehydes were unaffected. Data from adding

ethylene back to transgenic fruit with the inability to synthesize it on their own implied

that a continuous supply of ethylene is necessary for volatile synthesis (Defilippi et al.,

2004).

Abdi, McGlasson, Holford, Williams, and Mizrahi (1998) used 1-MCP to

determine whether parameters of plum ripening, such as the development of skin color

and production of aroma volatiles, were ethylene dependent or independent. The cultivar

'Beauty' was treated with 1-MCP (26 and 39 ppm, 24 h) at 200C and developed aroma









volatiles one week later than untreated plums, according to a taste panel. When the

cultivar 'Shiro' received the same treatment, only the untreated fruit developed aroma

volatiles at all.

Mitcham, Mattheis, Bower, Biasi, and Clayon (2001) found that European pears

responded to various concentrations of 1-MCP with suppressed production of aroma

volatiles, assessed subjectively but no quantities were recorded. Once the effects of the

treatment wore off, volatiles began to form normally.

The processes regulated by ethylene are complex, and the response of a particular

species or cultivar to 1-MCP, a powerful growth regulator, is not always predictable. This

limits its commercial application and indicates that it is not suitable for every

circumstance.

Factors Affecting Tomato Volatiles

The quantities and composition of volatiles in tomato fruit are highly dependent on

genetic and environmental factors active throughout fruit development. Volatiles can

vary between cultivars. For example, the variety 'FL-47' is known to be low-volatile, and

consequently, low-quality (Tandon et al., 2003).

Preharvest conditions such as fertilizer regime can also affect the tomato's volatile

production. The amount of aroma volatiles including hexenal, phenylacetaldehyde,

benzaldehyde, 8-ionone, and 6-methyl-5-hepten-2-one was increased by elevated levels

of nitrogen and potassium (Wright and Harris, 1985). This increase had a negative impact

on tomato flavor (Wright and Harris, 1985).

Fruit maturity at harvest can influence the volatile profile. Maul et al. (1998)

determined that tomato fruit that were harvested immature green, as indicated by the fruit

taking 5 days to reach breaker stage in ethylene treatment, displayed lower levels of









many aroma volatiles, including cis-3-hexenal, 6-methyl-5-hepten-2-one, 2-

isobutylthiazole, and ethyl vinyl ketone, compared with tomato fruit that were harvested

mature green and only took one day to break in ethylene treatment.

After the tomato is harvested, ideally, care should be taken by the handlers and

packers to ensure that an ideal volatile composition is preserved. Appropriate storage

temperature is an important component of postharvest handling. The temperature during

the final stages of ripening has the greatest effect on the fruit volatile profile once ripe.

Maul et al. (2000) found that too low of a storage temperature will cause an increase in

off-flavors and a decrease in aroma. Storage at a temperature below 12.50C for even two

days in the case of cv. 'BHN-189' or four days in the case of cv. 'Solimar' resulted in a

loss of flavor quality, as evaluated by a trained taste panel. The quality was further

reduced if the storage temperature was 50C. The main loss in aroma volatiles consisted of

a reduction in the lipid-derived volatiles (hexanal, cis-3-hexenal, ethyl vinyl ketone,

trans-2-hexenal, trans-2-heptenal, and cis-3-hexenol) that accompanied the lower storage

temperatures. The carotenoid-derived volatiles (B-ionone and 6-methyl-5-hepten-2-one)

were also reduced but only slightly. Tomatoes held at 20C achieved higher ratings from

the taste panel in aroma, flavor, and intensity of sweetness, even after twelve days of

storage.

In another study, Boukobza and Taylor (2002) compared low temperature (60C),

moderate temperature (210C and 350C), and high temperature (450C) storage

environments. The tomatoes were held at the trial temperature for three days (in the low

temperature vs moderate temperature experiment) or six hours (in the high temperature vs

moderate temperature experiment) before being transferred to 210C for 4-6 hours to









mimic retail conditions. Low-temperature storage caused an eventual decrease in the

amounts of isobutylthiazole, methylbutanal, and methylbutanol, possibly due to a

reduction in enzyme activity or a decreased level of available amino acid precursors.

High-temperature storage reduced the amounts of hexanal, hexenal, hexenol,

isobutylthiazole, methylbutanal, methylbutanol, and 6-methyl-5-hepten-2-one, an effect

believed to be due to the denaturing effect of the heat on the enzymes of the lipid

oxidation pathway from which the volatiles would have formed. The reduction in aroma

volatiles was aggravated by increasing the temperature further or increasing the duration

of holding at 210C.

Bruising can cause changes in a tomato's volatile profile, most notably a reduction

in the amounts of cis-3-hexenal, 6-methyl-5-hepten-2-one, ethyl vinyl ketone, cis-3-

hexenol, and 2-isobutylthiazole. These changes were identified by comparing bruised

locular tissue to unbruised locular tissue. Placental tissue responded to bruising with

increased levels of ethyl vinyl ketone and 8-ionone compared with unbruised placental

tissue, and the amount of trans-2-hexenal in bruised pericarp tissue was higher than in

unbruised pericarp tissue. These alterations are due possibly to the disruption of the

enzymatic schedule by the damage resulting from the physical impacts. (Moretti,

Baldwin, Sargent, and Huber, 2002).

A relationship between ethylene and the synthesis of many tomato volatiles is

evident from a number of studies. Oeller, Wong, Taylor, and Pike (1991) investigated

tomato plants which expressed an antisense construct for ACC synthase, suppressing the

ethylene biosynthesis pathway. Ethylene production in the transformant was reduced

nearly 200-fold, and the fruit failed to ripen beyond an orange color. Their aroma was









never developed. Antisense fruits ripened upon exposure to exogenous ethylene, and

ripening was accompanied by the development of aroma volatiles. Baldwin et al. (2000)

reported that ethylene is required for carotenoid synthesis, possibly increasing ethylene's

effects on at least those volatiles that are derived from carotenoids. Kausch and Handa

(1997) found that the ethylene-insensitive Never-ripe (Nr) mutant accumulated the

mRNA for lipoxygenase, an enzyme important in the synthesis of volatiles from linolenic

and linoleic acids, but did not accumulate the lipoxygenase protein. This implies that

ethylene is not necessary for the accumulation of the mRNA for lipoxygenase, but that it

is necessary for the translation or stability of the lipoxygenase protein and, consequently,

for the formation of lipid-derived volatiles, which is supported by Ealing's (1994) work

showing lipoxygenase activity increasing and then decreasing along with ethylene

production in the early stages of tomato ripening (Ealing, 1994). Another important

enzyme, alcohol dehydrogenase (ADH), catalyzes the conversion of hexanal to hexenol.

ADH activity was reported to be ethylene-independent, which is supported by its elevated

activity, possibly due to decreasing oxygen concentration, in ripening grape fruit, which

are nonclimacteric (Zhu et al., 2005).

During normal tomato ripening, Baldwin, Nisperos-Carreido, and Moshonas (1991)

found that the amounts of most aroma volatiles increased, including cis-3-hexenol, cis-3-

hexenal, trans-2-hexenal, 6-methyl-5-hepten-2-one, and 2-isobutylthiazole. The increase

in many of the volatiles followed the same trend as the climacteric increase in respiration

and production of ethylene and lycopene during ripening. Ethyl vinyl ketone decreased

during ripening, but in only one of the cultivars investigated, 'Sunny'. In the cultivar

'Solimar', a small increase in ethylene production during the breaker stage corresponded









with the appearance or significant increase in the amounts of cis-3-hexenol, cis-3-

hexenal, trans-2-hexenal, 6-methyl-5-hepten-2-one, and 2-isobutylthiazole.

The production of 1-hexenal, trans-2-hexenal, and 6-methyl-5-hepten-2-one was

reduced in table ripe tomato fruit ('308' and '311') treated at mature green stages with 1-

MCP (2 ppm, 15 h) and stored at 21 C, compared with untreated fruit. Production of

ethyl vinyl ketone and cis-3-hexenal was enhanced in 1-MCP-treated fruit compared with

the controls (Mir, Canoles, and Beaudry, 2004). The amount of 1-hexanal and 2-

isobutylthiazole was higher in turning-harvested fruit than in green-harvested fruit, but

the amount of ethyl vinyl ketone and cis-3-hexenal was greater in green-harvested fruit

than in turning-harvested fruit. The volatiles altered by treating tomatoes with 1-MCP

were the same as the volatile altered by harvesting fruit at an earlier developmental stage.

This implies that 1-MCP treatment may encourage flavors associated with harvesting

tomatoes at an earlier stage of maturity (Mir et al., 2004), which often has a deleterious

effect on flavor (Maul et al., 1998). However, the magnitude of the changes in aroma

caused by 1-MCP in this study is small enough to indicate that it may not be a concern to

the perception of consumers.

Tieman et al. (2006) monitored the production of 'M82' tomato volatiles as the

fruit progressed from immature green to light red stage. Emissions of cis-3-hexenal,

trans-2-hexenal, B-ionone, and 6-methyl-5-hepten-2-one remained low during early

ripening and increased dramatically between turning stage and light red stage. The largest

increase in 2-phenylethanol was at breaker stage. Maximum emission of 3-methyl-i-

butanol was at turning stage, and 2-isobutylthiazole decreased from immature green

onwards. Mir et al. (2004) examined the volatile profile of '308' and '311' tomatoes









during the mature green, turning, and full ripe stages. However, neither work

quantitatively addressed the changes occurring in tomato volatile emissions during the

period following the attainment of full ripe stage, nor has the volatile profile of 1-MCP

treated fruit been compared with fruit past this initial attainment of full ripe. The

objective of the present study was to examine the development of overripe tomatoes and

compare them with tomatoes of the same age which had undergone 1-MCP treatment to

determine if 1-MCP continues to affect tomatoes past the initial point of ripening, or if

changes in volatile emissions between 1-MCP ripened and untreated ripe tomatoes were

due to the 1-MCP treatment directly or to the age of the fruit. It was also of interest to

examine untreated tomatoes past full ripe stage because tomatoes are often purchased

when full ripe or nearly full ripe, and subsequently held for a period of days prior to

consumption, and it was of interest to discover for how many days is the volatile profile

associated with the first day of "full ripe" (as defined by attaining a hue angle of 50)

maintained before being altered by overripening.














CHAPTER 3
MATERIALS AND METHODS

Plant Material

Tomato (Lycopersicon esculentum) fruit, cvs. Florida 47, Florida 91, and BHN586,

employed for the studies performed were obtained from West Coast Tomato, Inc., in

Palmetto, FL, North Florida Tomatoes in Quincy, FL, and University of Florida North

Florida Research & Education Center Suwannee Valley extension stations in Live Oak,

FL. Depending on the specific experiment, fruit were obtained at several developmental

stages, including mature-green, breaker, turning, pink and full red, as defined on the basis

of surface lycopene distribution (USDA 1991:

http://www.ams.usda.gov/standards/tomatfrh.pdf). Each stage reflects further breakdown

of chlorophyll and accumulation of lycopene. Breaker fruit are mostly green but show a

blush of red or pink around the blossom end that constitutes less than ten percent of the

fruit surface. Turning fruit have a larger area of red development, between 10% and one

third of the surface. Pink fruit are between one and two thirds red, and light red are

between two thirds and 90% red. When the tomato reaches the full red stage, it is

generally considered by consumers to be ripe and ready to eat.

Fruit used in all experiments were obtained on the day of harvest, and transported

immediately to Postharvest facilities at the University of Florida. Upon arrival, fruit were

washed in 1.34 millimolar Na-hypochlorite prepared from household bleach, selected for

uniformity in color and freedom from blemishes, and placed in storage facilities at 20C

until treated. No fruit used in any of these studies were treated with exogenous ethylene.









Specific conditions of 1-MCP treatment and storage duration are described in separate

sections below.

Fruit Color Measurements

Fruit external color was measured daily using a Minolta Chroma Meter CR-400

(Minolta Camera Co Ltd, Japan) calibrated using a white tile as the standard. The chroma

meter records lightness (on a scale of black to white, where black is numerically zero),

chroma (color saturation), and hue angle, a spectral scale in which 450 represents red and

1800 represents green. The measurements were taken at three equatorial locations and the

three hue angle values were averaged.

Analysis of Whole Fruit Firmness

Fruit firmness was measured every other day nondestructively using an Instron

Universal Testing Instrument (model 4411; Canton, Mass). The Instron was fitted with a

5 kg load cell and a blunt-ended, convex 7.5 mm probe. After establishing zero force

contact with the surface of the tomato, the probe was driven with a cross-head speed of

50 mm/min, targeting an area over locular tissue and not internal walls. The force of

compression was recorded in Newtons (N) at the point at which the tomato skin had been

compressed a distance of 2 mm. The same fruit were measured for the duration of the

experiment. Firmness was measured at three equatorial points for each tomato, and the

values averaged.

Evaluation of Volatile Profile of Ripe to Overripe Fruit

This experiment was performed to determine the changes in volatile profile of

tomatoes between the onset of full ripeness (as designated by an average hue angle of

below 500) and over the subsequent 16 d as the tomato becomes overripe.









Fruit Selection and Cleaning

Tomato (cv. BHN586) fruit ranging from breaker to pink stage were obtained from

a packinghouse in February 2006. From these, 110 turning fruit were selected, dipped in

1.34 millimolar hypochlorite, rinsed, and arranged on paper towels to dry. Each fruit was

individually marked with a ballpoint pen on a white adhesive label. Turning fruit were

selected for this experiment so that the precise day of ripening (as designated by an

average hue angle below 500) could be identified through daily monitoring.

Identifying Onset of Ripe Stage Using Color Measurements

Fruit external color was measured daily using a Minolta Chroma Meter CR-400

(Minolta Camera Co Ltd, Japan) as described in the section 'Fruit Color Measurements'.

The measurements were taken at three equatorial locations designated by the points 90,

1800, and 2700 in reference to the white label sticker. The three hue angle values were

averaged, and those fruit that reached an average hue angle of below 500 were selected

and divided into two identical samples. The day on which the hue angle reached this

starting point was designated 'Day 0'. On Day 1, 2, 6, 11, and 16 during storage at 200C,

five fruit were selected from each sample and removed for volatile analysis, with the Day

1 and 2 fruit representing ripe fruit and the Day 16 fruit representing significantly

overripe fruit. Volatiles were analyzed using the method described above in the section

'Analysis of tomato volatiles'.

Determination of Fruit Developmental Stage for 1-MCP Treatment

This experiment was designed to determine the developmental stage at which 1-

methylcyclopropene (1-MCP) would be most effective at delaying, but not preventing,

the attainment of a full-ripe condition, as defined on the basis of fruit reaching a hue

angle of 450, representing full-red external pigmentation. Fruit (cv. FL-47) were obtained









from the University of Florida North Florida Research & Education Center in Quincy,

FL. Upon return to Gainesville, fruit were cleaned and washed as described under 'Plant

Material'. One experiment was performed using fruit that was obtained at the 'green'

stage. Fruit for other experiments were obtained after color break.

Fruit at green, breaker, and pink stages were subsequently treated with 250 ppb 1-

MCP for 12 h at 200C in 174-L containers as described in detail under 'Exposure of

Tomato Fruit to 1-MCP'. Following treatment, fruit were transferred to 200C storage

facilities for up to 14 d and evaluated daily for color using a Minolta Chroma Meter CR-

400 (Minolta Camera Co Ltd, Japan) as described in detail under 'Fruit Color

Measurements'. Fruit were also measured every other day for firmness using an Instron

Universal Testing Instrument) model 4411; Canton, Mass) as described in detail under

the section 'Analysis of whole fruit firmness'. Volatile analysis was performed on the

breaker-stage treated fruit, once they had reached full ripe, using the method described in

the section 'Analysis of tomato volatiles'.

Exposure of Tomato Fruit to 1-MCP

For all 1-MCP experiments in this study, 1-MCP gas was generated by weighing

the appropriate quantity of SmartFresh powder (1-Methylcyclopropene, 0.14% A.I.)

(AgroFresh, a division of Rohm & Haas) to produce the desired atmosphere inside 174-L

and 20-L treatment chambers. The chamber selected was based on the number of

tomatoes being treated. The powder (26 mg for the 20-L chambers and 112 mg for the

174-L chambers) necessary to produce an atmosphere of 250 ppb (for the 174-L

chambers) or 500 ppb (for the 20-L chambers) 1-MCP in the treatment chambers was

added to a 125-mL Erlenmeyer flask containing 40 mL of tap water. After swirling to

dissolve the 1-MCP powder, the flask was placed in the treatment chambers that were









immediately sealed. The chambers were sealed by clamping a clear Plexiglas cover and

foam gasket to the open end of each treatment chamber (174-L chambers) or by inserting

a threaded Gamma-Seal (B&A Products Ltd. Co., Oklahoma) (20-L containers). Control

fruit were placed in a treatment chamber with a flask containing 40 mL of water alone.

Treatment was performed at 200C for twelve hours, after which time the containers were

opened and the fruit transferred to 200C normoxic storage facilities. At intervals during

storage at 200C, the fruit were removed and evaluated for color, firmness, and volatile

production.

Analysis of the Effects of 1-MCP on Tomato Ripening and Volatile Profiles

Tomato fruit (cv. 'FL-91') were obtained in October 2005 from a packinghouse at

the green stage and transported to Gainesville the same day. Fruit were dipped in 1.34

millimolar Na-hypochlorite, rinsed, and dried at room temperature. Fruit were held at

200C until color break and then selected for color uniformity at breaker stage.

Two experiments were performed. For each experiment, fifteen identical breaker-

stage fruit were selected and these fifteen fruit were subdivided into three groups of five

fruit each. One group was treated with 500 ppb 1-MCP for 12 h at 200C in 20-L

treatment chambers as described earlier in the section 'Exposure of Tomato Fruit to 1-

MCP'. The other two groups were designated as untreated controls. The extra control

group was needed as described below.

Following treatment with 1-MCP, the fruit were transferred to 200C storage

facilities for up to 14 d and evaluated daily for color using a Minolta Chroma Meter CR-

400 (Minolta Camera Co Ltd, Japan) as described in detail under 'Fruit Color

Measurements'. Fruit were also measured every other day for firmness using an Instron









Universal Testing Instrument) model 4411; Canton, Mass) as described in detail under

'Analysis of Whole Fruit Firmness' above.

The untreated tomatoes reached a full red condition, as designated by the hue angle

of each fruit averaging below 500, after 7 to 10 d. Within 1 day of this, one of the two

groups of untreated fruit was sampled to determine the aroma volatile constituency as

described below. At this point, the 1-MCP treated fruit were still at a pink stage.

1-MCP treated fruit were held until they reached full red (average hue angle < 50),

approximately 4 to 11 d later than the control fruit, and then sampled to determine their

volatile profile as described below. The other untreated control group, which had been

full red (average hue angle < 500) for 4 to 11 d, was analyzed at the same time. Thus for

this experiment, the three treatments consisted of: 1. Fruit treated with 1-MCP at the

breaker stage and ripened at 200C, 2. Control fruit ripened at 200C, and 3. Control fruit

held at 200C until the 1-MCP-treated fruit reached a full-ripe condition.

The experiment was repeated using fruit from another harvest from the same source

three weeks later.

Analysis of Tomato Volatiles

In both the ripening evaluation and 1-MCP experiment described in these studies,

fruit with an average hue angle of below 500 were used to determine the aroma volatile

profile. To compose a single 100 g sample, a longitudinal wedge of approximately 60

degrees was removed from each of the five fruit and the tissue diced on a plastic tray

using a sharp knife into cubes of approximately 1 cm3. All tissues, including the

pericarp, locular gel, juice, and seeds were retained in order to obtain a representative

whole-fruit volatile profile. Each sample was held between 5 min to 1 h in a hexagonal









weigh-boat (Fisher, Canada) and then loaded into glass tubes measuring 2.54 cm x 61 cm

(Volume: 309.1 mL). Both ends of the tubes were sealed with a rubber septum.

Volatiles were collected on Super-Q resin columns for 1 h using the method

described by Schmelz, Alborn, and Tumlinson (2001). Filtered air was passed through

the glass tubes and across the tomato samples at a flow rate of 618 mL/min. After 1 hour,

a 5 [il nonyl acetate internal standard was injected into each resin column using a glass

syringe. The contents of the columns were eluted with 150 [l methylene chloride, and

stored in 300 [L glass inserts inside 1.8 mL glass jars at -800C to preserve volatile

integrity.

The volatiles within the glass jars were analyzed using an Agilent Technologies

6890N gas chromatograph with a DB-5 column (Agilent Technologies) and a flame

ionization detector, using helium as the carrier gas. The injector temperature was 2200C,

the detector temperature was 2800C, and the oven temperature was between 40 and

2500C. The carrier gas flow rate was 1.2 mL/min. The software package ChemStation

(Agilent Technologies) was used to analyze the peaks produced by the volatiles, where

retention times were compared with known standards and used to identify peaks. The

peak areas were then exported into Microsoft Excel.

Statistical Analysis

Volatile production data was analyzed using the least squares method with a

confidence interval of 95% in JMPIN, the student version of the JMP program made by

SAS. Firmness and color data was analyzed using standard errors calculated by Microsoft

Excel.














CHAPTER 4
RESULTS AND DISCUSSION

Evaluation of Volatile Profile of Ripe to Overripe Fruit

The changes in volatile emissions as tomato fruit ('M82') progress from immature

green, through mature green, breaker, turning, and pink, to light red stage, have been

observed and recorded in Tieman et al. (2006). The experiments in the present study were

designed to determine the changes in tomato volatile emissions in the successive stages,

starting at the day on which fruit reach a full ripe stage (as defined by an average hue

angle below 500) and continuing until 16 d following ripening.

Tomato fruit ('BHN586') were obtained at turning stage and ripened at 200C. Color

was monitored daily until individual fruit attained an individual average hue angle below

500. Figure A-1 shows the hue angles of the fruit at the start of the experiment and each

consecutive day during storage. The hue angle quickly decreased as the tomato ripened in

200C storage.

The tomatoes upon reaching hue angle values below 500 (Figure A-1, day 4) were

held for up to 17 days at 200C (Figure A-2), during which time fruit were periodically

measured for volatile emissions. According to these results, a tomato, once reaching red,

does not experience further significant color change even as many as seventeen days after

ripening.

Figure A-3 shows the average firmness of the tomatoes sampled. A ripe tomato is

usually around 5 N, and for the first three days after ripening (using hue angle < 500 as an

indicator of ripening) the tomatoes maintained a firmness comparable to the normal









firmness of full ripe tomatoes. However, firmness continued to decrease as the tomatoes

progressed from ripe to overripe, declining to between 3 and 4 N by 11 d following

ripening.

Changes in volatile emissions of the 'BHN586' tomato fruit during ripening and

over-ripening are shown in Figures A-4 to A-8. Volatiles are discussed in order of their

importance to tomato aroma, as noted by other researchers (Buttery and Ling, 1992;

Buttery, 1993; Krumbein and Auerswald, 1998).

The "C6" compounds hexanal and cis-3-hexenal greatly impact the aroma of

tomato leaves and fruit, producing a "green, grassy" scent (Buttery and Ling, 1992) or a

sweet or "fresh green" flavor (Krumbein and Auerswald, 1998). The flavor of hexanal

alone has also been referred to as "sweet", "ripe", and "tomato flavor" (Maul et al.,

2000), identifying it as a key component of tomato aroma and taste. Cis-3-hexenal and

hexanal are derived from the fatty acids linoleic and linolenic acids and increase in

concentration as the tomato ripens (Buttery and Ling, 1992). Tieman et al. (2006)

observed low levels of cis-3-hexenal in 'M82' tomato fruit up until the turning stage, at

which point a dramatic increase in production occurred through the light red stage.

According to Buttery and Ling (1992), cis-3-hexenal emission in tomato fruit increases

slightly at color break, but experiences a fifty-fold increase at the full-ripe stage. In the

present study, 'BNH586' tomato fruit experienced a decrease in cis-3-hexenal production

as fruit progressed from full ripe to 16 d overripe (Figure A-4). Since cis-3-hexenal is a

key component of ripe tomato aroma, it is not surprising that its production rises to the

highest point 1 or 2 d after attaining a hue angle below 500 and then drops again.

Interestingly, Mir et al. (2004) found that cis-3-hexenal production decreased in '308'









and '311' tomatoes as they progressed from mature green to turning and then to full red,

but this contrasting data may be explained by the difference in cultivar. Hexanal

production in 'BNH586' tomatoes fruit fell between 1 and 2 d after attaining full red, but

thereafter increased and remained high during the 16 d of observation, as shown in Figure

A-5.

Ethyl vinyl ketone, or 1-penten-3-one, is another 'C6' volatile derived from fatty

acids and found in both vegetative and reproductive tissues of the tomato plant. As with

the other 'C6' volatiles, it imparts a 'green' aroma (Krumbein and Auerswald, 1998). The

'308' and '311' tomatoes investigated in Mir et al. (2004) were found to produce less

ethyl vinyl ketone at the turning stage than when mature green, after which production

remained constant through the full ripe stage. As shown in Figure A-6, production of

ethyl vinyl ketone in 'BNH586' tomato fruit remained constant within the first three days

after attaining a full ripe stage, after which it rose over the course of the next week. Ethyl

vinyl ketone emissions peaked on day 11 and remained statistically constant for the next

5 d. This pattern is consistent with the trend noted by Mir et al. (2004).

8-ionone is a cyclic compound synthesized from the metabolism of carotenoids

(Buttery and Ling, 1992). Carotenoid-derived volatiles are formed in the fruit but not in

the vegetative tissues of tomato plants,. 8-ionone has been characterized as imparting a

sour taste to taste panelists (Tandon et al., 2003). Production of 3-ionone increased

dramatically as the 'BHN586' tomato fruit progressed from ripe to overripe (Figure A-7).

3-ionone-production was relatively constant within the first three days after fully

ripening, and increased sharply at 11 d. At 11 and 16 d after ripening, 8-ionone

production had increased approximately six-fold compared with values observed at the









full-ripe stage. Tieman et al. (2006) reported a dramatic increase in 8-ionone production

between the turning and light red stages, at which time their observations ceased. The

data in Figure A-7 illustrate that this volatile continues to increase through and after full

ripening. There was no significant difference in 8-ionone production between the two

identical groups sampled on any of the days measured.

Other tomato flavor volatiles are synthesized from amino acids. Isovaleraldehyde,

also called 3-methylbutanal, is formed from the amino acid leucine. It has been

associated with an unpleasant flavor (Krumbein and Auerswald, 1998). Tieman et al.

(2006) observed a steady increase in isovaleraldehyde production in 'M82' tomato fruit

in the time from immature green to turning stage, after which point emissions of

isovaleraldehyde decreased again as the tomato progressed to light red. The 'BNH586'

tomatoes used in the present experiment experienced steady isovaleraldehyde production

for the first three days after attaining full ripe stage, after which production increased

slightly. By day 11, however, it had fallen to well below the full-ripe rate of production,

and emissions of isovaleraldehyde remained low over the next 5 d (Figure A-8).

Determination of Fruit Developmental Stage for 1-MCP Treatment

The purpose of these experiments was to determine the optimal stage for treating

the tomato fruit with 1-MCP in order to be certain the 1-MCP would sufficiently delay

ripening. Tomato fruit (Lycopersicon esculentum 'FL-47') at green, breaker, turning, and

pink stages of development were treated with 1-MCP (250 ppb, 12 h, 200C) or air and

ripened at 200C. Fruit surface color was recorded daily and firmness every other day.

Fruit at the green stage were found to be unsuitable for 1-MCP experiments, due to

the unpredictable numbers of immature-green fruit in the samples. As noted by Hurr et

al., (2005), 'green' tomato fruit treated with 1-MCP eventually diverge into 2 distinct









populations in terms of the degree of suppression of softening and surface red color

development. Softening and color development of immature green fruit (an assignment

made retrospectively based on the number of days to color break of the untreated

controls) were acutely and irreversibly inhibited by the ethylene antagonist. Mature-

green fruit, on the other hand, eventually softened and colored to a degree exhibited by

control fruit, though both ripening parameters were significantly delayed. Aside from the

difficulties in properly classifying green fruit, there is ample evidence that even mature-

green fruit, once detached, never develop the characteristic volatiles associated with high

quality. Definitive classification of green fruit as immature green or mature green

requires either nuclear magnetic resonance technology (NMR) (Abbott, 1999) or X-ray

technology (Brecht, Shewfelt, Garner, and Tollner, 1991) which are expensive and time-

consuming, or cutting the fruit open. In view of the inability of distinguishing mature

versus immature-green fruit, subsequent experiments were performed using fruit at the

breaker stage and beyond.

Treating tomato fruit with 1-MCP at the pink or turning stage was also problematic

because in the first five days following treatment, no difference in color or firmness was

observed between the ripening of control fruit and 1-MCP treated fruit. This parallel

ripening pattern is shown in Figures B-I through B-4. Color of fruit treated at the pink

stage did not appear to have been affected by the 1-MCP treatment, and the difference in

average hue angle between 1-MCP treated and control fruit was less than one degree

throughout the six days following ripening, indicating statistically similar values. No

difference was observed between the firmness of treated and untreated pink fruit over the

course of the observation. Fruit treated with 1-MCP at either the pink or turning stages









softened just as rapidly as air-treated control fruit. It is possible that differences may have

emerged after the six days observed, but since at the time the experiment was terminated

all fruit had already softened and reddened sufficiently to indicate full ripe within a

similar period of time, there was nothing to indicate that 1-MCP had any effect on their

development during ripening.

These results are in contrast with those of Hurr et al. (2005), who found that pink

and turning 'FL-47' tomato fruit did respond to 1-MCP treatment (1 ppm, 24 h, 200C), as

did all other stages of tomato fruit development through full-ripe, with delayed softening

and color development. This may be due to the fact that the concentration used was four

times what was used in these experiments (1 ppm vs 250 ppb) and the duration of

treatment was twice as long (24 h vs 12 h), and a treatment of that magnitude may be

required to affect the development of pink or turning fruit. However, an initial

concentration of only 150 ppb successfully produced a delay in ripening parameters such

as softening and development of red color in 'Prisca' tomato fruit after 20 h of treatment

(Hoeberichts, Van der Plas, and Woltering, 2002). It is possible that the difference in

cultivar may be responsible for the difference in 1-MCP effectiveness. Blankenship and

Dole (2003) stated that there is evidence of cultivar differences in response to 1-MCP for

apple and banana fruits. This might be due to cultivar differences in the magnitude of

ethylene production or in inherent sensitivity to ethylene. Another possible explanation

for the deviation of the present results from previous work is that the fruit used in this

experiment were stressed due to being transported in the trunk of a car for 1 h just after

harvest, where high temperatures may have had a negative effect. This is unlikely,









however, since fruit color development, which is highly sensitive to high-temperature

injury, was not impaired.

Due to the problems with green, pink, and turning fruit enumerated above, breaker

fruit were employed for all further experiments. Fruit ('FL-47') treated with 1-MCP (250

ppb, 12 h, 200C) at the breaker stage were monitored for changes in color and firmness

until they attained a full ripe stage, the stage at which 90% or more of the fruit is covered

by red pigmentation and the hue angle is below 500. In this experiment, the length of time

required for fruit to reach full red was significantly affected by 1-MCP. As shown in

Figure B-5, control fruit reached an average hue angle below 450 eight days earlier than

1-MCP-treated fruit. Additionally, development of red pigmentation occurred with more

uniformity over the surface in control fruit, although uniform red pigmentation did

eventually develop in all fruit. Fruit treated at breaker stage with 1-MCP also exhibited a

delay in loss of firmness compared with control fruit.

1-MCP-treated fruit experienced a delay in development of the ripening parameters

of color and firmness compared with untreated fruit. It was of interest to discern whether

this delay would affect volatile emissions. With that in mind, volatile analysis was

performed as described earlier on ripe fruit derived from air- or 1-MCP-treated breaker

fruit, and on breaker fruit taken before 1-MCP treatment.

Fatty-Acid Derived Volatiles

The amounts of cis-3-hexenal and cis-3-hexenol produced in ripe control fruit were

higher than the amounts produced in breaker fruit (Figures B-6 and B-9). This increase in

cis-3-hexenal during ripening in tomato fruit corroborates the data from Tieman et al.

(2006), which indicated an increase in cis-3-hexenal between the turning and light red

stages, but not Mir et al. (2004), who reported a decrease in cis-3-hexenal between









mature green, turning, and ripe fruit. The amount of both cis-3-hexenal and cis-3-hexenol

produced in ripe fruit derived from 1-MCP-treated breaker fruit was higher than the

amounts in ripe control fruit (Figs B-6 and B-9). In the case of cis-3-hexenal, this is

consistent with the increase observed by Mir et al. (2004) in '308' and '311' tomatoes

treated with 2 ppm 1-MCP for 15 h at 210C. However, Mir et al. (2004) did not observe

any change in the amount of cis-3-hexenol throughout their experiment. This difference

might be attributed to cultivar differences, or difference in 1-MCP treatment or volatile

sampling procedure. Mir measured volatiles in fruit homogenates whereas in the present

study the volatiles were measured from a heterogenous mixture of skin, pericarp, locule

gel, juice, and seeds diced into cubes of approximately 1 cm3.

Hexanal and trans-2-hexenal were found in statistically similar amounts in breaker

and ripe control fruit, and higher levels in ripe fruit derived from 1-MCP-treated breaker

fruit (Figures B-7 and B-10). This contradicts the decrease observed in Mir et al. (2004)

between ripe control and ripe fruit derived from 1-MCP-treated mature green fruit, and

also the increase observed in that study in hexanal production between the mature green

and turning stages. However, once the tomatoes in Mir et al. (2004) had reached turning

stage, their hexanal emissions remained constant, and it is possible that the conclusion

can be drawn from these combined results that the increase in hexanal associated with

tomato ripening is initiated at the breaker stage, and not turning stage. Mir et al. (2004)

did not measure tomatoes at breaker stage, and the tomatoes observed in that study

retained constant hexanal emissions from the turning through full-ripe stage. An increase

in the amount of trans-2-hexenal produced in ripe control fruit compared with breaker

fruit does appear at the 90% confidence level, although not at a 95% confidence level.









Tieman et al. (2006) had found an increase in the amount of trans-2-hexenal produced as

'M82' tomato fruit ripen, but this increase was not observed in 'FL-47'. The increase in

trans-2-hexenal may have occurred between the turning and light red stages, but

remained undetected because the only stages at which volatiles were sampled were

breaker and full red. A possible explanation is that the increase may have occurred

transiently, only between the turning and light red stages, and this trend is corroborated

by the data from Tieman et al. (2006). Additionally, the trans-2-hexenal production data

presented in Figure B-10 is not consistent with the observations made by Mir et al.

(2004), in which trans-2-hexenal was reported to have been produced at higher levels in

untreated fruit, although in both experiments there was an increase in the amount

produced in ripe fruit compared with fruit in the early stages of development.

Mir et al. (2004) had observed a decrease in ethyl vinyl ketone production from

mature green to turning, after which production levels remained constant. Treatment with

1-MCP (2 ppm, 15 h, 21C) reduced the levels of ethyl vinyl ketone in '308' and '311'

tomatoes to the level they had exhibited when mature green. However, in this experiment,

there was no difference in the amount of ethyl vinyl ketone produced by 'FL-47'

tomatoes between any of the treatments/stages (Figure B-8).

Carotenoid Derived Volatiles

The amount of B-ionone produced was statistically similar for all treatments (Figure

B- 11). The amount of 6-methyl-5-hepten-2-one produced was much higher in both ripe

control and ripe fruit derived from 1-MCP-treated breaker fruit than in breaker fruit

(Figure B-12). An increase in both of these volatiles was observed in Tieman et al. (2006)

between the turning and light red stages and in Mir et al. (2004) between mature green,

turning, and full ripe. In the case of 6-methyl-5-hepten-2-one, at least, this increase upon









ripening seems not to have been compromised by exposure to 1-MCP at the breaker

stage. In contrast to the results reported here, Mir et al. (2004) reported decreased levels

of both volatiles in response to 1-MCP. In this case it may be more practical to attribute

the differences in observations to the difference between the 1-MCP concentrations in

each experiment [2 ppm in Mir et al (2004) vs 250 ppb in the present study] rather than

cultivar differences because the untreated 'FL-47' fruit in Figure R behaved consistently

with the 'M82' fruit in Tieman et al. (2006) and the '308' and '311' fruit in Mir et al.

(2004).

Amino Acid Derived Volatiles

The amounts of isovaleraldehyde and 3-methyl-l-butanol produced in ripe control

fruit were higher than the amounts in both breaker fruit and ripe fruit derived from 1-

MCP-treated breaker fruit, which were statistically similar at the 95% confidence level

(Figures B-13 and B-15) but different at the 90% confidence level, where ripe fruit

derived from 1-MCP-treated breaker fruit were found to produce higher emissions of 3-

methyl-1-butanol than breaker fruit. Tieman et al. (2006) observed maximum emissions

of 3-methyl-1-butanol at the turning stage, after which it decreased, which may seem to

contradict these results. However, comparing only the breaker and light red stage data

from Tieman et al. (2006), there was a small overall increase in 3-methyl-1-butanol.

Whether or not the 1-MCP-treated fruit observed here experienced this transient increase

is unknown because turning fruit were not measured for volatile content. In contrast with

the results presented in Figure P, untreated '308' and '311' fruit observed in Mir et al.

(2004) maintained a constant level of 3-methyl-1-butanol production throughout the

mature green, turning, and full ripe stages, while 1-MCP treated fruit produced higher

levels of 3-methyl-1-butanol.









2-methylbutyraldehyde was produced in higher amounts in ripe control fruit than in

breaker fruit. Neither was statistically different from the amount produced in ripe fruit

derived from 1-MCP-treated breaker fruit at the 95% confidence level (Figure B-14) but

at the 90% confidence level ripe control fruit was observed to produce higher levels of 2-

methylbutyraldehyde than ripe fruit derived from 1-MCP-treated breaker fruit.

1-nitro-2-phenylethane was produced in higher amounts in ripe fruit derived from

1-MCP-treated breaker fruit than in breaker or ripe control fruit (Figure B-17). There was

no statistical difference observed in the amounts of 2-phenylethanol produced between

any of the treatments/stages (Figure B-16). Since 1-nitro-2-phenylethane and 2-

phenylethanol are both derived from phenylalanine, it is reasonable to assume that they

would respond similarly to 1-MCP if their production trends were dependent on ethylene.

This expectation was not fulfilled by the results in this study. However, as noted in

Figures V and W, the volatiles exhibited different patterns of response to 1-MCP. Buttery

and Ling (1992) stated that amino acid-derived volatiles such as 1-nitro-2-phenylethane

and 2-phenylethanol are formed between the breaker and full ripe stages, indicating that

an increase should have been detected at least between breaker and ripe control fruit.

That increase was not observed in this study for either volatile.

Volatiles of Other Derivation

The derivation of 2-isobutylthiazole is unknown (Goff and Klee, 2006), although

Schutte (1974) has suggested that 2-isobutylthiazole may originate from leucine using

isovaleraldehyde as an intermediate. The flavor attributed to 2-isobutylthiazole is sweet

and fruity and it was observed to cause a 'bite' feeling in the mouth (Krumbein and

Auerswald, 1998). Both ripe control and ripe fruit derived from 1-MCP-treated breaker

fruit produced higher levels of 2-isobutylthiazole than those produced by breaker fruits,









and there was no difference observed in the amounts produced between ripe control and

ripe fruit derived from 1-MCP-treated breaker fruit (Figure B-18). In Tieman et al.

(2006), the maximum emission of 2-isobutylthiazole was during the immature green

stage, thereafter declining as tomato development proceeded. However, Tieman et al.

(2006) only measured fruit volatiles up until light red stage, so it is possible that the

increase observed in the present study, which appears not to have been impacted by

exposure to 1-MCP, occurred entirely after the fruit attained a full red color. The increase

observed by Tieman et al. (2006) is contradicted by the data in Mir et al. (2004), which

shows an increase in 2-isobutylthiazole production between mature green and turning,

after which the amount produced remains constant in untreated '308' and '311' tomato

fruit. Mir et al. (2004) also shows a decrease in the amount of 2-isobutylthiazole in 1-

MCP fruit compared with ripe control fruit, but this was not observed in the present

study.

Response of Tomato Fruit Color and Firmness to 1-MCP

Tomato fruit (Lycopersicon esculentum 'FL-91') at the breaker stage were treated

with 1-MCP (12 h, 200C, 500 ppb) or air and ripened at 200C. Firmness decreased more

rapidly in air-treated fruit, as shown in Figures C-i through C-4. This is consistent with

previous studies involving the effect of 1-MCP in delaying 'FL-47' tomato softening,

such as Hurr et al. (2005), in which a week to 10 d was required for 1-MCP treated fruit

to soften to the same firmness as control fruit. Hoeberichts et al. (2002) also

demonstrated that 1-MCP treatment causes tomato fruit ('Prisca') to attain a softness

associated with ripening after a delay, compared with controls.

Development of red coloration occurred more slowly in 1-MCP -treated fruit than

in control fruit, as shown in Figure C-5, and with less uniformity. Since color was









measured at three equatorial positions, it is not evident that for some 1-MCP treated fruit,

patches of green or yellow remained at the stem end of the fruit for several days. These

areas did eventually develop red color, resulting in a uniform full red fruit.



Tomato Volatile Production in Ripe, Overripe, and 1-MCP-treated 'FL-91' Tomato
Fruit

These experiments were designed to determine whether or not emission of the

thirteen volatiles considered most important to tomato aroma would be affected by

ethylene suppression. In addition, these experiments sought to compare the volatile

emissions of 1-MCP treated fruit not only with control fruit of a similar ripening stage,

but with control fruit of similar age, and which were therefore overripe. The four trials

performed within this experiment will be discussed together due to the fact that they

involved tomatoes of the same cultivar ('FL-91'), obtained at the same stage (breaker)

from the same packing house, treated at the same stage (breaker) with the same

concentration of 1-MCP (500 ppb) for the same time period (12 h). Experiments 3 and 4

were performed on fruit obtained three weeks later than Experiments 1 and 2. Where no

interaction is described, no significant differences were found between treatments at the

95% confidence level using the least squares method.

Fruit designated as 'ripe control' were sampled upon reaching a hue angle of 500

after treatment in air. Fruit designated as 'I-MCP' were treated at breaker stage with 500

ppb 1-Methylcyclopropene (1-MCP), 12 h, 200C) and then sampled upon reaching a hue

angle of 500. Since 1-MCP delays the change in pigmentation associated with tomato

ripening by suppressing the ethylene response, this change in pigmentation occurred 4 to

11 d later than the 'ripe control' fruit. Fruit designated as 'overripe' were treated in air, but









sampled concurrently with the 1-MCP-treated fruit, between 4 to 11 days after the 'ripe'

reached a hue angle of 500. The rationale behind the sampling of overripe air-treated fruit

as well as ripe air-treated fruit was to rule out the possibility that differences in the

tomato volatile profile between air-treated and 1-MCP-treated ripe fruit were due to fruit

age rather than a response to delayed ripening. The 1-MCP-treated fruit were 4 to 11 d

older than the air-treated fruit when they reached the 500 hue angle, and sampling the

overripe fruit was necessary to distinguish between volatile differences due to fruit age

and not to any direct effects of 1-MCP on ethylene-mediated processes.

Fatty-Acid Derived Volatiles

Cis-3-hexenal was found in higher amounts in ripe control fruit than in overripe

fruit in Experiments 2 and 3, and higher amounts in ripe control fruit than in ripe fruit

derived from 1-MCP-treated breaker fruit in experiment 2, which contradicts the results

observed earlier in this study (Figures C-7 and C-8) and also the observations of Mir et

al. (2004). There were no differences in cis-3-hexenal production between treatments in

Experiments 1 or 4 (Figures C-6 and C-9).

Hexanal was found in higher amounts in overripe fruit than in ripe control fruit in

Experiment 1, but no significant differences were found between ripe fruit derived from

1-MCP-treated breaker fruit and either ripe control or overripe fruit in this experiment

(Figure C-10). There were no differences between treatments in Experiments 2 and 3

(Figures C-11 and C-12). In Experiment 4, higher amounts of hexanal were found in ripe

fruit derived from 1-MCP-treated breaker fruit than in ripe control fruit, supporting the

results observed earlier in this study (Figure C-13). There were no differences between

ripe fruit derived from 1-MCP-treated breaker fruit and overripe fruit or between overripe

fruit and ripe control fruit at the 95% confidence level, although at the 90% confidence









level, ripe fruit derived from 1-MCP-treated breaker fruit produced more hexanal than

ripe control fruit in experiment 1, and overripe fruit produced more hexanal than ripe

control fruit in experiment 4. These indications that 1-MCP treatment may cause some

'FL-91' tomato fruit to produce higher levels of hexanal contradicts data presented in Mir

et al. (2004), in which '308' and '311' tomato fruit treated withl-MCP showed a 50%

reduction in hexanal production, resulting in emission rates comparable to those of

mature green fruit.

Ethyl vinyl ketone production was statistically similar for all treatments in

Experiments 1, 2, and 4 (Figures C-14, C-15, and C-17). In Experiment 3, greater

amounts of ethyl vinyl ketone were found in overripe fruit than in ripe fruit derived from

1-MCP-treated breaker fruit (Figure C-16). No differences were found between ripe

control fruit and either overripe fruit or ripe fruit derived from 1-MCP-treated breaker

fruit at the 95% confidence level, but overripe fruit produced more ethyl vinyl ketone

than ripe control fruit at the 90% confidence level. This lack of statistical difference

mirrors the results for the ethyl vinyl ketone measured earlier in this study.

There were no statistical differences in the production of trans-2-hexenal between

any of the treatments at the 95% confidence level in all four experiments (Figures C-18

through C-21). However, at the 90% confidence level, overripe fruit produced more

trans-2-hexenal than ripe control fruit in Experiments 3 and 4.

Cis-3-hexenol production was statistically similar for all treatments at the 95%

confidence level in Experiments 1, 2, and 4 (Figures C-22, C-23, and C-25). However, at

the 90% confidence level, overripe fruit produced higher levels of cis-3-hexenol than ripe

control fruit in Experiments 1 and 4. Overripe fruit in experiment 3 produced higher









levels of cis-3-hexenol than ripe fruit or ripe fruit derived from 1-MCP-treated breaker

fruit (Figure C-24). No differences were found between cis-3-hexenol production in ripe

control fruit and ripe fruit derived from 1-MCP-treated breaker fruit. This similarity

between treatments is fairly consistent with the observations of Mir et al. (2004), in

which no differences were found in the amounts of cis-3-hexenol produced by mature

green, turning, ripe control, and ripe fruit derived from 1-MCP-treated mature-green fruit.

Carotenoid Derived Volatiles

8-ionone was found in higher amounts in both overripe and ripe fruit derived from

1-MCP-treated breaker fruit than in ripe control fruit in Experiment 3 (Figure C-28).

There were no differences in 8-ionone production between treatments in Experiments 1,

2, or 4 (Figures C-26, C-27, and C-29), corroborating results presented earlier in this

study. Both findings contradict the observations ofMir et al. (2004), in which ripe fruit

derived from 1-MCP-treated mature green fruit produced much lower levels of 8-ionone

than ripe control fruit.

6-methyl-5-hepten-2-one production was statistically similar for all treatments in

Experiments 1 and 2 at the 95% confidence level (Figures C-30 and C-31), although at

the 90% confidence level, ripe fruit derived from 1-MCP-treated breaker fruit produced

more 6-methyl-5-hepten-2-one than ripe control fruit in Experiment 1. Higher levels of 6-

methyl-5-hepten-2-one were identified in overripe fruit than in both control ripe and ripe

fruit derived from 1-MCP-treated breaker fruit in Experiment 3 (Figure C-32). In

Experiment 4. higher levels of 6-methyl-5-hepten-2-one were found in overripe fruit than

in ripe fruit derived from 1-MCP-treated breaker fruit, and at the 95% confidence level,

production of 6-methyl-5-hepten-2-one in ripe control fruit was statistically similar to

both overripe fruit and ripe fruit derived from 1-MCP-treated breaker fruit. (Figure C-33).









At the 90% confidence level, however, overripe fruit produced more 6-methyl-5-hepten-

2-one than ripe control fruit in Experiment 4. The statistical similarity between ripe fruit

derived from 1-MCP-treated breaker fruit and ripe control fruit echoes the similarity

observed earlier in this study and contradicts the findings of Mir et al. (2004), in which 1-

MCP was observed to produce a decrease in 6-methyl-5-hepten-2-one emissions in '308'

and '311' tomato fruit.

Amino Acid Derived Volatiles

Higher levels of isovaleraldehyde were observed for ripe control fruit than in both

overripe fruit and from ripe fruit derived from 1-MCP-treated breaker fruit in Experiment

2 (Figure C-35). In Experiment 3, isovaleraldehyde was found in higher amounts in ripe

fruit derived from 1-MCP-treated breaker fruit than in both overripe and ripe control fruit

(Figure C-36). In Experiment 4, isovaleraldehyde was found in higher amounts in the

overripe fruit compared with ripe fruit derived from 1-MCP-treated breaker fruit, both of

which were statistically similar to ripe control fruit (Figure C-37). There were no

differences in isovaleraldehyde production between treatments in Experiment 1. (Figure

C-34)

3-methyl-1-butanol was found in higher amounts in ripe control fruit than in

overripe fruit, both of which were statistically similar to the amount in ripe fruit derived

from 1-MCP-treated breaker fruit at the 95% confidence level, in Experiment 1 (Figure

C-38). At the 90% confidence level, however, ripe control fruit produced higher levels of

3-methyl-1-butanol than ripe fruit derived from 1-MCP-treated breaker fruit in

Experiment 1. In Experiment 2, higher amounts of 3-methyl-1-butanol were found in ripe

control fruit than in both overripe fruit and in ripe fruit derived from 1-MCP-treated

breaker fruit (Figure C-39). Higher levels of 3-methyl-l-butanol were identified in ripe









fruit derived from 1-MCP-treated breaker fruit than in both overripe and ripe control fruit

in experiment 3 (Figure C-40), corroborating results from Mir et al. (2004). There were

no differences in 3-methyl-1-butanol production between treatments in Experiment 4

(Figure C-41).

1-nitro-2-phenylethane was found in higher amounts in both overripe fruit and in

ripe fruit derived from 1-MCP-treated breaker fruit than in ripe control fruit in

Experiments 1 and 3, corroborating observations described earlier (Figures C-42 and C-

44). In Experiment 4, higher levels of 1-nitro-2-phenylethane were identified in overripe

fruit than in both ripe fruit derived from 1-MCP-treated breaker fruit and in ripe control

fruit (Figure C-45). There were no differences in production of 1-nitro-2-phenylethane

between treatments in Experiment 2 (Figure C-43).

Higher levels of both 2-phenylethanol and 2-methylbutyraldehyde were identified

in ripe control fruit than in both overripe fruit and in ripe fruit derived from 1-MCP-

treated breaker fruit in Experiment 2 (Figures C-47 and C-51). No differences were

observed in the production of either 2-phenylethanol or 2-methylbutyraldehyde in

overripe fruit and in ripe fruit derived from 1 -MCP-treated breaker fruit in experiment 2

at the 95% confidence level, although at the 90% confidence level, higher levels of 2-

methylbutyraldehyde were observed in ripe fruit derived from 1-MCP-treated breaker

fruit than in overripe fruit. There were no differences in 2-phenylethanol or 2-

methylbutyraldehyde production between treatments in Experiments 1, 3, or 4 (Figures

C-46, C-48, C-49, C-50, C-52, and C-53).









It is interesting to note that as with the earlier experiment in this study, the reaction

of 1-nitro-2-phenylethane and 2-phenylethanol, both derived from phenylalanine, to

ethylene suppression was not similar.

Volatiles of Other Derivation

2-isobutylthiazole was found in higher amounts in ripe fruit derived from 1-MCP-

treated breaker fruit than in both overripe and ripe control fruit in Experiment 3 (Figure

C-56), consistent with the report of Mir et al. (2004). There were no differences in 2-

isobutylthiazole production between treatments in Experiments 1, 2, or 4 (Figures C-54,

C-55, and C-57). There were no differences observed between ripe control and ripe fruit

derived from 1-MCP-treated breaker fruit in the study discussed earlier, and the decrease

observed in Mir et al. (2004) was minor, indicating that ethylene action may not be an

important factor in the formation of 2-isobutylthiazole.

General Discussion

It is evident from the results of these studies that factors other than ethylene

recognition affect volatile emissions. Numerous preharvest factors can influence volatile

production in tomato fruit, such as fertilizer regime (Wright and Harris, 1985), choice of

cultivar (Tandon et al., 2003), and climatic conditions during the growing season (cite).

Harvesting conditions, such as fruit maturity at harvest (Maul et al., 2000) and storage

temperature (Boukobza and Taylor, 2002; Maul et al., 2000), can also play a role in

altering the volatile profile of tomato fruit. Bruising is also a factor (Moretti et al.,

2001), which means that postharvest handling practices are likely influential. These

natural variations were minimized by obtaining the fruit for the experiments in this study

from the same packing house during the same season, and from the same cultivar.

However, in the 'FL-91' experiment, the tomato fruit for experiments 3 and 4 were









obtained three weeks later than the fruit for experiments 1 and 2. A significant increase

was found in the total volatile emissions (pooling data from all three treatments) from

Experiments 1 and 2 compared with Experiments 3 and 4. Upon examination of the

weather in October 2005 from Quincy, FL, the source of the tomatoes for this

experiment, a possible explanation arises from the temperature data (Florida Automated

Weather Network, http://fawn.ifas.ufl.edu). In the first three weeks of October, during

which the fruit from the first harvest were obtained, the temperature averaged around

200C. During the last week of October, approximately half a week before the start of

experiments 3 and 4, temperatures dropped to between 1 and 40C and remained there for

9-12 h per night for three consecutive days. Johnson, Gould, Badenhop, and Johnson

(1968) observed tomatoes of the same varieties differing in the amount of cis-3-hexenol

emitted in their juice depending in which week the tomatoes were harvested, showing

that there is precedent in the literature for such variation.

Another source of volatile variation lies in the method of sample processing. In

order to obtain a representative whole-fruit volatile profile, all tissues of the tomato

fruit-including the pericarp, locular gel, and seeds-were retained after dicing and their

volatile emissions collected and analyzed. Since each sample represents tissue taken from

five fruit, precise amounts of locule gel, or any other fruit tissue, would not be consistent

from sample to sample. In addition, sometimes as many as fourteen samples were

prepared on one day before being loaded into the glass tubes and sealed with septa. The

last sample diced was on the lab bench in an open weigh-boat for only five minutes,

whereas the first sample diced might have remained in the open air for an hour, during









which time some volatiles may have escaped, or experienced changes in

synthetic/degradation pathways.

There is evidence in the literature of volatile emissions being affected by the

amount or method of tissue processing involved in the volatile sampling technique.

Carbonell-Barrachina, Agusti, and Ruiz (2006) compared hexanal production in blended

tomatoes with hexanal production observed in tomato paste by Buttery et al. (1990) and

hexanal production observed by Maul et al. (1998) using a gas chromatograph headspace

analysis technique. Carbonell-Barrachina et al. (2006), who used a dynamic headspace

technique, found that hexanal, as well as the other compounds analyzed, was higher in

their study than in tomato paste and lower than in the tissues analyzed by gas

chromatograph headspace analysis. In an effort to develop a non-destructive method of

volatile sampling, they also compared aroma volatile emissions from blended tomatoes

with emissions from whole fruit. Blended tomatoes exhibited much higher aroma

production than whole tomatoes, which supports the statement that tissue disruption can

lead to additional production of some volatiles. Since the present study involved tomatoes

that were never blended, only finely chopped, some differences between these results and

previous studies may be attributed to divergent techniques.

Evidence in Zhu et al. (2005) would indicate that ethylene suppression would

diminish production of fatty-acid derived volatiles and have no direct impact on

production of volatiles derived from amino acids. While in one of the four 'FL-91'

experiments cis-3-hexenal content was higher in untreated ripe fruit than in ripe fruit

derived from 1-MCP-treated breaker fruit, the rest of the data from the fatty-acid derived

volatiles in this study did not indicate that ethylene action inhibition had caused a









decrease in fatty-acid derived volatile production. In fact, in some experiments, it

produced an increase in hexanal emissions, contradicting previous studies (Mir et al.,

2004). As for amino acid derived volatiles, whose production was not expected to be

directly affected by ethylene suppression, there were isolated cases of ripe control fruit

exhibiting higher production of amino acid derived volatiles than ripe fruit derived from

1-MCP-treated breaker fruit, but the reverse was also observed, as well as an observation

of no difference between treatments. Since the results were inconsistent between samples

and between experiments, it is possible that taste differences existed between individual

samples, but it is difficult to formulate a broad declaration about what kind of difference

in taste would be present.

Although no taste panels were used in the present study, one worker in the

laboratory could distinguish between treated and untreated ripe fruit by odor. In the

future, it may be productive to augment quantitative volatile analysis, such as the gas

chromatography employed within this study, with parallel taste tests performed by a

trained taste panel. Such panels have already examined the volatile content of tomatoes,

but have yet to explore potential differences in subjective taste due to ethylene inhibition.

















APPENDIX A
'BNH586' HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS


70.00



65.00



60.00



55.00



50.00



45.00



40.00


Days at 20* C


Figure A-1. Hue angle of 'BHN586' tomato fruit during storage at 200C. Fruit were
selected at the turning stage and placed directly in 200C storage facilities. Hue
angle was determined daily at the equatorial region of twenty-five individual
fruit. Values represent the means (n= 75. 3 measurements each on each of 25
individual fruit), +/- standard error


-- Group I
-0-Group 2












46.00



44.00



42.00
Ch


40.00

z

38.00



36.00



34.00


Figure A-2-a


47.00


46.00


45.00 -


44.00-


43.00


42.00 --


41.00


40.00 -


39.00


E-Hue angle (C) at time of volatile
sampling


3 5 12 17
Days after ripening


Chart Area


DE-Hue angle (0) at time of volatile
sampling


2 4 11 16
Days after ripening


Figure A-2-b


Figure A-2. Hue angle of 'BHN586' tomato fruit during storage at 200C after attaining a
hue angle of 500. Fruit were obtained at the turning stage and hue angle
determined daily at the equatorial region of five individual fruit. On the days
indicated, 5 fruit were removed and volatiles measured. 'Days after ripening'
refers to days following the date on which a 500 hue angle was observed on all







53


five individual fruit measured. Figures A-2-a and A-2-b depict the two
identical groups of fruit from the same source, selected and stored at the same
time and at the same stage, and belonging to the same cultivar. Values
represent the average of those five fruit, +/- standard error.


1 2 4 11 16
Days after ripening


Figure A-3. Fruit firmness (N) of 'BHN586' tomato fruit during storage at 200C after
attaining a hue angle of 500. Fruit were obtained at the turning stage and hue
angle determined daily at the equatorial region of five individual fruit. On the
days indicated, 5 fruit were removed and volatiles measured. 'Days after
ripening' refers to days following the time at which a 500 hue angle was
observed on all five individual fruit measured. Values represent the average of
those five fruit, +/- standard error.


Uk

E 4.5

a
u.
4



3.5



3







54


cis-3-hexenal


El Group 1
E Group 2


4
Days after ripening


Figure A-4. Cis-3-hexenal production in ripe 'BNH586' tomato fruit. Fruit were ripened
at 200C, and volatile production was measured on 1, 2, 4, 11, and 16 days
after fruit reached an average hue angle of 500. Group 1 and Group 2 are
identical samples taken from fruit obtained at the same time from the same
source and stored for the same duration. Values represent the average of five
fruit, +/- standard error.











Hexanal

40O


350





I-

200 -Group I
1 E1 Group 2

150 -





0



1 2 4 11 16
Days after ripening


Figure A-5. Hexanal production in ripe 'BNH586' tomato fruit. Fruit were ripened at
200C, and volatile production was measured on 1, 2, 4, 11, and 16 days after
fruit reached an average hue angle of 500. Group 1 and Group 2 are identical
samples taken from fruit obtained at the same time from the same source and
stored for the same duration. Values represent the average of five fruit, +/-
standard error.







56



ethyl vinyl ketone


1.5


1.3


1.1


.' 0.9

C0
01 0.7


0.5


0.3


0.1


El Group 1
* Group 2


4
Days after ripening


Figure A-6. Ethyl vinyl ketone production in ripe 'BNH586' tomato fruit. Fruit were
ripened at 200C, and volatile production was measured on 1, 2, 4, 11, and 16
days after fruit reached an average hue angle of 500. Group 1 and Group 2 are
identical samples taken from fruit obtained at the same time from the same
source and stored for the same duration. Values represent the average of five
fruit, +/- standard error.







57



B-ionone


E Group 1
[I Group 2


1 2 4 11 16
Days after ripening


Figure A-7. B-ionone production in ripe 'BNH586' tomato fruit. Fruit were ripened at
200C, and volatile production was measured on 1, 2, 4, 11, and 16 days after
fruit reached an average hue angle of 500. Group 1 and Group 2 are identical
samples taken from fruit obtained at the same time from the same source and
stored for the same duration. Values represent the average of five fruit, +/-
standard error.











Isovaleraldehyde


35


30


25


C 20


*1 15


10


5


0


El Group 1
E Group 2


4
Days after ripening


Figure A-8. Isovaleraldehyde production in ripe 'BNH586' tomato fruit. Fruit were
ripened at 200C, and volatile production was measured on 1, 2, 4, 11, and 16
days after fruit reached an average hue angle of 500. Group 1 and Group 2 are
identical samples taken from fruit obtained at the same time from the same
source and stored for the same duration. Values represent the average of five
fruit, +/- standard error.


















APPENDIX B
'FL-47' HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS


[] Control
E 1-MCP


1 4 6
Days after treatment


Figure B-1. Firmness (N) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250
ppb, 12 h, 200C) at the pink stage and ripened at 200C. Days represent time
since treatment. Values represent the average of fifteen fruit, +/- standard
error.


16


14


12


Z 10
U


6

I.








60



20


18


16


14

z
"-12
M
II El Control
E 10
10 1-MCP


L-

6



4--
2



1 4 6
Days after treatment


Figure B-2. Firmness (N) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250
ppb, 12 h, 200C) at the turning stage and ripened at 200C. Days represent time
since treatment. Values represent the average of fifteen fruit, +/- standard
error.








61


90

85

80

75 -

70 --

y aeEl Control
65 -6-
5 1-MCP

Z 60

55


50 -

45 -

40
1 3 4 6
Days after treatment


Figure B-3. Hue angle (0) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250
ppb, 12 h, 200C) at the pink stage and ripened at 200C. Days represent time
since treatment. Values represent the average of fifteen fruit, +/- standard
error.







62


100



90 -



80 -


C 70 aControl
70 --
m m-MCP
















Figure B-4. Hue angle Vaof 'FL-47' tomato fruit after treatment with air or 1-MCP (250



error.












100


90o I


80 --




70 --



60 --


50 -


40 --
1.00


E Control
0 1-MCP


7.00
Days after treatment


Figure B-5. Hue angle (0) of 'FL-47' tomato fruit after treatment with air or 1-MCP (250
ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Days represent time
since treatment. Values represent the average of fifteen fruit, +/- standard
error.


30
C


25 -




20 -

b
-, [E Breaker
S15- Ripe Control
M Ripe 1-MCP


10 -


/







64



Figure B-6. Cis-3-hexenal production in 'FL-47' tomato fruit. Fruit were treated with air
or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
45. 1-MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.


180
b
160

140

120-

100- E Breaker
E Ripe Control
O o BRipe 1-MCP

60 -
a
40 -

20 -




Figure B-7. Hexanal production in 'FL-47' tomato fruit. Fruit were treated with air or 1-
MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 45. 1-
MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.












0.3



0.25



0.2

-.

0.15



0.1



0.05



0


E Breaker
* Ripe Control
*Ripe I-MCP


Figure B-8. Ethyl vinyl ketone production in 'FL-47' tomato fruit. Fruit were treated with
air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
45. 1-MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.












60



50



40



-30



20



10



0


[] Breaker
N Ripe Control
E Ripe 1-MCP


Figure B-9. Cis-3-hexenol production in 'FL-47' tomato fruit. Fruit were treated with air
or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
45. 1-MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.


I -


C 0.8


0.6

0.4

0.2


] Breaker
* Ripe Control
E 1-MCP







67




Figure B-10. Trans-2-hexenal production in 'FL-47' tomato fruit. Fruit were treated with
air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
45. 1-MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.


0.09


L Breaker
* Ripe Control
E] Ripe 1-MCP


0.01 I -L I-


Figure B-11. B-ionone production in 'FL-47' tomato fruit. Fruit were treated with air or 1-
MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Volatile
production was measured when fruit reached an average hue angle of 45. 1-
MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.


0.08

0.07

0.06

. 0.05

0.04

0.03

0.02











8


7


6


5

aL
I4


3


2




1


Figure B-12. 6-methyl-5-hepten-2-one production in 'FL-47' tomato fruit. Fruit were
treated with air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and
ripened at 200C. Volatile production was measured when fruit reached an
average hue angle of 45. 1-MCP treated fruit required an additional 8 days to
reach this hue value. Breaker fruit were sampled without treatment. Values
represent the average of fifteen fruit, +/- standard error.


D Breaker
* Ripe Control
1Ripe 1-MCP




















O Breaker
* Ripe Control
URipe 1-MCP


Figure B-13. Isovaleraldehyde production in 'FL-47' tomato fruit. Fruit were treated with
air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
450. 1-MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.




















E Breaker
* Ripe Control
* Ripe 1-MCP


Figure B-14. 2-methylbutyraldehyde production in 'FL-47' tomato fruit. Fruit were
treated with air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and
ripened at 200C. Volatile production was measured when fruit reached an
average hue angle of 45. 1-MCP treated fruit required an additional 8 days to
reach this hue value. Breaker fruit were sampled without treatment. Values
represent the average of fifteen fruit, +/- standard error.












70


60


50


. 40


C 30


20


10


0


Figure B-15. 3-methyl-l-butanol production in 'FL-47' tomato fruit. Fruit were treated
with air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 45. 1-MCP treated fruit required an additional 8 days to reach this
hue value. Breaker fruit were sampled without treatment. Values represent
the average of fifteen fruit, +/- standard error.


] Breaker
N Ripe Control
MRipe 1-MCP










Cha


















___I___


0.16


0.14


0.12


0.1
.C

0.08
U'
C
0.06


0.04


0.02


0


Figure B-16. 2-phenylethanol production in 'FL-47' tomato fruit. Fruit were treated with
air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
450. 1-MCP treated fruit required an additional 8 days to reach this hue value.
Breaker fruit were sampled without treatment. Values represent the average of
fifteen fruit, +/- standard error.


E Breaker
* Ripe Control
SRipe 1-MCP


EMERIMMMEW




















[] Breaker
* Ripe Control
M Ripe 1-MCP


Figure B-17. 1-nitro-2-phenylethane production in 'FL-47' tomato fruit. Fruit were
treated with air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and
ripened at 200C. Volatile production was measured when fruit reached an
average hue angle of 45. 1-MCP treated fruit required an additional 8 days to
reach this hue value. Breaker fruit were sampled without treatment. Values
represent the average of fifteen fruit, +/- standard error.




















0 Breaker
* Ripe Control
* Ripe 1-MCP


Figure B-18. 2-isobutylthiazole production in 'FL-47' tomato fruit. Fruit were treated
with air or 1-MCP (250 ppb, 12 h, 200C) at the breaker stage and ripened at
200C. Volatile production was measured when fruit reached an average hue
angle of 45. 1-MCP treated fruit required an additional 8 days to reach this
hue value. Breaker fruit were sampled without treatment. Values represent
the average of fifteen fruit, +/- standard error.
















APPENDIX C
'FL-91' HUE ANGLE, FIRMNESS, AND VOLATILE EMISSIONS

16









11 Overripe
U 1-MCP









3 5 7 8 11
Days after treatment

Figure C-1. Firmness (N) of'FL-91' tomato fruit after treatment with air or 1-MCP (500
ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Days represent time
since treatment. Values represent the average of five fruit, +/- standard error.




















Z 13

-0- Ripe
|t -- Overripe
-0- 1-MCP



7





3 5 7 9 11 13 IS 18
Days after tratmnt


Figure C-2. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500
ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Days represent time
since treatment. Values represent the average of five fruit, +/- standard error.

20.000


1B.OOO ^ "-------------------------
18.000


16.000


Z 14.000--
'-0- Ripe

E 12.000 -s- Overripe
\ 1-MCP

U.



6.000



4.000
1 3 6 8 10 12 14 16
Days after treatment


Figure C-3. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500
ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Days represent time
since treatment. Values represent the average of five fruit, +/- standard error.



















2
z


3


-0- Ripe
--6 Overripe
-- 1-MCP


2 4 6 a 10
Days after treatment


Figure C-4. Firmness (N) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500

ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Days represent time

since treatment. Values represent the average of five fruit, +/- standard error.




a b-



s. 0- ,


-- ~ff -


-C- .


Ut ... -
~ -


Figure C-5. Hue angle (0) of 'FL-91' tomato fruit after treatment with air or 1-MCP (500

ppb, 12 h, 200C) at the breaker stage and ripened at 200C. Days represent time

since treatment. Values represent the average of five fruit, +/- standard error.

C-5-a) Experiment 1. C-5-b) Experiment 2. C-5-c) Experiment 3. C-5-d)

Experiment 4.


-W I-p.
~-1 -D TK


13.000

11.WO I

g.O000

7-.OOG T.


21.=0o


It


12 14


'*-W w'C













If a


30
a
::.-^-


OE Ripe
HOverripe
S1-MCP


Figure C-6. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 5 days to reach this hue value,
at which time an additional control (hue angle at 500 + 5 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error.



a



50
,0 ORipe
S40 Overripe
SI-MCP
30
b
20
10



Figure C-7. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 11 days to reach this hue
value, at which time an additional control (hue angle at 500 + 11 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.




















30 -


b


O Ripe
*Overripe
E1-MCP


Figure C-8. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 6 days to reach this hue value,
at which time an additional control (hue angle at 500 + 6 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error.





4t4















Figure C-9. Cis-3-hexenal production in ripe 'FL-91' tomato fruit. Fruit were treated with
air or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 4 days to reach this hue value,
at which time an additional control (hue angle at 500 + 4 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error.
















120 I


b


ab
T


EL Ripe
Overripe
i-____________ MCP


-1-


Figure C-10. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 5 days to reach this hue value,
at which time an additional control (hue angle at 500 + 5 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error.


SRipe
*Overripe
Si-MCP


Figure C-11. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 11 days to reach this hue
value, at which time an additional control (hue angle at 500 + 11 days) sample
was analyzed. Values represent the average of five fruit, +/- standard error.


Nn R

















.- -A ORipe
SOverripe
.. 1-MCP








Figure C-12. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 6 days to reach this hue value,
at which time an additional control (hue angle at 500 + 6 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error.

250 -
b
2o0 ab


E Ripe
M Overripe
*1-MCP








Figure C-13. Hexanal production in ripe 'FL-91' tomato fruit. Fruit were treated with air
or 1-MCP (500 ppb, 12 h, 200C) at the breaker stage and ripened at 200C.
Volatile production was measured when fruit reached an average hue angle of
500. 1-MCP treated fruit required an additional 4 days to reach this hue value,
at which time an additional control (hue angle at 500 + 4 days) sample was
analyzed. Values represent the average of five fruit, +/- standard error.