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Suppressing Ethylene Action: The Influence of 1-Methylcyclopropene on Ripening Responses of Banana Fruit, Musa acuminata

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Title:
Suppressing Ethylene Action: The Influence of 1-Methylcyclopropene on Ripening Responses of Banana Fruit, Musa acuminata
Creator:
STANLEY, DANIEL A.
Copyright Date:
2008

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Subjects / Keywords:
Bananas ( jstor )
Chills ( jstor )
Cooling ( jstor )
Fruiting ( jstor )
Fruits ( jstor )
Low temperature ( jstor )
Metrorrhagia ( jstor )
Peels ( jstor )
Ripening ( jstor )
Sugars ( jstor )
City of Gainesville ( local )

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University of Florida
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University of Florida
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Copyright Daniel A. Stanley. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2010
Resource Identifier:
660156459 ( OCLC )

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SUPPRESSING ETHYLENE ACTION: THE INFLUENCE OF 1-METHYLCYCLOPROPENE ON RIPENING RESPONSES OF BANANA FRUIT, Musa acuminata By DANIEL A. STANLEY 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 2007

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2007 Daniel A. Stanley

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To my mother Edith, my father Edric and my sister Amanda for each of them has inspired me in their own unique and special wa y to always strive for the highest heights.

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iv ACKNOWLEDGMENTS I would like to thank Dr. Donald Huber, my Committee Chair, and Dr. Steven Sargent for providing guidance from the beginni ng to the end of my research project and for all the knowledge they have generously pa ssed on to me. I would also like to thank Dr. Maurice Marshall and Dr. Jerry Bartz for th eir contributions as committee members. The help of Mr. James Lee, Mr. Brandon Hurr, Dr. Jiwon Jeong, Mrs. Kim Cordasco and Mrs. Adrian Berry are also greatly ap preciated. Mr. Jeff Wanner and Chiquita of Bradenton, Florida were extremely helpful and essential to the success of this project as they provided us with a rea dy supply of bananas on demand. Many other Professors in the Horticu ltural Sciences Department such as Dr. Carlene Chase, Dr. Bala Rathinasabapathi and Dr. Daniel Cantliffe, in addition to Dr. Hugh Popenoe from the Soil & Water Scie nce Department and Dr. Jonathan Earle from the Agricultural and Biological Engineer ing Department have contributed to my professional development in various ways and I want to send a special thank you to all of them.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES...........................................................................................................xi OBJECT......................................................................................................................... ....xv ABSTRACT.....................................................................................................................xvi CHAPTER 1 THE BANANA FRUIT................................................................................................1 Introduction: World Banana Production......................................................................1 Postharvest Considerations...........................................................................................1 2 LITERATURE REVIEW.............................................................................................3 Botanical Classification................................................................................................3 Fruit Development and Structure..................................................................................3 Inflorescence Development...................................................................................4 Banana Fruit Botanical Cl assification and Morphology.......................................5 Fruit Maturity Indices and Harvest........................................................................6 Banana Fruit Ripening..................................................................................................7 Climacteric Behavior: Ethylene...........................................................................7 Climacteric Behavior: Respiration.......................................................................9 Ethylene and Respiration Considerati ons in Commercial Postharvest...............11 Inhibitors of Ethylene Action..............................................................................12 The 1-Methylcyclopropene Molecule.................................................................13 Perceptions and Measurements of Fruit Quality........................................................16 Experimental Analysis of Fruit Quality..............................................................18 Peel Pigments......................................................................................................19 Textural Properties..............................................................................................19 Carbohydrates and Sugars in Banana Pulp..........................................................20 Aroma Volatiles and Fruit Flavor........................................................................20

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vi Fruit Browning: Polyphenol Oxid ase and Phenolic Compounds...............................21 Chilling Injury............................................................................................................24 Postharvest Pathology of Banana Fruit.......................................................................26 3 EFFECTS OF COMMERCIAL-SCALE APPLICATION OF 1-MCP ON BANANA RIPENING: SUSCEPTIBILI TY TO CHILLING INJURY AND SUITABILITY FOR FRESH-CUT PROCESSING..................................................35 Introduction.................................................................................................................35 Materials and Methods...............................................................................................36 Plant Material, Ethylene and 1-MCP Treatments................................................36 Fruit Ripening Parameters...................................................................................37 Storage Under Conditions th at Induce Chilling Injury........................................38 Fresh-Cut Banana Fruit and Chilling Injury........................................................38 Statistical Analysis..............................................................................................39 Results and Discussion...............................................................................................39 Peel Color............................................................................................................39 Peel Graying........................................................................................................41 Senescent Spotting...............................................................................................41 Incidence of Fungal Infection..............................................................................43 Whole Fruit Firmness..........................................................................................43 Incidence of Chilling Injury................................................................................44 Fresh-Cut Banana Fruit and Chilling Injury........................................................46 Conclusions.................................................................................................................46 4 EFFECTS OF COMMERCIAL ET HYLENE APPLICATION ON BANANA RIPENING TO DETERMINE TIMING OF 1-MCP APPLICATION AND EFFECTS OF CHILLING INJURY..........................................................................61 Introduction.................................................................................................................61 Materials and Methods...............................................................................................62 Plant Material and Ethylene Treatments.............................................................62 Fruit Ripening Parameters...................................................................................62 Storage Under Conditions th at Induce Chilling Injury........................................62 Total Soluble Sugars, Peel Sol uble Phenolic Compounds and Peel Polyphenol Oxidase Activity...........................................................................63 Statistical Analysis..............................................................................................65 Results and Discussion...............................................................................................65 Peel Color............................................................................................................65 Incidence of Chilling Injury................................................................................66 Peel Browning.....................................................................................................67 Total Soluble Sugars............................................................................................67 Peel Soluble Phenolic Compounds......................................................................67 Peel Polyphenol Oxidase Activity.......................................................................68 Conclusions.................................................................................................................69

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vii 5 EFFECTS OF 1-MCP CONCENTRAT ION ON BANANA RIPENING.................73 Introduction.................................................................................................................73 Materials and Methods...............................................................................................75 Plant Material and 1-MCP Treatments................................................................75 Fruit Ripening Parameters...................................................................................77 Total Soluble Sugars, Peel Sol uble Phenolic Compounds and Peel Polyphenol Oxidase Activity...........................................................................78 Statistical Analysis..............................................................................................81 Results and Discussion...............................................................................................82 Peel Color............................................................................................................82 Peel Graying........................................................................................................84 Incidence of Fungal Infection..............................................................................85 Whole Fruit Firmness..........................................................................................86 Total Soluble Sugars............................................................................................89 Peel Soluble Phenolic Compounds......................................................................92 Peel Polyphenol Oxidase Activity.......................................................................94 Conclusions.................................................................................................................96 6 EFFECTS OF ETHYLENE-AC TION INHIBITION AND LOW TEMPERATURE STORAGE ON BANANA RIPENING.....................................117 Introduction...............................................................................................................117 Materials and Methods.............................................................................................119 Plant Material and 1-MCP Treatments..............................................................119 Fruit Ripening Parameters.................................................................................120 Storage Under Conditions th at Induce Chilling Injury......................................121 Total Soluble Sugars, Peel Sol uble Phenolic Compounds and Peel Polyphenol Oxidase Activity.........................................................................121 Statistical Analysis............................................................................................124 Results and Discussion.............................................................................................124 Peel Color..........................................................................................................124 Incidence of Chilling Injury..............................................................................128 Senescent Spotting.............................................................................................132 Peel Graying......................................................................................................134 Incidence of Fungal Infection............................................................................136 Pulp Firmness....................................................................................................137 Total Soluble Sugars..........................................................................................140 Peel Soluble Phenolic Compounds....................................................................143 Peel Polyphenol Oxidase Activity.....................................................................148 Conclusions...............................................................................................................152

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viii 7 FINAL CONCLUSIONS..........................................................................................170 Conclusions...............................................................................................................170 Implications for Experiment al and Research Settings.......................................170 Implications for the Postharvest Industry..........................................................171 Implications for Consumers..............................................................................171 APPENDIX. POSTHARVEST SUPPLY CHAIN: PLANT PHYSIOLOGY, ECONOMICS AND FOOD SCIENCE....................................................................172 Introduction...............................................................................................................172 Commercial Postharvest Ha ndling of Banana Fruit.................................................172 Field Harvest.....................................................................................................172 Banana Processing Plant Activities...................................................................173 Storage and Transportation................................................................................173 Postharvest Ripening Faci lities and Procedures................................................173 LIST OF REFERENCES.................................................................................................183 BIOGRAPHICAL SKETCH...........................................................................................194

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ix LIST OF TABLES Table page 3-1 Banana treatment with 1-MCP under commercial and controlled conditions.........48 3-2 Storage regimes for banana hands destined for fresh-cut processing......................48 3-3 Whole fruit firmness values (Newtons)....................................................................49 3-4 Fresh-cut fruit firmness values (Newtons)...............................................................49 4-1 Control and ethylene treatment 5 C storage regimes beginning Day 3....................70 4-2 Color score of control and ethylene-treated fruit.....................................................70 4-3 Absorbance readings of banana peel PPO................................................................70 5-1 Extraction buffers and subs trates used to assay PPO...............................................99 5-2 Fruit pulp samples soluble solids content (Brix)....................................................99 5-3 Sucrose standards solubl e solids content (Brix).....................................................99 6-1 Horsfall-Barrett scale units used to quantify senescent spotting development......155 6-2 Control and 1-MCP treatment groups 5C storage regime s beginning Day 6.......155 6-3 Peel color comparisons be tween specified treatments...........................................156 6-4 CI development comparisons between specified treatments..................................156 6-5 Senescent spotting development comp arisons between specified treatments........157 6-6 Fruit firmness comparisons between specified treatments.....................................157 6-7 Maximum delays observed in soluble sugar accumulation....................................157 6-8 Soluble sugar content comparis ons between specified treatments.........................158 6-9 Peel soluble phenolics content comp arisons between specified treatments...........158

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x 6-10 Chlorogenic acid content comparis ons between specified treatments...................159 6-11 Polyphenol oxidase (PPO) activity comp arisons between specified treatments....159

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xi LIST OF FIGURES Figure page 1-1 Commercial plantation growing Musa acuminata .....................................................2 2-1 Banana pseudostem made up of petioles/leaf sheaths..............................................30 2-2 Banana fruit single si gmoidal growth curve............................................................31 2-3 Respiratory or ethylene pattern of climacteric vs. non-climacteric fruit..................31 2-4 The ethylene molecule’s chemical structure............................................................32 2-5 Ethylene biosynthesis (step 1 to 3) an d the Yang Cycle (step 1 to 2, 4 to 7)...........32 2-6 The 1-methylcyclopropene mo lecule’s chemical structure......................................32 2-8 Various phenolic substrates of PPO in banana fruit.................................................33 2-9 Proposed fruit browning pathway............................................................................34 3-1 Peel color of Control and 1-MCP-treated fruit (300 nLL-1)....................................50 3-2 Peel color of PU, P300 and P900 fruit.....................................................................51 3-3 Graying of banana fruit............................................................................................52 3-4 Peel senescent spotting.............................................................................................52 3-5 Close-up of peel senescent spotting.........................................................................52 3-5 Peel senescent spotting in Cont rol and 1-MCP-treated fruit (300 nLL-1)...............53 3-6 Peel senescent spotting in PU, P300 and P900 fruit................................................54 3-7 Incidence of fungal infection in C ontrol and 1-MCP-treated fruit (300 nLL-1)......55 3-8 Close-up of fungal infection.....................................................................................55 3-9 Incidence of fungal infecti on in PU, P300 and P900 fruit.......................................56 3-10 Whole fruit firmness of Control and 1-MCP-treated fruit (300 nLL-1)...................57

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xii 3-11 Whole fruit firmness of PU, P300 and P900 fruit....................................................58 3-12 Chilling injury symptoms in banana fruit................................................................58 3-13 Peel color of Control and 1-MCP-treat ed fruit stored at low temperatures.............59 3-14 Fresh-cut banana fruit...............................................................................................60 4-1 Variation in fruit ripening re sulting from ethylene treatment..................................71 4-2 Graying of ethylene-treated fruit 24 h after removal from chilling injury...............71 4-3 Banana fruit browning as a result of repetitive impact compression.......................72 5-1 Peel color of Control and 1-MCP-treated fruit (500 nLL-1)..................................100 5-2 Peel color of Control and 1-MCP-treated fruit (250 nLL-1)..................................101 5-3 Degree of graying in Control and 1-MCP-treated fruit (500 nLL-1).....................102 5-4 Degree of graying in Control and 1-MCP-treated fruit (250 nLL-1).....................103 5-5 Incidence of fungal infection in C ontrol and 1-MCP-treated fruit (500 nLL-1)....104 5-6 Incidence of fungal infection in C ontrol and 1-MCP-treated fruit (250 nLL-1)....105 5-7 Firmness of Control and 1MCP-treated fruit (500 nLL-1)...................................106 5-8 Firmness of Control and 1MCP-treated fruit (250 nLL-1)...................................107 5-9 Soluble sugar content of Contro l and 1-MCP-treated fruit (500 nLL-1)...............108 5-10 Soluble sugar content of Contro l and 1-MCP-treated fruit (250 nLL-1)...............109 5-11 Peel soluble phenolic content of C ontrol and 1-MCP-treated fruit (500 nLL-1)...110 5-12 Peel soluble phenolic content of C ontrol and 1-MCP-treated fruit (250 nLL-1)...111 5-13 Chlorogenic acid content of Cont rol and 1-MCP-treated fruit (500 nLL-1)..........112 5-14 Chlorogenic acid content of Cont rol and 1-MCP-treated fruit (250 nLL-1)..........113 5-15 Increasing polyphenol oxidase (PPO) activity.......................................................114 5-16 Non-increasing polyphenol oxidase (PPO) activity...............................................114 5-17 Total PPO activity of Control and 1-MCP-treated fruit (500 nLL-1)....................115 5-18 Total PPO activity of Control and 1-MCP-treated fruit (250 nLL-1)....................116

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xiii 6-1 Peel color in respons e to low-temperature.............................................................160 6-2 Peel chilling injury in response to low-temperature...............................................161 6-3 Peel senescent spotting in response to low-temperature........................................162 6-4 Non-chilling-induced peel graying.........................................................................163 6-5 Incidence of fungal infection..................................................................................164 6-6 Fruit mesocarp firmness in response to low-temperature......................................165 6-7 Soluble sugar content in response to low-temperature...........................................166 6-8 Peel soluble phenolics content in response to low-temperature.............................167 6-9 Peel chlorogenic acid content in response to low-temperature..............................168 6-10 Total PPO activity in response to low-temperature...............................................169 A-1 Perforated polyethylene bag covering banana fruit................................................174 A-2 Fruit harvest............................................................................................................174 A-3 Railway arrival at processing plant........................................................................175 A-4 Primary processing activities.................................................................................175 A-5 Secondary processing activities.............................................................................176 A-6 Hands removed from second fungicide dip and placed on drying tray..................176 A-7 Stem wound sealant application.............................................................................177 A-8 Fruit weighed and boxed for shipping....................................................................177 A-9 Boxed fruit palletized for shipping.........................................................................178 A-10 Fruit unloaded at ports and sh ipped by refrigerated truck.....................................178 A-11 Interior of fully loaded trailers...............................................................................178 A-12 Temperature probe meter used to sample fruit pulp temperature..........................179 A-13 Empty ripening room.............................................................................................179 A-14 Ethylene generators................................................................................................180

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xiv A-15 Ethylene gassing of palletized and boxed fruit......................................................180 A-16 Fruit pallets reloaded in to refrigerated trailers.......................................................181

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xv OBJECT Object page 1 Video of banana harvest.........................................................................................175

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xvi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SUPPRESSING ETHYLENE ACTION: THE INFLUENCE OF 1-METHYLCYCLOPROPENE ON RIPENING RESPONSES OF BANANA FRUIT, Musa acuminata By Daniel A. Stanley May 2007 Chair: Donald J. Huber Major: Horticultural Sciences Banana production areas in the tropics are geographically isolated from many of the major consumer markets in temperate regions. The disparity between harvest and consumption has prompted investigati ons utilizing the et hylene antagonist 1-methylcyclopropene (1-MCP) to delay the ripening process and prolong the marketable shelf life of the fruits. Banana fruit ( Musa acuminata , Cavendish subgroup, cv. Williams) used in this study were commerc ially harvested, transported and treated with ethylene (300 LL-1, 15C, 24 h) in Bradenton, Flor ida. Individual experiments examined the effects of applying 500 nLL-1 1-MCP, 250 nLL-1 1-MCP and 250 nLL-1 1-MCP in conjunction with low temperature storage (5C, 24 hours an d 5C, 9 days) to determine the effects on the following ripening parameters: peel color development, whole-fruit and pulp firmness, incidence of graying, incidence of senescent spotting,

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xvii incidence of fungal mycelia pr oliferation, pulp soluble suga r, peel total phenolics and peel polyphenol oxidase activity. Application of 500 nLL-1 1-MCP effectively delayed p eel color development, pulp soluble sugar accumulation, peel total phenol ics accumulation and polyphenol oxidase activity by an average of 3 to 5 days as comp ared with control fruit. 1-MCP-treated fruit also maintained fruit firmness levels a bove 30 Newtons (N) for the duration of the experiment while the controls continued to soften below 20 N. 1-MCP had no effect on the incidence of graying or fungal mycelia proliferation as thes e ripening associated events progressed normally with thei r respective ripening stages. Application of 250 nLL-1 1-MCP resulted in shorter de lays in color development and polyphenol oxidase activity yet pulp soluble sugar ac cumulation and peel total phenolics accumulation were not significantly affected. The lower 1-MCP concentration produced similar firmness results with 1-MC P levels remaining above 30 Newtons (N) while the controls softened below 20 N. Application of 250 nLL-1 1-MCP prior to low temperat ure storage (5C, 24 h or 5C, 9 days) produced visible delays in p eel color development and noticeable chilling injury (CI) symptoms not masked by 1-MCP application when compared with control fruit exposed to identical storage conditions. Significant delays in pulp firmness, pulp soluble sugar accumulation, peel phenolics accumulation and polyphenol oxidase activity were also observed. Measurements of se nescent spotting and graying were confounded by the presence of CI symptoms, while funga l appearance was not affected in any treatment.

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1 CHAPTER 1 THE BANANA FRUIT Introduction: World Banana Production The edible banana is one of the most important food crops cultivated throughout tropical regions of the world today. It is ranked as the fourth most consumed starchy crop worldwide after maize, rice and wheat (INIBAP 2005). Banana production in the western hemisphere is concentrated in Centra l and South American c ountries (Figure 1-1) while in the eastern hemisphere production ar eas are concentrated in Southeast Asia and neighboring Pacific nations (FAO 2005). In 2003, worldwide fruit exports totaled 15.5 million metric tons valued at over $4.7 billion from over 100 different nations (FAO 2005). Until the mid 1940s the ‘Gros Michel’ cultivar was the main banana exported to world markets until its suscepti bility to the fungal pathogen Fusarium oxsysporum (Fusarium wilt, Panama disease) rendered it obsolete. After the 1940s the industry developed and began to export cultivars of the Cavendish su bgroup that are the primary cultivars grown today (Kerbel 2004). Postharvest Considerations Banana fruit are harvested and shipped in the “pre-ripe” green st age. Upon arrival in domestic markets, fruit undergo induced ripening before retail display to provide consumers with ripe, ready-to-eat fruit. Once ripening is initia ted, the physiological processes advance so quickly that it becomes difficult for the retailer to sell the entire load and for the consumer to eat the en tire hand purchased. The lack of extended

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2 shelf-life poses problems for the retailer b ecause decreased sales result in economic losses. From the consumer’s perspective, the lack of “home” shelf-life may discourage future purchases. For these reasons, the purpose of this study was to determine the feasibility of utilizing the ethylene antagonist 1-methylcycl opropene (1-MCP) to extend the shelf-life of commercially handled ba nana fruit and in doing so the following objectives were achieved: Objective 1 : Contribute to the established body of knowledge pertaining to the physiological mechan isms of banana fruit ripening as affected by 1-MCP application by observing fruit under controlled conditions and analyzing fruit sample s for changes in intrinsic quality over time. Objective 2 : Provide data to help estab lish the practicality of delaying ripening and extending marketable sh elf-life of commercially handled banana fruit by applying 1-MCP. Figure 1-1. Commercial plantation growing Musa acuminata , Cavendish subgroup cv. Williams bananas. Photo taken with permission on location in Siquerres, Costa Rica by Daniel A. Stanley.

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3 CHAPTER 2 LITERATURE REVIEW Botanical Classification The banana plant has been described as a monocotyledonous “tree-like perennial herb” that consists of root tissue, an underground rhizome or corm, and a pseudostem made up of petioles/leaf sheaths that culminate in large al ternate leaves (Figure 2-1) (Seymour et al., 1993; Carr, 2005). Edible fr uit-producing banana pl ants belong to the botanical Order Zingiberales and Family Mu saceae (INIBAP, 2005). Within this family there are many species but the most relevant to modern-day banana fruit production is Musa acuminata . The Musa acuminata species (A genome) is nativ e to the tropical, hot humid lowlands of Malaysia and consists of dipl oid AA genomes and dipl oid-derived triploid AAA genomes, both of which began producing ed ible fruits through spontaneous genetic mutations (Seymour et al., 1993). Plants exhibiting the triploid AAA genome produce parthenocarpic fruit while fruits of the dipl oid AA genome group cont ain viable seeds. All subsequent descriptions and data in the Literature Review refer to the Musa acuminata of the AAA genomic group, Cavendish subgroup cv. Williams banana unless otherwise stated. Fruit Development and Structure Terms pertaining to fruit development mu st be defined for reference in the following text. Growth refers to irreversible increase in physical attr ibutes such as fruit weight, size. Maturation refers to stages of development leadi ng to attainment of

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4 physiological or horticultural maturity. Phys iological maturity refers to a stage of development when a plant or plant part continues normal ontogeny (physical or biochemical changes in the fruit) even if de tached (Watada, 1984). Horticultural maturity refers to a stage when a plant or plant pa rt possesses features for utilization for a particular purpose (Watada, 1984). In the cas e of banana fruit, horticultural maturity occurs when fruit are green (consumed as star chy vegetable-crop) or as they near the end of the linear growth phase and enter the m onomolecular growth phase (described further in Inflorescence Development and Fr uit Maturity Indices and Harvest). Understanding the development of the bana na fruit is important for determining final fruit quality, harvest techniques and pos tharvest handling. Ba nana fruit exhibit a single sigmoidal growth curve pattern (Figur e 2-2) with three stages of development (Rhodes, 1980). Inflorescence Development Prior to the first stage of fruit developmen t, the inflorescence develops in the corm and grows through the pseudostem of the leaf sheaths until it emerges through the apical meristem approximately nine months after pl anting suckers. The inflorescence produces characteristic maroon colored bracts whic h cover cymes of imperfect, zygomorphic female flowers, although no pollination need occur for fruit set. As bracts retract and eventually abscise, female flowers from the proximal end of the inflorescence emerge (Carr, 2005). This process continues toward th e distal end of the inflorescence with each successive bract revealing younger flowers (Figure 2-2). The female flower is composed of a compound pistil made up of three fused carpels, one style, and an infe rior ovary with 3 locules, ea ch containing numerous axile ovules which develop into seed remnants (C arr, 2005). By the time the bracts uncover

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5 the distal male flowers, the female flowers have past their receptive stage (Israeli et al., 1986). The male flower is composed of up to 5 fertile stamens, a staminode, and a tepal. In commercial production, the youngest distal fe male and all male flowers are removed to decrease carbon sequestration from the alrea dy developing fruit. This process decreases the sink:source ratio of bunc h size to leaf surface area (Israeli et al., 1986). Banana Fruit Botanical Classification and Morphology The banana fruit is classified as a berry. Each fruit consists of the exocarp (peel) which encompasses the mesocarp (pulp) a nd endocarp (inner pulp, seed remnants). While the mesocarp and endocarp are not clear ly differentiated in mature fruit, the distinction between the exocar p and endocarp manifests fully during fruit ripening. The exocarp tissue is composed of the following: a single layer of epidermal cells with an outer cuticle and stomate cells; 6 to 11 laye rs of chloroplast containing parenchyma cells; vascular bundles encompassed within “lactiferous elements”; air spaces that expand upon fruit development; an inner epidermis that differentiates into pulp cells upon fruit development (Israeli et al., 1986). Each tissue is derived from different part s of the female flower. The peel tissue evolves from the ovary wall. The endocarp/ pulp tissue primarily consists of large parenchyma cells and evolves from pericarp cell division (Turner, 1997) derived from the inferior ovule of the female flower (Rhodes, 1980). These cells usually have high water retention capacity to facilitate accumulation of soluble sugars and organic compounds. Seed remnants developed from axile ovules ar e visible in the endocarp tissue yet are not viable (Carr, 2005).

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6 Fruit Maturity Indices and Harvest Bagging of the bunch is practiced during frui t development to protect the fruit from cosmetic damage (personal communicati on, Corbana Banana Co., Costa Rica). A perforated polyethylene bag is tied around the peduncle and left open at the bottom to prevent creating modified atmosphere condi tions within the bag (see the Appendix for images). Fruit become physiologically mature 3 to 4 months after anthesis and at this point the fruit is near the end of its linear growth phase and entering its monomolecular phase (Figure 2-2C). When harvested, hands at the proximal end of the stem are more mature than those at the distal end due to the order of emergen ce of the fruit. Fruit age exhibits a negative relationship to length of the pre-climacteric period (i.e. the older the bunch, the shorter the pre-climacteric). Fruit are harvested ye ar-round when they are both physiologically and horticulturally mature. Caliper grade of the second hand from the proximal end the diameter of the fruit is used along with days after anthesis to dete rmine appropriate fruit for harvest (Israeli et al., 1986). At this point the fruit are green, in a pre-ripe, preclimacteric stage. Harvest maturity is a tradeoff between allowing fruit extended time for development and allowing enough “green lif e” to facilitate shipping and marketing (Kerbel, 2004). Harvest of the banana fruit results in bot h the initiation and termination of many biological processes. As the fruit are rem oved from the plant, th ere is a cessation of carbon import. Being climacteric fruit, this cessation does not affect the final sugar content of the fruits if ha rvested at a physiological matu re stage. For a detailed description of the commercial posthar vest handling chain see the Appendix.

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7 Banana Fruit Ripening Ripening is a series of physiological changes caused by the synchronized upregulation of ripening-related genes. The ripening process occurs along a continuum from the latter stages of growth through ear ly senescence resulting in aesthetic and/or edible quality (Watada, 1984). Divisions of the continuum are based on initiation and termination of biological pro cesses including the occurrence of the climacteric period, softening, and altered pigmentation and volat ile profiles. The climacteric period is characterized by a rise in respiration and au tocatalytic ethylene production and includes: the pre-climacteric minimum occurring after ha rvest and before ripening processes have begun; the climacteric rise and peak occurr ing as ripening processes begin; the postclimacteric decline occurring as ripening continues (Figure 2-3) (Saltveit, 2004b). In banana fruit the ethylene peak precedes the respiratory peak (Seymour, 1993). Climacteric Behavior: Ethylene Ethylene is an olefin hydrocar bon with the chemical formula C2H4 (Figure 2-4). It is known that ethylene plays a role as a plant hormone by regulating various developmental processes such as the initiati on of fruit ripening (Abeles et al., 1992). Ethylene is produced at basal sy stem I levels in developing fruits before harvest and after harvest in the preclimacteric state. System II autocatalytic ethylene production occurs during the climacteric while fruit are ripe ning (Seymour, 1993). Ethylene is also produced as a wounding response in severed or damaged areas during fruit harvest and transport (Abeles et al., 1992).Bananas stored in atmospheric CO2 and O2 levels are sensitive to ethylene concentrations as low as 0.3 to 0.5 LL-1 (Peacock et al., 1972) which may occur at physiological levels as developing fruit produce approximately 0.2 LL-1 ethylene until the climacteric is initiated (Israeli et al., 1986). Studies have

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8 shown internal ethylene concentrations of 0.1 LL-1 in pre-climacteric fruit, 1.5 LL-1 during the initiation of the clim acteric rise and up to 40 LL-1 at the climacteric peak (Burg and Burg, 1962). Decreased gas diffusi on through the peel tissu e allows internal ethylene concentrations to accumulate to higher levels than external storage atmosphere concentrations which have been re corded at peak rates of 3 L kg-1h-1 (McMurchie et al., 1972). The buildup of in ternal ethylene from the pulp pl ays a role in initiating peel degreening and other ripening associated processes (Abeles et al., 1992; Dominguez et al., 1993). Application of exogenous ethylene or ethyl ene analogs such as propylene in high concentrations elicit ripening responses in banana fruit similar to those observed during natural system II production (Golding et al., 1998) . It has been shown that application of 1.0 nLL-1 ethylene to pre-climacteric Gros Michel banana fruit decreased the time required for initiation of the respiratory climacteric by 3 days as compared with the control (no ethylene) (Rhodes, 1981). Once th e climacteric is init iated, application of exogenous ethylene does not accelerate or incr ease the magnitude of the respiratory or ethylene climacteric. On the contrary, studies have shown that high levels of exogenous ethylene or propylene can suppress et hylene biosynthesis (Vendrell and McGlasson, 1971; Golding et al., 1998). Ethylene biosynthesis begins with the enzymatic conversion of the amino acid methionine to intermediates s-adenosyl methionine (SAM), 1-aminocyclopropane1-carboxylic acid (ACC) and finally to ethyl ene (Figure 2-5) (Seymour, 1993; Adams et al., 1979) while methionine is biological ly recycled through the Yang Cycle (Yang et al., 1984). ACC-synthase (ACS), and ACC-oxidase (ACO) are the main enzymes

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9 regulating the ethylene biosynthetic pathway. ACO activity in banana pulp tissue has been detected as early as 4 h after continuous ethylene ap plication while increases in ACS activity were not apparent until 12 h of ethylene treatment (Inaba and Nakamura, 1986). MA-ACS1 mRNA accumulation in the cytosol results in the conversion of SAM to ACC. As ACC cont ent increases, ACO activity increases and climacteric production of ethylene begins (L ui et al., 1999). The perception of higher ethylene concentrations causes more ethylene to be produced and hence the positive feedback system II mechanism ensues. Ethylene is perceived by transmembrane r eceptors located in the surface of the plasma membrane of the banana fruit peel and pulp cells (Abeles et al., 1992). The default status of the unbound ethylene receptor is in an active mode. Upon exposure of developmentally receptive fruit to ethylene, the molecule binds with the receptor protein inactivating it. The binding results in si gnal transduction from the receptor to the nucleus, allowing ethylene biosynthesis to be gin (Yang, 1987). As fruit ripen, synthesis of new ethylene-binding sites occurs (Sisler et al., 1997). Ethylene is utilized throughout the banana industry to stimulate early ripening and manage commercial supply chains to ensure customer demand is met with ripening or ready-to-sell fruit (see the Appendix for deta ils regarding commercial ethylene gassing). Climacteric Behavior: Respiration The process of aerobic respiration requires the biochemical reaction of atmospheric O2 with carbohydrates, lipids or organic acids in the tissue to generate energy, intermediate compounds and produce CO2 and water as by-products. Factors affecting respiration include storage temperature, at mospheric gas composition, physical stress, and stage of development. Increased temperature increases respiration rates

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10 exponentially until the crop incurs heat stress while low O2 concentrations (<2 to 3%) inhibit respiration (S altveit, 2004b). Banana fruit exhibit a rise in respiration as the ethylene climacteric subsides during the onset of ripening (Liu et al., 2004; Palmer , 1971). In the preclimacteric state the fruit consume approximately 13 mL O2kg-1h-1, consumption then decreases to less than 10 mL O2kg-1h-1 at the climacteric minimum, and may increase to over 43 mL O2kg-1h-1 at the climacteric peak (Rhodes et al., 1981). Banana respiratory climacteric at 20C has been reported to be between 4 to 10 times the climacteric minimum depending on variety (Rhodes, 1980; Palmer, 1971). Respiration rates have been recorded at 125 mg CO2kg-1h-1 during the climacteric peak (Palmer, 1971). The relationship between the respiratory climacteric peak and ripening have not been totally solidified. Studies done by Ve ndrell, and McGlasson (1971) where banana fruit was exposed to ethylene then placed in controlled atmosphere storage of 0.5 to 1.0% O2 did not reveal a rise in re spiration yet the fruit did ch ange color indicating some degree of ripening. In other studies, protein synthesis in hibitors prevented softening, chlorophyll degradation and ethylene production, while the respiratory peak continued to occur (Brady, 1976). For these reasons, it ha s been suggested that the respiratory climacteric occurs independently of certain ripening events and vice versa. The respiratory quotient (RQ) is defined as the ratio of the CO2 produced to O2 consumed (measured in moles or volumes) durin g a respiration reaction (Saltveit, 2004b). In aerobic respiration, RQ valu es of less than 1 indicates me tabolism of lipids, RQ values of approximately 1 indicates metabolism of carbohydrates, while RQ values of more than 1 indicates metabolism of organic acids. Hi gh RQ values indicate anaerobic respiration

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11 (Saltveit, 2004b). Experimentally, banana RQ decreased from 1.0 in pre-climacteric fruit to approximately 0.76 during the respirati on increase and returned to 1.0 at the climacteric peak (Rhodes 1980, Palmer 1971). Sim ilar results were achieved in a study employing propylene (McM urchie et al., 1972). Ethylene and Respiration Considerations in Commercial Postharvest Understanding ethylene production, respiration rates and their effects are important for maintaining fruit quality in commercial shipping and storage. Banana, along with other fruits such as mango, guava and tomato are classified into the “moderate” group of ethylene producers which range from 1.0 to 10.0 L C2H4kg-1h-1 at 20C (Kader, 2002b). At 15C, ripe banana fruit were reported to produce 5.0 L C2H4kg-1h-1 (Kerbel, 2004). This is important for shippi ng and storage requirement s because if stored with “high” (10 to 100 L C2H4kg-1h-1 at 20C) or “very high” (>100 L C2H4kg-1h-1 at 20C) producers, premature ripe ning may be induced. Yet if stored with “low” (0.1 to 1.0 L C2H4kg-1h-1 at 20C) or “very low” (<0.1 L C2H4kg-1h-1 at 20C) producers, the sensitive fruit may deteriorate prematur ely as well (Kader, 2002b). Commercial fruit are gassed at ethylene concentr ations between 150 to 1000 nLL-1 at 15C for 24 to 48 hours to induce uniform ripening. Higher concen trations are used to ensure uniform gas concentrations and saturati on in facilities where the chambers are not airtight (Kerbel, 2004). Respiration rates are inversely associated with shelf life. Banana, along with other fruits such as blueberry, peach, mango and to mato are classified as having “moderate” respiration rates (Kader, 2002b). One notable by-product of respiration of concern to postharvest handling is the generation of vital heat. At 15 to 16C, CO2 generated by green bananas measured between 21 to 23 mg CO2kg-1h-1 while ripe bananas at 13C

PAGE 29

12 generated 140 mg CO2kg-1h-1. These values calculate1 to approximately 1,285 to 1,408 kcalmetric ton-1day-1 for green fruit while ripe bananas at 13C generated approximately 8,568 kcalmetricton-1day-1. At 20C respiration values for ripe fruit were reported to be 280 mg CO2kg-1h-1 which is equivalent to 17,136 kcalmetricton-1day-1 (Kerbel, 2004). Decreased storage temperature (above ch illing injury temperatures) decrease biological metabolism and respiration rates and hence the evolution of heat in the storage system. Optimum storage and transport temp erature is 14 to 15C and 90 to 95% relative humidity (RH), while optimum ripening temp erature is between 15 to 20C and 90 to 95% RH. (Kader, 2002b; Kerbel, 2004). Knowing these average values allow postharvest facilities to design and maintain adequate refrigerati on capacity, ventilation and air circulation (Saltveit, 2004b). Inhibitors of Ethylene Action The ability to regulate ethylene synthesis and perception has become a useful tool for postharvest managers and scientists. Prevention of ethylene perception has become an invaluable tool used to study ethylene’s re gulatory effects. Commercial anti-ethylene treatments may prove useful in crops that exhibit decreased qua lity upon exposure to ethylene while delayed ripening is desired in climacteric tissue that respond positively to ethylene. There are a wide variety of inhi bitors of ethylene action that range from gaseous 1-substituted cyclopropenes (Sisler et al ., 2003) to silver thiosulfate utilized in cut flower preservation (Liao et al., 2000). The following section will focus on 1-MCP since it has been approved by the FDA and its app lications are the focus of this research. 1 conversion: 1 mg CO2kg-1h-1 x 61.2 = 1 kcalmetricton-1day-1

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13 The 1-Methylcyclopropene Molecule The molecule 1-methylcyclopropene (1-MCP) is a cyclic olefin (Figure 2-6). It functions as an ethylene antagonist by bindi ng to the receptor site designated for the ethylene molecule effectively preventing the signal transduction regul ating ethylene action (Sisler et al., 1997). The binding is considered noncompetitive and has an affinity for the receptor 10 times that of ethylene (Blankenship et al., 2003). The effects of 1-MCP have been explored for their ability to delay the ripening process and prolong the marketable shelf life of a number of temperate, tropical and sub-tropic al fruits, vegetables and flowers (Blankenship et al., 2003). Initial studies applying ethylen e antagonists to banana fr uit have provided valuable results. Golding et al., (1998) treated pr eclimacteric, mature green bananas with 500 LL-1 propylene then 45 LL-1 1-MCP was applied at 6 or 24 h after propylene to determine the effects of ethyl ene on various ripening paramete rs. 1-MCP application 6 h after propylene treatment delayed the ethyl ene and respiratory climacteric peaks by 24 and 25 days, respectively. The magnitude of the delayed ethylene climacteric peaked at 2.0 Lkg-1h-1, approximately double that of the control fruit’s level of 0.8 Lkg-1h-1. The magnitude of the delayed respirat ory climacteric peaked at 27 mL CO2kg-1h-1 while the control fruit peaked at 48 mL CO2kg-1h-1. The onset of peel color change was delayed by 23 days in 1-MCP treated fruit and non-uniform yellowing of peel was also noted. Volatile production began 30 days after that of control fruit where peak levels were 400 gL-1kg-1, or 75% of the control fruit levels of 525 gL-1kg-1 (Golding et al., 1998). 1-MCP application 24 h after propylene treatment delaye d peel color change and total volatiles production by 4 days compared to the control fruit. P eak levels of volatile

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14 production occurred at 250 gL-1kg-1, or 47% of the control fruit (525 gL-1kg-1). The onset of climacteric ethylene production and respiratory activ ity were not inhibited as the initiation of these processes was already underway at the 24 h 1-MCP treatment time. Similar patterns occurred where the et hylene production ra tes were higher (1.25 Lkg-1h-1) versus those of control fr uit control fruit (1.8 Lkg-1h-1) and the peak respiration rates were lower (39 mL CO2kg-1h-1) compared to the control fruit (48 mL CO2kg-1h-1) (Golding et al., 1998). The time delays observed in various ripe ning parameters after 1-MCP application indicate that functional ethylen e receptors play a role during the first 24 h after ripening is initiated and disruption of regulatory processes governing ethyl ene biosynthesis and respiration result in increases in ethylene rate s and decreases in respiration rates. Another notable find was that unbound 1-MCP continued to diffuse from treated bananas for days after initial treatment which may indicate a “binding saturation point ” for 1-MCP and the active ethylene receptors (Golding et al., 1998). The authors concluded that “once autocatalytic ethylene production is initiated certain pr ocesses become independent of further ethylene action” (Golding et al., 1998). Jiang et al., (1999) reported an inverse relationship between 1-MCP concentration and exposure duration required to achieve protection against ethylene-induced (100 LL 1, 24 h, 20C) ripening and softening of ba nana fruit, confirming the findings of Sisler et al., (1997). 1-MCP application was effective if applied 1 day after ethylene treatment yet was not effective when applied 3 or more days after et hylene treatment. The effect of ethylene on increased softening of 1-MCP-treated fruit was greater as time elapsed between 1-MCP and ethylene treatments. The authors attribute this behavior to

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15 synthesis of new ethylene binding sites as fruit ripen that concurs with similar conclusions drawn from initial studies of the inhibitor (Sisle r et al., 1997; Golding et al., 1998). Studies involving 1-MCP treatment in co mbination with polyethylene bag storage have been conducted to facilitate postha rvest storage and shipping in areas where refrigeration is not available (Jiang et al ., 1999). The modified atmosphere packaging (MAP) in combination with 0.5 LL-1 1-MCP increased time required for fruit to reach peel color stage 6 to 41 days compared with MAP control fruit (no 1-MCP treatment) that required 28 days and the non-MAP contro l fruit that required 16 days. Sisler et al., (2003) tested the effect of eleven different 1-substituted cyclopropene molecules on banana fruit ripening. Th e study aimed to determine the minimum concentration required for effective protec tion against immediate ethylene treatment (333 LL 1, 18 h) and to determine the time required for fruit to regain sensitivity to ethylene after exposure to saturating levels of each cyclopropene. At a concentration of 0.7 nLL-1, 1-MCP provided fruit with 12 d of protection from ethylene while 0.3 nLL-1 1-decylcyclopropene provided fr uit with 36 d of protection. Other 1-substituted cyclopropenes exhibited simila r results in blocking ethyl ene perception with varying degrees of efficacy depending on the carbon ch ain length of the functional group (Sisler et al., 2003). 1-MCP has become the choice cy clopropene utilized in many experimental and commercial settings because it is F DA-approved and commercially available as SmartFresh from AgroFresh, Inc., a subsidiary of Rohm and Haas (Spring House, PA). Pelayo et al., (2003) report ed that application of 1MCP (varying concentrations, temperatures and durations) to stage 3 or 4 bananas 36 to 48 h after commercial ethylene

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16 treatment yielded inconsistent ripening result s in various experiments leading the authors to conclude that treatment of partially ripe bananas with 1-MCP is not a viable commercial treatment. A related study c onducted by Harris et al., (2000) addressed the effects 1-MCP on fruit harvested at three di fferent dates after bunch emergence. The importance of this study rela tes to commercial harvest prac tices where fruit bunches are harvested at varying days after bunch emerge nce which leads to variation in maturities between bunches in addition to the variati on within each bunch (due to developmental factors discussed earlier). When treated with 500 nLL-1 1-MCP and stored at 20C in 0.1 LL-1 ethylene, the earliest harvested (least mature) fruit required 39.7 3.0 days to ripen, while the most mature fruit requir ed 27.9 2.3 days to ripen. The difference observed in days required to ripen is rela ted to the differences in maturity and responsiveness to ethylene and 1-MCP. The results of the Harris et al., (2000) experiment may relate to why Pelayo et al., (2003) observed the vari ability in ripening responses of partially ripe fruit. Recent studies have explored the procedure of applying 1-MCP prior to ethylene treatment on unripe green bananas and its e ffects on cell wall hydrolases involved in ripening, yet peel color score data were not presented and the procedure’s viability as a commercial treatment was not discussed (Lohani et al., 2004). As with other, more thoroughly examined fruits, the exact timing of 1-MCP treatment relati ve to the previous ethylene exposure, concentration, exposu re duration, and temperature of 1-MCP application will need to be further explored in banana. Perceptions and Measurements of Fruit Quality Banana fruit quality parameters are defined by the destination market of the fruit. Two distinct markets exist in commercial ba nana production: regiona l markets located in

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17 the country of origin and inte rnational markets primarily located in the U.S. and E.U.; the latter will be the focus of all subsequent di scussion. Individual standards of quality are based on buyer preferences and the consumer’s position in th e supply chain i.e. whether they are the producer, postharvest ha ndler, consumer or researcher. Quality attributes can be divided into th e general categories of external visual appearance and internal fruit texture, flavor and nutritive value. Visual appearance may be the broadest category as it encompasse s many aspects consumers use to judge food quality in markets. At the retail level, per ceptions of fruit quality are judged by size and shape of the fruit bunch in addition to the appe arance of peel color, absence of bruises, blemishes, insect herbivory, latex stains and fungal pathogens (Kerbel, 2004; Israeli et al., 1986). Tolerance of peel disorders is minimal ther efore cosmetically “perfect” fruit are desired by retailers and consumers. The USDA has no official guidelines for minimum banana quality standards and/or grades so banana producing companies implement internal standards to en sure product quality and uniformity. Sensory evaluations such as fruit firmness, texture and flavor components play less of a role in determining fruit quality wh en retail displayed. Upon consumption, these attributes play major roles in consumer acceptance and repurchasing consideration (Baldwin, 2004). Nutritive valu e is difficult to judge subjec tively by consumers so U.S. Food and Drug Administration nutritional labe ls are generally accepted and used for guidance (U.S. FDA, 2005). Food safety concerns relate to the presence of contaminants ranging from biological (bacteria, fungi, insect parts, human hair, etc.) to chemical (pesticide residues, processing by products etc.) to mechanical parts (plastic, metal in fresh cut)

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18 (Gorny, 2004). Food safety has become of in creasing concern due to the presence of imported produce in local markets. Other cons iderations affecting repurchasing behavior include duration of post-purchase shelf-life and price due to the fact that two-thirds of the retail cost of produce is in curred in the postharvest ha ndling chain (Kader, 2002b). Experimental Analysis of Fruit Quality Objective scientific determination of indi vidual quality attributes comprise most data derived from postharvest studies. Quality determinations may be either destructive or non-destructive. Experimental considera tions require adequate sampling to predict “average quality” and also the “quality dist ribution” of the harvest or imported load (Abbot, 1999). Quality measurem ents derived from instrument s and laboratory analysis are preferred over subjective human evaluation to reduce variation and facilitate the ease of information exchange between researchers (Abbott, 1999). Fruit size, shape and presence/absen ce of peel disorders and biological contamination are evaluated visually while weight is quantified by physical measurement. Peel color development is direc tly related to internal fruit texture, firmness and starch:sugar ratios and is quantified using electroma gnetic radiation measurements. Banana peel color development is based on chlorophyll degradation and simultaneous carotenoid synthesis and exposure (Seymour , 1993). Peel color change has been incorporated into industry ripening charts a nd are used throughout the supply chains as the primary ripening reference (Figure 2-7). Less subjective experimental methods of quantifying color change require conversion of optical color r eadings to numeric values. Colorimeters define color using hue, chroma and value readings taken from an 8 mm aperture. In banana fruit, colorimeter accuracy diminishes as the fruit ripens due to the appearance of dark brown sene scent peel spots that may sk ew readings. Another color

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19 measurement utilizes Colorvision technology which measures similar values as the colorimeter but performs a composite color reading for the entire surface of the fruit while the fruit is in a light chamber (Yoruk et al., 2003). Peel Pigments Early research on banana peel pigments revealed decreases in chlorophyll from 50 to 100 ggram fresh weight-1 (ggfw-1) in green fruit to non-det ectable levels in ripe yellow fruit (Seymour, 1993; Gross and Fluge l, 1982). The two chlorophyll degrading pathways are known as the chlorophyllase and chlorophyll oxidase pathways. At temperatures above 24C chlorophyll degradatio n in Cavendish cultivars is decreased due to assumed inhibition of specific enzymes in these pathways (Janave and Sharma, 2004). Chlorophyll breakdown reveals underlying caro tenoid composition in the peel (Gross et al., 1976). Gross and Flugel (1982) listed more than 14 pigments contributing to total carotenoid composition. As fruit ripen there is a reduction in tota l carotenoids between stage 2 and stage 3 from 20.0 to 10.0 ggfw-1. As fruit continue to ripen from stage 4 onward, there is an increase in carotenoi d synthesis causing levels to peak at 19.0 ggfw-1 in full yellow stage 6 fruit (Gross and Flugel, 1982). Melanin pigments are also synthesized during peel se nescence and/or cellular ruptur e and will be discussed in the Fruit Browning section. Textural Properties Texture relates to mechanical properties and can be quantified by correlation with other ripening parameters such as fruit firmness or starch:sugar ratios. Fruit firmness is measured using instruments such as pene trometers or Instron Universal Testing Instruments that base their readings on co mpression forces required to deform the fruit

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20 over a specified distance and time (Jeong et al., 2003). These measurement devices can be used to test unpeeled whole fruit, peeled whole fruit or fr esh-cut fruit slices depending on the objectives of the research. Carbohydrates and Sugars in Banana Pulp Fruit soluble sugar levels are preferably measured using a colorimetric assay correlated to known standards (Dubois et al., 1956). These values are related to carbohydrate metabolism, starch:sugar ratio s, firmness, texture and flavor. A refractometric index as percent or degree Brix (Brix) can be used to quantify soluble solids content but may not accurately estimat e the sweetness of the fruit because other soluble compounds such as ascorbic acid, orga nic acids, amino acids, pectins, pigments and phenolic compounds can contribute to the readings (Kader et al., 2003). At physiological maturity, green banana fr uit are composed of approximately 80% starch by dry weight, equivalent to 25% st arch by fresh weight (Israeli et al., 1986; Rhodes, 1980). Soluble sugar c ontent in the fruit remains below 1% until ripening is initiated and starch-sugar conversions begin th at may lead to final values between 18 to 25% sugar content (Prabha and Bhagyalakshm i, 1998; Israeli et al ., 1986). The main sugar present in banana fruit pulp is sucr ose while the reducing sugars fructose and glucose also contribute to total soluble sugar content (Israe li et al., 1986). Aroma Volatiles and Fruit Flavor Aroma volatiles are produced during fr uit ripening or upon tissue maceration (Buttery, 1993). More than 200 different co mpounds have been identified in ripening banana fruit in the parts per billion to parts per million range. The majority of banana volatiles are composed of aromatics includi ng esters, alcohols, aldehydes, ketones, organic acids, hydrocarbons and carbonyl co mpounds (Nursten, 1970). Israeli et al.,

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21 (1986) noted that esters compose approxima tely 70% of total aromatic compounds in banana fruit. The “banana-like flavor” is due to the presence of pentyl esters, “fruitiness” due to butyl esters, and the astringent, “gre en woody flavor” of unripe fruit is due to pentyl and hexyl alcohols, aldehydes and ket ones. Volatile compounds are synthesized from precursor molecules including the unsatur ated fatty acids linolenic and linoleic acid, the amino acids leucine, isoleucine an d valine, (Rhodes, 1980), and ethanol and acetaldehyde (Hyodo et al., 1983). Peak producti on of volatiles occurs approximately 7 to 10 days after the climacteric pe ak depending on variety (Rhodes, 1980; Golding et al., 1998). Astringency and acidity also contribute to fruit flavor. Astringency is a result of soluble tannins in th e pulp tissue (Pesis, 2005) and d ecreases as fruit ripen through reduction of these compounds’ concentrati on by polymerization with acetaldehyde (Matuso and Ito, 1982; Esguerra et al., 1992). Banana pulp pH decreases from 5.4 to 4.5 due to the increase in organi c acids such as malate and citrate (Rhodes, 1980; Wyman and Palmer, 1964). Nutritive components are measured using chemical compound quantifications and are listed in detail at the USDA Agriculture Research Service (USDA ARS) Nutrient Data base Laboratory website (http://www.nal.usda.gov/ fnic/foodcomp/search/ last accessed January, 2007). Although subjective instrument/laboratory an alysis is important for determining individual quality components, there is no substitution for human perception of overall quality as this is the deciding factor in repurchasing decisions (Abbot, 1999). Fruit Browning: Polyphenol Oxidase and Phenolic Compounds Fruit browning occurs in fresh and proce ssed fruit and vegetable products and is considered detrimental to aes thetic and nutritional food quality . Causes of fruit browning

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22 include abrasion and hypersens itive responses during growth and development, exposure to extreme preand postharvest environmen tal conditions, postharvest mishandling and senescence of fruit tissue. The characteris tic brown color exhibited results from the accumulation of insoluble melanin pigments from enzymatic and non-enzymatic oxidation of phenolic compounds (Martinez et al., 1995; Yoruk et al., 2003). Polyphenol oxidase (PPO) is the enzyme responsible for catalyzing enzymatic browning in plants and fruits (Yoruk et al., 2003). Banana PPO (EC 1.10.3.2.) is located primarily in plastids and associated with th ylakoid membranes of intact plant tissue yet once senescence or cellular damaged occur th e enzyme may be found in the cytoplasm (Vaughn and Duke, 1984; Gooding et al., 2001). PPO isozymes are generally encoded by multigene families (Gooding et al., 2001) and vary within and between species of plants yet all perform the general function of oxidizing phenolic compounds (Yoruk et al., 2003). Phenolic compounds are aromatic rings w ith hydroxide and other functional groups and composition in fruits varies with species , cultivar, degree of ripening, environmental conditions of growth and storage conditions (Figure 2-8) (Yoruk et al., 2003). These compounds contribute to the colo r, astringency, bitterness and overall flavor in edible fruits and exhibit fungicida l properties in plan t cells during pathogen infection and hypersensitive responses. Phenolics such as dopamine also function as antioxidants therefore adding to the nutri tional value of the fruit (Kanaz awa and Sakakibara, 2000). Phenolics are synthesized in the cytoplasm but stored in plant cell vacuoles that physically separates them from the PPO and prevents their oxidation in intact tissue (Vamos-Vigyazo, 1981; Walker and Ferrar, 1998).

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23 The proposed browning pathway begins when phenylalanine is converted to various free phenolics by phenylalanine ammoni um lyase (PAL). Free phenolics may be monoor diphenols and can be oxidized by PPO to form quinones which spontaneously polymerize to form melanin pigments (Figure 2-9) (Pealver et al ., 2002; Marshall et al., 2000; Yoruk et al. 2003). The polymerized melanin pigments function as chemical bond barriers to prevent spread of infections and bruising in pl ant tissues and also exhibit antimicrobial properties. Postharvest experiments have begun to addr ess the prevention of senescent spotting (browning) in banana peel that begins as small brown dots (<0.5 mm) and in time become larger and eventually begin to coalesce in ex treme cases. Delay of senescent spotting is desirable from a marketing standpoint as consumer purchasing and consumption preference decrease by 33% and 52% resp ectively as spotting becomes apparent (Chiquita, 2000). Choehom et al. (2004) addre ssed inhibition of senescent spotting of banana fruit, cv. Sucrier, utilizing modified atmosphere packaging (MAP). The MAP consisted of covering fruit on trays with “Sun wrap” polyvi nyl chloride film and allowing fruit to ripen at 29 to 30C. Over the si x day experiment, carbon dioxide (CO2), ethylene and relative humidity levels increased while oxygen (O2) levels decreased compared to the control fruit. Addition of CO2 scrubbers or ethylene absorbents had no effect on spotting levels so it was conclu ded that the decreased in vivo browning observed was due to low oxygen. Further experiments utilizing CA treatments of 5, 10 and 15% O2 indicated normal development of sene scent spotti ng required O2 concentrations higher than 5%.

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24 The conclusion is consistent with reports that oxidizing enzymes requi re the presence of molecular oxygen to be catalytical ly active (Peal ver et al., 2002). Choehom et al., (2004) noted in vitro PPO activity increased over the six day period from 2 activity unitsmg protein-1 to over 20 activity unitsmg protein-1 in MAP fruit while maximum control fruit levels were 10 activity unitsmg protein-1. Total phenolics increased from 3.75 mggfw-1 to 4.5 mggfw-1 in control fruit and 6.0 mggfw-1 in MAP fruit. The inverse relationship between decreased in vivo spotting and increased in vitro activity was assumed to be due to “a feedback mechanism whereby more active protein is produced in the face of PPO inhibition” (Choehom et al., 2004). The in vitro activity was assumed to measure the poten tially active protein under adequate O2 conditions. These findings do not coincide with those of Gooding et al. (2001) who measured high levels of PPO activity in the peel of developing cv. Williams fruit, subsequent decreases in activity as fruit matured and constant activity levels during ripening. Chilling Injury Chilling injury (CI) is a disorder that o ccurs when banana fruit are stored at temperatures below 13C for durations of more than a few hours (Kerbel, 2004). Symptoms including peel and pulp discoloration, increased sensitivity to mechanical injury, and inability of fruit to ripen ad equately are manifested 18 to 24 hours after removal from injurious temperatures (Turner, 1997). The water soaking and browning of the peel is assumed to be caused by ruptur e of cellular component s, specifically the vacuole, and exposure of this compartment’s phenolic compounds to endogenous PPO. The exact mechanism of how low temperatur es affect fruits is currently being studied. Marangoni et al. (1996) has presented evidence of lo w temperature’s affect on

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25 cellular membranes while Matsui et al., (2003) suggested that lowtemperature induced free radicals contribute to cell death. In banana fruit stored at temperatures < 8C, increased electrolyte leakage from peel ti ssue was assumed to be caused by increased membrane permeability (Jiang et al., 2004). The study also utilized a 14C-ethylene release assay (Sisler et al., 1979; Hall et al., 1999) which showed decreased ethylene binding in banana fruit during low temperatur e storage. Jiang et al., (2004) therefore assume that the symptoms of inadequate fr uit ripening observed afte r exposure to chilling temperatures are associated with the d ecreased ethylene perception by membrane-bound receptors disrupted by the low temperature. Receptor protein disruption by the low temperature was also noted by Macnish et al., (2000) where 1-methylcyclopropene, an inhibitor of ethylene acti on, was applied at 10 nLL-1 (12 h, 20C) and binding to the ethylene receptors was not achieved at temper atures of 2C compared to the control at 20C. Nguyen et al. (2004) measured PPO activity in relation to banana fruit (cv. Sucrier) held in MAP at chilling injury (CI) temperatur es. Fruit were placed in polyethylene bags (30 days, 10C) with ethy lene absorbers and a CO2 scrubber resulting in the MA concentrations equilibrating at 0.4 LL-1 ethylene, 12% O2 and 4% CO2 after the first 7 days of storage. Fruit stored in MAP exhibited no CI symptoms before day 18 while control fruit began exhibiting CI symptoms by day 6. MAP fruit exhibited less peel discoloration, decreased loss of peel total free phenolics and lower PPO activity levels compared with control fruit not held in MA P. It was proposed that the phenolic compounds may be used as substrates in brow ning reactions during chilling injury thus the decrease in total phenolics was due to free phenolic “turnover by PPO and other

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26 enzymes [being] more rapid than synthesi s” (Nguyen et al., 2003). The authors concluded that MAP in combination with ethylene absorbers and a CO2 scrubber may be utilized to reduce chillin g-induced peel browning. Postharvest Pathology of Banana Fruit The detection and control of postharvest pa thogens and diseases is of importance due to the economic costs of lost produce shipments and for food safety concerns. Between 10 to 30% of all harvested crops are lost to pathogens during postharvest handling and storage (Sholberg et al., 2004). Fungal and bacteria plant pathogens cause the majority of postharve st disease problems. Anthracnose is one of the most common dis eases occurring in preand postharvest fruit. In banana this disease is caused by Colletotrichum musae and appears as salmon colored infections as the pathogen proliferates . Evidence of latent preharvest infections is seen on green banana as small dark marks sometimes mistaken as wounding or senescent spotting. These specs are localized hypersensitive respons es from infections occurring during development. Stem-end rot is caused by Lasiodiplodia theobromae and/or Thielaviopsis paradoxa , both of which may infect the fruit through wounds at the cut stem or hand. Symptoms include brown, soft flesh that may become water-soaked. Cigar-end rot caused by Verticillium theobromae and/or Trachysphaera fructigena occurs but is not of much concern in the U.S. as many of the infected fruit never reach export markets (Kader, 2002b; Kerbel, 2004). Crown rot is a generic name for disease caused by a composite of fungal pathogens where one or more of the following may be present: Colletotrichum musae, Thielaviopsis paradoxa, Fusarium roseum, Fusarium semitectum, Fusarium pallidoroseum,

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27 Botryodiplodia theobroame, Ceratocystis paradoxa, Curvularia sp Verticillium sp,. Acremonium sp., Lasiodiplodia theobromae, and Deightoniella torulosa (Kader, 2002b; Kerbel, 2004). Crown rot usually occurs on cut surfaces of the fruit that include the proximal “crown” area and on wounded tissue. As the infection progresses the neck and fruit itself may become colonized. Most ba cterial infections are caused by the pathogen Erwinia carotovora and result in characteristic soft rot symptoms. On-farm sanitation practices diminish posth arvest losses to pa thogen proliferation due to the decreased amount of inoculum pres ent. Packinghouse sanitation practices such as ensuring appropriate water temperature, sa nitizer levels, and fungi cide dips for ample time further reduce losses. Proper handling proc edures designed to minimize mechanical damage, bruising and wounding allow the fruit to maintain its structural integrity and decrease susceptible surface areas. Adequate cooling after processing and maintenance of 14 to 15 C temperatures during shipping and storage decreases the chances for spore germination (Kerbel, 2004). The objective of this study is to analyze the effects of ethylene suppression via 1-MCP treatment on the whole fruit ripening pa rameters of color development, senescent spotting development, fungal proliferation, fr uit firmness (whole and pulp), total soluble sugars and browning as related to the enzy me-substrate interaction of PPO and total phenolics. Previous studies have shown 1-MCP tr eatment (various concentrations and durations) to unripe, preclimacteric banana fruit reta rds color development where the onset of peel color change was delayed by 23 days in 1-MCP treated fruit and nonuniform yellowing of peel was also noted (G olding et al., 1998, Jia ng et al., 1999). Other

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28 studies involving 1-MCP application (vario us concentrations and durations) to climacteric banana fruit at stage 3 or 4 within 36 to 48 h afte r commercial ethylene treatment yielded inconsistent ripening resu lts where some fruit progressed normally through their color development while others did not (Pelayo et al ., 2003). The purpose of this study was to analyze the effects of ethylene suppression on commercially gassed fruit color development while monitoring th e development of banana peel senescent spotting. Banana peel senescent spotting has been reported to be inhibited by modified atmosphere packaging (Choehom et al., 2004) ye t no studies have explored the effects of ethylene suppression on this ripening parameter.. Previous studies done by Jiang et al., ( 1999) have shown 1-MCP treatment (various concentrations and durations) retards whole fruit softening whilestudies done by VilasBoas and Kader (2006) have shown similar result s with fresh-cut banana fruit. Softening of 1-MCP-treated (1 LL-1, 12 h) fruit accelerates with storage in increasing O2 concentrations (21 to 100%) as compared to 1-MCP treated fruit held in air (Jiang et al., 2001) while softening of 1-MCP-treated fruit (various concentrations, 12 h) decreases when held in temperatures above 30C for 7 d. This study explored the softening of 1-MCP-treated fruit as it’s affected by low te mperature (5C) storage. Other studies have explored the effects of low temperature stor age (6 and 10C) on br owning of Kluai Khai and Kluai Hom Thong banana varieties by an alyzing PPO activity and total phenolics content (Nguyen et al. 2003). Yet no study has been undertaken involving 1-MCP treatment and low temperature storage effects on PPO activity and total phenolics content of the Williams banana variety. Similarly, no study has addressed 1-MCP treatment and low temperature storage effects on total soluble sugars. Ther efore this study has explored

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29 these untouched issues and in order to contribute to th e established body of knowledge pertaining to the physiological mechanisms of banana fruit ripening as affected by ethylene suppression via 1-MCP.application. Ripening parameter measurements were determined by observing and analyzing sample s for changes over time. The conclusions determined if 1-MCP application offered any benefits for delaying ripening and extending the shelf-life of commer cially handled banana fruit.

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30 Figure 2-1. Banana pseudostem made up of pe tioles/leaf sheaths with young plants in background. Photo taken by Daniel A. St anley on location at the University of the Virgin Islands Agriculture E xperiment Station, St. Croix, U.SV.I.

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31 A B C Figure 2-2. Banana frui t single sigmoidal growth curve. A) Initial stage of active cell division and slow growth. B) Stage of increasing fruit size and weight associated with cell expansion. C) Stag e of declining fruit growth rate and initiation of ripening. [Photos reprin ted with permission from Maguire, I. 2004. Tropical fruit photogrtaphy, http://tfphotos.ifas.ufl.edu/ , last accessed November, 2006. U.F. TREC/IFAS, Homestead, FL.] Figure 2-3. Respiratory or ethylene pattern of climacter ic vs. non-climacteric fruit during ripening. [Adapted from Saltveit, M. 2004b. Respiratory Metabolism (Page 4, Figure 1). In: USDA ARS Agricu lture Handbook Number 66: The Commercial Storage of Fruits, Vegetabl es, and Florist and Nursery Stocks, http://www.ba.ars.usda .gov/hb66/contents.html , last accessed December 2006. USDA ARS, Washington D.C..] Time Growth C B A Pre-climacteric minimum Climacteric rise Post-climacteric phase Climacteric peak Non-climacteric pattern Carbon Dioxide or Ethylene Production Time

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32 Figure 2-4. The ethylene molecule’s chemical structure. 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID CARBON DIOXIDE S-ADENOSYL MATHIONINE HCN ETHYLENE 5’-METHYLTHIORIBOSE-1-P METHIONINE 5’-METHYLTHIORIBOSE 5’-METHYLTHIOADENOSINE 2-KETO-4-METHYTHIOBUTYRATE 2 1 4 3 6 5 7 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID CARBON DIOXIDE S-ADENOSYL MATHIONINE HCN ETHYLENE 5’-METHYLTHIORIBOSE-1-P METHIONINE 5’-METHYLTHIORIBOSE 5’-METHYLTHIOADENOSINE 2-KETO-4-METHYTHIOBUTYRATE 2 1 4 3 6 5 7 Figure 2-5. Ethylene biosynthesis (step 1 to 3) and the Yang Cycle (step 1 to 2, 4 to 7) [Adapted from Seymour, G.B. 1993. Bana na. In: Seymour G.B., Taylor J.E., Tucker G.A. (Eds.), Biochemistry of Fruit Ripening, (Page 83, Figure 9) Chapman and Hall, London, U.K.] Figure 2-6. The 1-methylcyclopropene molecule’s chemical structure.

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33 Figure 2-7. Commercial ripe ning chart correlating peel color with numerical scores (stages) 2 through 7. [Reprinted with permission from Wanner, J. 2000. Chiquita ripening char t. Chiquita, N.A.] A B C Figure 2-8. Various phenolic substrates of PPO in banana fruit. A) Dopamine. B) Leucodelphinidin. C) Leuc ocyanidin. [Reprinted with permission from Marshall et al. 2000. Enzymatic Browning in Fruits, Vegetables and Seafoods (Figure 13), http://www.fao.org/ag/ags/agsi/EN ZYMEFINAL/Enzymatic%20Browning.ht ml , last accessed December, 2006. FAO, Rome, Italy.]

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34 Figure 2-9. Proposed fruit browning pathway. [Schematic adapted from, and chemical st ructures reprinted with permission from Marshall et al. 2000. Enzymatic Browning in Fr uits, Vegetables and Seafoods (Figure 13), http://www.fao.org/ag/ags/agsi/ENZY MEFINAL/Enzymatic%20Browning.html , last accessed December, 2006. FAO, Rome, Italy.]Phenylalanine Free Phenolics Phen y lalanine ammonium l y ase Phenolic compounds Quinones Pol yp henol oxidase O2Melanin Pigments Quinones S p ontaneousl y p ol y merize

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35 CHAPTER 3 EFFECTS OF COMMERCIAL-SCALE AP PLICATION OF 1-MCP ON BANANA RIPENING: SUSCEPTIBILITY TO CHI LLING INJURY AND SUITABILITY FOR FRESH-CUT PROCESSING Introduction Banana fruit exhibit more desirable qualit y characteristics when allowed to mature fully on the plant (Kader, 2002a), yet as ma turation proceeds postharvest handling and transportation ability decreas e (Kerbel, 2004). The time a nd distance disparity between harvest in Central America and consumption in the U.S. and E.U. has prompted the banana industry to utilize the artificially sy nthesized plant growth regulator, ethylene, to induce ripening upon demand. The process of ripening fruits using exogenous ethylene allows distributors to harvest unripe fruits (g reen), transport them to consumer markets and present fruit to the consumer ripe a nd ready for consumption (Kerbel, 2004). Recent developments in packaging scien ce and food safety have allowed banana fruit (among others) to be marketed as freshcut, usually in a fruit salad arrangement or along with deserts served in restaurants, ice cream parlors etc. Fresh-cut fruit usually require light processing (peeli ng, slicing) which may cause tissue damage and lead to accelerated loss of fruit quality (Vilas-B oas and Kader, 2006). For this reason, immediate low temperature storage is utilized to decrease the fruit’s metabolism and slow the decay process (Jiang et al., 2004). Low temperature storage is also necessary for prevention of detrimental pathogenic proliferation. Current research efforts are focused on us ing ethylene analogs to prevent ethylene perception and delay fruit ripening (Jiang et al., 1999) and 1-methylcyclopropene

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36 (1-MCP) is one such ethylene action inhibi tor that has become widely used. The objectives of this study were to evaluate the effects of commercial scale application of 1-MCP on banana ripening characteristics and suitability as a fresh-cut product. Materials and Methods Plant Material, Ethylene and 1-MCP Treatments Banana fruit used in this experiment orig inated from Chiquita banana plantations in Honduras. All postharvest handling and treatm ents were arranged and/or supervised by Dr. Tony Beltran of AgroFresh Inc. before fr uit arrived at the Un iversity of Florida Postharvest Horticulture Laboratory in Gainesville, Florida. Commercially packed cartons were ethylene treated duri ng sea shipment to Miami (see The Appendix, Commercial Handling for de tails on packaging). Upon arrival in Miami, fruit in one marine container were treated with 300 nLL-1 1-MCP (12 h, 18C) and labeled according to their respective positions in the c ontainer (Table 3-1). Fruit without 1-MCP treatment from another marine container were held for the same duration (12 h, 18C) to serve as cont rols. A simultaneous treatment involved 1-MCP gassing of two randomly selected cartons of fruit at 300 and 900 nLL-1 1-MCP (12 h, 18C) in sealed, 117.3 L plastic container (Rubbermaid Inc., Fairlawn, Ohio) while other fruit were sealed in identical plastic container and left untreated (12 h, 18C) to serve as controls (Table 3-1). After treatment, fru it from the plastic containers treatment were repacked in their original cartons for transport to Gainesville. Six control cartons, six 1-MCP cartons and three cartons from th e plastic container treatment were then shipped by refrigerated truck (15C) from Miami to the Postharvest Horticulture Laboratory at the University of Florida in Gainesville. The six control cartons (labeled U1-U6 fo r “Untreated” number 1 thr ough 6) represented control

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37 replicates while the six 1-MCP cartons (l abeled T-) represented 1-MCP treatment replicates (Table 3-1). In the plastic c ontainer treatment, PU, P300 and P900 represent the control and the two 1-MCP trea tment concentrations of 300 nLL-1 and 900 nLL-1, respectively. Upon arrival in Gainesville (Day 0), ther e was no apparent spatial separation of treatment cartons within the ship ping truck. Eight hands were selected from each carton (four from the top la yer and four from the bottom layer), labeled according to their respective cartons (Table 3-1) and placed on trays previously sanitized with 200 ppm commercial bleach. Fruit were allowe d to ripen (18C, 80 to 90% RH) over a 15-d period. Fruit Ripening Parameters Sixty hands of bananas were examined and digitally photographed daily on black velvet cloth under food-grade evaluation lights simulating th e full visible spectrum. These selected, nondestructive quality parameters listed in this sec tion were determined daily (except fruit firmness that was determ ined every other day) over a 15-d period. After data were recorded, banana fruit we re returned to the ripening facilities. Peel Color : Individual hands were visually rated on a scale of 2 to 7 based on industry color score charts (see th e Appendix, Figure A-17 for ripening chart). Half values (i.e. 3.5) we re utilized for greater accuracy. Incidence of Senescent Spotting : The area of senescent spotting (browning) was reported as a percent of total visible surface area of the middle fruit of each hand as seen by an observer looking down on the fruit (see Results and Discussion for detailed images). Incidence of Graying : Determination of presence or absence of graying on any finger of a hand representing the entire hand (see Results and Discussion for detailed images). Th e number of hands exhibiting graying was reported as a percentage of the total hand count for each treatment.

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38 Incidence of Fungal Infection : Determination of presence or absence of fungal mycelia on the stem-end surfa ce of a hand. The number of hands exhibiting fungal mycelia was reported as a percentage of total hand count for each treatment. Fruit Firmness : Determined on four individual fruit per treatment using an Instron Universal Testing Instrument (Model 4411, Instron Instruments Inc. Norwood, MA). A 50-kg load cell fitted with a 50-mm flat, stainless steel probe was positioned at zero-force contact on the equatorial region of each banana fruit positioned horizontally on its side on a solid platform. Fruit was compressed to a depth of 6 mm at a rate of 10 mmmin-1. The maximum load encountered over the dist ance of probe travel was recorded and expressed in Newtons (N). Frui t were discarded after destructive firmness tests. Storage Under Conditions that Induce Chilling Injury On Day 3, twelve hands from the control and twelve hands from the 1-MCP-treated groups were subjected to chilli ng injury (CI) temperatures (Tab le 3-2). After a period of 24 h, six hands from the control and six hands from the 1-MCP treated group were transferred to 20C (Day 4). The remaini ng six control and six 1-MCP-treated hands were kept at 5C for an additional 24 h (48 h total) then transferred to 20C on Day 5. Digital photographs were taken and color score data were recorded on these fruit for the duration of the study. On Day 10 and Day 15 firmness tests were c onducted on replicates from each treatment group. Fresh-Cut Banana Fruit and Chilling Injury On Day 5, three individual banana fruit from each CI treatment group were removed from the hand using a laboratory kn ife, labeled and prepared as fresh-cut samples (peeled and sliced longitudinally from proximal to distal end). Halves were laid on plastic film-covered trays with one half expos ing the interior surfac e and the other half exposing the exterior surface of the fruit. After being digitally photographed, fruit were covered with clear plastic a nd transferred to 5C for the duration of the study.

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39 On Days 10 and 12 firmness tests were c onducted on three banana halves (stored with cut surface down) from each treatment gr oup as described above, with the following modifications: a 5-kg load cell, 10 mm per minute travel rate, 10-mm diameter convex probe, and 3-mm probe depth, measured at the fruit apex on the equator. Statistical Analysis Data were analyzed using the Statis tical Analysis Software version 9.1.3 (SAS Institute Inc. Cary, NC) “Proc Mixed” procedure. In applicable cases a Tukey adjustment was utilized for the Day x Treatment interaction to adjust the experiment-wise error rate for multiple compar isons within a treatment group. Results and Discussion Peel Color Upon arrival at the Postharvest Horticulture Laboratory at the University of Florida in Gainesville, control fruit color score of 4.0 was significantly different (P < 0.05) from the 1-MCP fruit color score of 3.0 (Figure 3-1). The difference in peel color indicates the fruit had been 1-MCP treated at least 24 h pr ior to fruit arrival, thus allowing the nontreated control fruit to ripen normally. Color score 6 (the ideal marketing color) was attained by control fruit on Day 3 while 1-MC P fruit attained score 6 on Day 8 indicating a delay of ripening between control and 1-MCP fruit of 5 days. Total time required to reach score 7 for control and 1-MCP fruit was 10 and 13 days, respectively. The delay in ripening along with the increased time required to reach score 7 indicate that the 1-MCP application had a marked effect on peel color development. All fruit monitored in the plastic container treatment arrived at U.F. at higher color scores than fruit in the marine container treatme nt. This may be due to the fact that fruit selected for the plastic containe r treatment were initially riper or the modified atmosphere

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40 conditions created in the plastic containers allowed ethylene build-up which may have induced earlier ripening in both untreated and treated fruit. Fruit peel color score in the plastic contai ner treatment exhibited similar patterns of color change with the untreated control fruit from the plastic container treatment (PU) beginning slightly above 4.0 while the 300 nLL-1 and 900 nLL-1 1-MCP treated fruit from the plastic container treatment (P300 a nd P900, respectively) be gan slightly above 3.5 (Figure 3-2). Between the P300 and P900 groups, the P900 peel wa s slightly greener upon arrival and throughout the experiment indicating the three times higher 1-MCP concentration did affect fruit color change ye t values never became significantly different (P < 0.05). It was therefore concluded that th e lower concentration was more effective. Between Day 1 and Day 2 fruit color change increased rapidly in all groups due to the increase in storage temper ature to 18C. For the durat ion of the study fruit followed typical ripening patterns with PU fruit arriving at score 6 by Day 3. P300 and P900 fruit arrived at score 6 by Day 4 and Day 5, resp ectively. Although delays between PU and P300/P900 peel color began on Day 0 it was between Day 6 and Day 11 that PU peel color became significantly different (P < 0.05) from both P300 and P900. The effect lasted until Day 11 where peel color sc ore values were no longer significantly different (P < 0.05). Comparison of time required to reach score 6 between fruit from the marine container and plastic container treatment reveal ed that the plastic containers caused more rapid ripening than the marine containers. Th e increased rate of ripening may be due to ethylene buildup in the contai ners during fruit gassing as it is known that ethylene initiates fruit ripening (Saltviet, 1999). This combined with the co st of transferring and

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41 treating fruit in small containers renders the plastic containers not viable for large scale commercial applications. Peel Graying Fruit peel graying is a diso rder of unknown origin and developed at various times during ripening in fruit from both marine container and plas tic container treatments. Graying first appeared near the neck a nd/or distal ends of individual fingers (Figure 3-3A) and in certain cases spread towards the center over time. Graying where noted was not always present on all fingers of an affected hand. The incidence of graying was greatest in 1-MCP-treated fruit from the marine containers and the degree of graying developed on the entire fruit surface rather than being limited to the neck and/or distal ends . Graying appeared da rker in certain areas than others on fruit with large portions of the surface affected (Figure 3-3B). Senescent Spotting Senescent spotting is an in evitable consequence of ba nana fruit ripening and was observed in all fruit monitored in from th e marine container and plastic container treatments (Figure 3-4). Qualitatively, the spot ting in control fruit appeared as individual spots that enlarged over time while 1-MCP fr uit spots developed more like lesions rather than classic senes cent spotting due to their darker pigmentation, oblong shape and localized occurrence (Figure 3-5). Removal and plating of these spots on PDA did not yield fungal colonies yet a full scale study was not undertaken. Control and 1-MCP fruit in the marine c ontainer treatment began to develop equal amounts of spotting between Day 3 and Day 6. By Day 7 control fruit spotting began developing more rapidly than 1-MCP-treate d fruit (Figure 3-6) and by Day 8 values

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42 became significantly higher (P < 0.05). By Day 14 control fruit exhibited 30% more spotting than 1-MCP-treated fruit. The spotting mechanism may be relate d to ethylene perception which, when blocked by 1-MCP, causes altered appearance of the spots (Figure 3-5). The delay of senescent spotting development is a desirable ch aracteristic of treated fruit that may be of commercial importance due to the fact that ma rketers routinely remove fruit from retail display when spotting becomes apparent. The plastic container treatment did not affect the onset of senescent spotting (compared to the marine container treatment) as spotting occurred as early as Day 3 in all fruit (Figure 3-5, Figure 3-6). Spotting of untreated fruit from the plastic container treatment diverged and remained significan tly different (P < 0.05) from the 300 nLL-1 and 900 nLL-1 treatment on Day 7 and continued to develop until spotting reached levels greater than 60%, a value similar to that of control fruit in the marine container treatment (Figure 3-5, Figure 3-6). Leve ls of spotting in 300 nLL-1 and 900 nLL-1 treatments fluctuated between 10% and 20% and were not significantly differe nt (P < 0.05) by the end of the experiment. The results reveal that treating in plastic c ontainers does not offer any advantage in decreasing senescent spot ting nor does increasing 1-MCP concentration above 300 nLL-1. Fluctuations such as those observed in the 900 nLL-1 treatment on Day 9 and Day 10 are attributed to overestima tions and the difficulty of asse ssing percentage surface area by visual means. Senescent spotting begins as minute brown specs increasing in diameter over time until small spots eventually coales ce. Aggregating the total surface area of these spots as compared to the visible surface area of each fruit (or hand) permits a wide

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43 margin of error when performing subjective visual analysis because two observers may view identical specimens and measure different percentage values. For this reason, this method of data collectio n may not be readily transferable to other experiments or easily reproduced by other experimenters and wa s not used in further experiments. Incidence of Fungal Infection Fungal infections of control and 1-MCP groups in the marine container treatment appeared on Day 3 and continued to spread at approximately equal rates over the duration of the experiment (Figure 3-7). Infections us ually began on the stem end or distal tip of the fruit and were limited to these regions in intact fruit (Figure 38). In some cases, bruised or damaged fruit became infected, which over time then spread to healthy tissue. By Day 15 over 90 fruit had decay. The degree of infection varied among fruit with the earliest infected exhibiting the most mycelia and the latest exhibiting the least. The severity of infection per hand was not quantif ied as this was outside the scope of this study. Similar results were observed in the plas tic container treatment where the control, 300 nLL-1 and 900 nLL-1 fruit exhibited infections by Day 4 and progressed to greater than 95% fruit infected by Day 15 (Figure 3-9). The results indicate that neither 1-MCP nor plastic containers had any effect on decreasing th e occurrence of pathogens. Whole Fruit Firmness Fruit firmness in control and 1-MCP-trea ted fruit from the marine container treatment were initially near 75 newtons (N). The two treatment groups began to diverge on Day 5 and by Day 7 values were significantly different (P < 0.05) (Figure 3-10). The control group continued soften ing at a rate of approximate ly 7 to 8 N per day while 1-MCP firmness remained in the 55 to 65 N range until Day 10 when softening resumed

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44 and concluded at 40 N at Day 15. Final cont rol group firmness levels were 23 N, nearly 20 N softer than the 1-MCP group. These resu lts show 1-MCP had desirable effects in maintaining higher firmness levels of whole fruit. Fruit in the plastic container treatment we re initially approximately 5 N lower in firmness than fruit in the marine containe r treatment (Figure 3-10, Figure 3-11). These data correspond to the color score data wher e fruit from the plastic container treatment were slightly more advanced in color score development (riper) upon arrival than were fruit from the marine container treatment (F igure 3-1, Figure 3-2). Firmness levels of control and 300 nLL-1 fruit were initially below 70 N while firmness of 900 nLL-1 fruit began below 65 N yet these values were not si gnificantly different (P < 0.05). By Day 9 control levels dropped to 40 N and became significantly different (P < 0.05) from the 300 nLL-1and 900 nLL-1 which remained above 50 N. Control firmness levels continued to be significantly differe nt (P < 0.05) from 300 nLL-1 and 900 nLL-1 fruit for the duration of the experiment with final values of 20 N, virtually equivalent to that of the controls of the marine contai ner treatment. Final 300 nLL-1 and P900 nLL-1 fruit firmness values remained between 35 and 50 N, values comparable to those of the 1-MCP-treated fruit in the mari ne container treatment. 900 nLL-1 fruit maintained slightly higher firmness levels than the 300 nLL-1 fruit yet the levels were not significantly different (P < 0.05). The results indicate treating fruit in plastic containers offers no added benefit in retention of firmness of whole fruit. Incidence of Chilling Injury Storage of banana fruit at temperatures below 13C induces chilling injury (CI) which is detrimental to peel appearance yet beneficial for delaying ripening in certain commercial circumstances. Peel chilling in jury symptoms were manifested on fruit

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45 stored for both 24 and 48 h dur ations at 5C, therefore 24 h at 5C is sufficient to induce CI symptoms (Figure 3-12). After 8 Days at 20C, CI symptoms appeared different between control and 1-MCP-treated fruit. Control fruit peel de veloped a grayish tint along with blotchy brown areas. 1-MCP fru it also developed the grayish tint yet the browning areas appeared in random locat ions and in more angular patterns. Peel color development of control an d 1-MCP-treated fruit groups remained significantly different (P < 0.05) before, duri ng and after CI until Day 12. There was no significant difference (P < 0.05) in peel color development within control fruit stored at 5C for 24 or 48 h (Figure 3-13). Within the 1-MCP-treated fruit, peel color development exhibited a similar pattern where CI storage duration did not significantly (P < 0.05) alter color development (Figure 3-13). Subjective analysis revealed control fru it emitted more characteristic banana odors than 1-MCP-treated fruit upon peeling after re moval from CI temperatures on Day 5 and Day 6. The suppressed volatile evolution of treated fruit may be due to the delayed ripening stage of the fruit or due to seconda ry effects of 1-MCP on volatile production as Golding et al., (1998) reporte d fruit treated with 45 LL-1 1-MCP (a concentration over ten times higher than used in this experi ment) commenced volatile production nearly 30 days after control fruit with peak production levels at 75% of the control fruit levels. Firmness values of control and 300 nLL-1 1-MCP-treated fruit stored at low temperatures (24 h, 5C) were not significantly different (P < 0.05) than the control or 1-MCP fruit, respectively, on Day 10. Fruit from control and 300 nLL-1 1-MCP treatments stored at low temperatures (48 h, 5C) were significantly firmer (P < 0.05) than those of the control, control (24 h, 5C), 300 nLL-1 1-MCP-treated fruit and

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46 300 nLL-1 1-MCP-treated fruit (24 h, 5C) gr oups on Day 10 (Table 3-3). Firmness retention in the 48 h chilled groups is likely due to a delay in ripeni ng not apparent in the peel color development data as it is known th at low temperature storage slows ripening (Turner, 1997). By Day 15 comparisons betw een the control, cont rol (24 h, 5C) and control (48 h, 5C) groups re vealed firmness values were not significantly different (P < 0.05) as did the same comparison between the 300 nLL-1 1-MCP-treated fruit, 300 nLL-1 1-MCP-treated fruit (24 h, 5C) and 300 nLL-1 1-MCP-treated fruit (48 h, 5C) groups. Fresh-Cut Banana Fruit and Chilling Injury Fresh cut banana fruit exhibit different storage characterist ics and requirements than whole fruit due to the damage incurred during processing (peel ing and slicing) and the pulp’s subsequent direct exposure to oxyge n. Obvious detriments to fresh cut fruit quality include loss of firmness and browning which were exhibited in pulp of both C24 and C48 fruit (Figure 3-14). M24 and M48 fruit pulp maintained a whiter appearance, remained approximately 1 to 2 N firmer and exhibited less signs of browning and water soaking than identically chilled control fr uit (Table 3-4, Figure 3-14). The results indicate that applying 1-MCP prior to subj ecting fruit to CI temperatures may offer beneficial uses for fresh-cut industries. Conclusions The results of the marine container treat ment lead to the conclusion that 1-MCP application had significant e ffects on delaying fruit ripening. However, the position of the cartons in the vessel while undergoing 1-MC P treatment did not affect fruit exposure due to the highly diffusive of the applications in the gaseous state. The results of the plastic container treatment indi cate that treating fruit in sma ll plastic containers did not

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47 offer added benefit for maintenance of fruit qua lity as compared to the marine container treatment. The results also indicated that when treating fruit in plastic containers, 900 nLL-1 1-MCP offered no added benefit over treatment at 300 nLL-1 1-MCP. These results combined with the cost of transferring and treating fruit in small containers rendered the plastic containers not viable fo r large scale commercial applications.

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48 Table 3-1. Banana treatment with 1-MCP under commercial and controlled conditions. Treatment labels Treatment and location in marine container Marine container U1 Control, ethylene treated, no 1-MCP U2 Control, ethylene treated, no 1-MCP U3 Control, ethylene treated, no 1-MCP U4 Control, ethylene treated, no 1-MCP U5 Control, ethylene treated, no 1-MCP U6 Control, ethylene treated, no 1-MCP TLF 300 nLL-1 1-MCP, left front of marine container TRF 300 nLL-1 1-MCP, right front of marine container TRM 300 nLL-1 1-MCP, right middle of marine container TLM 300 nLL-1 1-MCP, left middle of marine container TRB 300 nLL-1 1-MCP, right back (door ) of marine container TLB 300 nLL-1 1-MCP, left back (door) of marine container Plastic container PU Plastic container, ethylene treated, no 1-MCP P300 Plastic container, 300 nLL-1 1-MCP P900 Plastic container, 900 nLL-1 1-MCP Table 3-2. Storage regimes for banana hands destined for fresh-cu t processing (control and 1-MCP treatments) beginning Day 3. Treatment Day 3 Day 4 Day 5 Treatment label 6 hands to 20C at 20C C24 Control 12 hands to 5C 6 hands remain at 5C to 20C C48 6 hands to 20C at 20C M24 1-MCP 12 hands to 5C 6 hands remain at 5C to 20C M48

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49 Table 3-3. Whole fruit firmness values (Newtons) of Control and 1-MCP-treated (300 nLL-1) fruit on Day 3 (20C) and followi ng transfer to chilling injury temperatures (5C). Treatment Day 3 Day 10 Day 15 Control 63.27 2.17 34.96 2.16 22.97 1.20 1-MCP 66.46 5.95 51.19 5.32 41.19 4.34 C24 36.75 4.84 24.75 .04 M24 53.55 12.71 42. 25 7.88 C48 47.76 4.43 21.74 6.77 M48 58.43 8.18 41.58 1.29 Table 3-4. Fresh-cut fruit firmness values (Newtons) of Control and 1-MCP-treated (300 nLL-1) fruit after 5C CI temperatures. Treatment Day 10 Day 12 C24 4.75 0.21 4.49 0.42 M24 6.32 0.72 5.72 0.56 C48 4.59 1.31 4.31 0.19 M48 5.99 0.24 5.30 0.34

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50 2 3 4 5 6 7 02468101214 Days in Storage (18C, 80 to 90% RH)Color Score Commercial Ripening Chart Control 300 nL/L 1-MCP Figure 3-1. Peel color of Control and 1-MCP-treated fruit (300 nLL-1) during storage (18C, 80 to 90% RH). (n=48). Vert ical bars represent standard error.

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51 2 3 4 5 6 7 02468101214 Days in Storage (18C, 80 to 90% RH)Color Score Commercial Ripening Chart PU P300 P900 Figure 3-2. Peel color of PU, P300 and P900 fr uit during storage (18C, 80 to 90% RH). (n=8). Vertical bars represent standard error.

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52 A B C Figure 3-3. Graying of banana fr uit on Day 8. A) Graying in c ontrol fruit. B) Graying in 1-MCP-treated fruit. C) Graying in P300 fruit. A B C Figure 3-4. Peel senescent spotting. A) Init iation of spotting in control fruit Day 3. B) Advanced spotting in control fruit Day14. C) Advanced spotting in 1-MCP-treated fruit Day 14. A B Figure 3-5. Close-up of peel senescent spotting to reveal different spotting characteristics. A) Control fruit Da y 14. B) 1-MCP-treated fruit Day 14.

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53 0 10 20 30 40 50 60 02468101214 Days in Storage (18C, 80 to 90% RH)Senescent Spotting Surface Area Affected (%) Control 300 nL/L 1-MCP Figure 3-5. Peel senescent spotting in Control and 1-MC P-treated fruit (300 nLL-1) during storage (18C, 80 to 90% RH). Each point represents the percent of total visible surface exhibiting spotti ng per hand (n=48). Vertical bars represent standard error.

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54 0 10 20 30 40 50 60 70 02468101214 Days in Storage (18C, 80 to 90% RH)Senescent Spotting Surface Area Affected (%) PU P300 P900 Figure 3-6. Peel senescent spotting in PU, P300 and P900 fruit during storage (18C, 80 to 90% RH). Each point represents the percent of total visible surface exhibiting spotting per hand (n=8). Vertic al bars represent standard error.

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55 0 10 20 30 40 50 60 70 80 90 100 02468101214 Days in Storage (18C, 80 to 90% RH)Fruit Exhibiting Fungal Infection (%) Control 300 nL/L 1-MCP Figure 3-7. Incidence of funga l infection in Control and 1-MCP-treated fruit (300 nLL-1) during storage (18C, 80 to 90% RH). E ach point represents the percentage of the total hands exhibi ting mycelia (n=48). A B Figure 3-8. Close-up of fungal infection on stem ends of Control and 1-MCP-treated fruit. A) Control fruit Day 14. B) 1-MCP fruit Day 14.

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56 0 10 20 30 40 50 60 70 80 90 100 02468101214 Days in Storage (18C, 80 to 90% RH)Fruit Exhibiting Fungal Infection (%) PU P300 P900 Figure 3-9. Incidence of fungal infection in PU, P300 and P900 fruit during storage (18C, 80 to 90% RH). E ach point represents the pe rcent of hands exhibiting mycelia (n=8).

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57 0 10 20 30 40 50 60 70 80 02468101214 Days in Storage (18C, 80 to 90% RH)Firmness @ Max Load (Newtons) Control 300 nL/L 1-MCP Figure 3-10. Whole fruit firmness of C ontrol and 1-MCP-treated fruit (300 nLL-1) during storage (18C, 80 to 90% RH). (n=4 ). Vertical bars represent standard error.

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58 10 20 30 40 50 60 70 80 02468101214 Days in Storage (18C, 80 to 90% RH)Firmness @ Max Load (Newtons) PU P300 P900 Figure 3-11. Whole fruit firmness of PU, P300 and P900 fruit during storage (18C, 80 to 90% RH). (n=4). Vertical bars represent standard error. A B C D Figure 3-12. Chilling injury symptoms in banana fruit. A) C24 fruit Day 13. B) C48 fruit Day 14. C) M24 fruit Day 13. D) M48 fruit Day 14.

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59 2 3 4 5 6 7 02468101214 Days in Storage (18C, 80 to 90% RH)Color Score Commercial Ripening Chart C24 C48 M24 M48 Figure 3-13. Peel color of C ontrol and 1-MCP-treated fruit stored at low temperatures during storage (18C, 80 to 90% RH); C24 (24 h, 5C), C48 (48 h, 5C), M24 (24 h, 5C), M48 (48 h, 5C). (n=6). Vertical bars represent standard error.

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60 A a B Figure 3-14. Fresh-cut banana fruit during storage (18C, 80 to 90% RH; 5C, 80 to 90% RH where applicable). A) C24 (top) versus M24 (bottom) after 4 days at 5C. B) C48 (top) vers us M48 (bottom) after 5 days at 5C.

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61 CHAPTER 4 EFFECTS OF COMMERCIAL ET HYLENE APPLICATION ON BANANA RIPENING TO DETERMINE TIMING OF 1-MCP APPLICATION AND EFFECTS OF CHILLING INJURY Introduction Ethylene is required for the commenceme nt of normal ripening processes in climacteric fruit (Saltveit, 2004a). Applic ation of exogenous ethylene in the range of 300 to 1000 LL-1 for 12 to 36 h at varying temp eratures and relative humidity (parameters vary by country and ripening facil ity) is utilized in commercial ripening of dessert bananas while it is forgone in certain markets to allow fruit to remain green for consumption as a starchy vegetable. Previ ous experiments have applied 1-MCP within 48 h after ethylene application to determine the efficacy of the i nhibitor’s prophylactic effects (Golding et al., 1998; Jiang et al., 1999; Pelayo et al., 2003). This experiment explored the differences in ripening behavior occurring between fruit that underwent commercial ethylene treatment and fruit that did not. The primary purpose of this experiment was to determine if 1-MCP treatment before ethylene application is feasible. Th e secondary purpose was to determine if the selected soluble solids (Dubios et al., 1956), total phenolic compounds, chlorogenic acid, and polyphenol oxidase (PPO) assa ys (Coseteng and Lee, 1987) were viable methods for measuring their respective components in banana pulp and peel tissue. The low temperature storage treatment explored exposur e of fruit to 5C storage (24 or 48 h) to further analyze chilling injury symptom devel opment in order to derive a rating scale to

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62 measure chilling injury and differentiate chilling-induced graying from non-chilling induced graying. Materials and Methods Plant Material and Ethylene Treatments Four cartons of banana fruit were obta ined from a commercial supermarket in Gainesville, FL and therefore underwent standa rd postharvest handling. Two cartons of fruit (sixteen hands per carton) were not et hylene treated (markete d as green bananas) and considered controls, while the other tw o cartons were ethylene treated at 300 LL-1 (24 h, 15C, 90% RH). Upon arrival at the Po stharvest Horticulture Laboratory at the University of Florida, fruit were sort ed for uniformity of size and shape. Fruit Ripening Parameters Forty-four hands of bananas (twenty-tw o ethylene-treated hands and twenty-two control hands) were allowed to ripen ( 15C, 80 to 90% RH) over a 14-day period and were examined and digitally photographed every day on black velvet cloth under foodgrade evaluation lights simulating the full visi ble spectrum. Color score and the location and extent of fruit browning were the main nondestructive quality parameter monitored. Storage Under Conditions that Induce Chilling Injury On Day 3 eleven hands from each treatment were transferred to 5C for 48 h to induce chilling injury (Table 4-1). On Day 5 the eleven hands from each treatment were then transferred to 20C for the duration of the study. Data were recorded on color development according to procedures outlined in Chapter 3.

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63 Total Soluble Sugars, Peel Soluble Phen olic Compounds and Peel Polyphenol Oxidase Activity Three individual fruit replicates were samp led every other day from the control and ethylene treatments for laboratory assays. A three-inch length of pulp and peel tissue from the center, equatorial region of the fru it was cut using a knife (proximal and distal ends discarded). Pulp and peel tissue were separated by peeling and frozen in liquid nitrogen (N2). Pulp tissue samples were st ored in plastic bags (Ziploc, SC Johnson Inc., Racine, Wisconsin) at -30C and used to de termine if the phenol-s ulfuric assay (Dubios et al., 1956) was a viable option for measuring total soluble sugars. For total soluble sugar determinations, 2 g banana pulp tissue were homogenized in 8 mL 95% ethanol using a Polytron homoge nizer (Brinkman, PT 10-35 Lenz Kruenz, Switzerland) (1 min., speed 4) after which sa mples were placed at -20C for 2 hours. The homogenate (10 mL) was centrifuged ( 15,000 rpm, 5 min., 20C), the supernatant decanted and saved, the pellet washed and resuspended with 10 mL 80% ethanol and centrifuged again (15,000 rpm, 5 min.) finally combining supernatants and adjusting to 20 ml final volume with 80% ethanol. Samp les were diluted 1:100 with 80% ethanol, combined with 0.5 mL 5% phenol and 2.5 ml H2SO4 and allowed to cool as the color developed. Samples were assayed colorometric ally according to Dubi os et al., (1956) at 490 nm and compared to glucose standards ranging from 0 to 200 gmL-1. Peel tissue samples were stored in plastic bags (Ziploc, SC Johnson Inc., Racine, Wisconsin) at -80C to determine the feasibility of measuring total phenolics and chlorogenic acid content in peel tissue accord ing to an apple tissue protocol (Coseteng and Lee, 1987). Preparation for the peel total phenolics and chlorogenic acid assays based on Coseteng and Lee, (1987) involve d homogenizing 5 g peel in 20 mL 95%

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64 ethanol (2 min., speed 9) using a Waring blender (Waring Laboratory Science Inc., Torrington, CT). The homogenate was boi led for 10 min., centrifuged (8000 rpm, 10 min., 20C), and filtered through Miraclot h (Calbiochem, EMD Biosciences, Inc., San Diego, CA) into storage tubes. The pellet was re-suspended in 20 mL 80% ethanol, boiled, centrifuged and filtered again, finally combining supernatants and adjusting to 50 mL final volume with 80% ethanol. The total phenolics assay involved sample d ilution with water (1:5) and combining 0.5 mL of this dilution with 1 mL DI-wat er, 2.5 mL 0.2 N Folin-Ciocalteu reagent and 2 mL 0.7 M NaCO3 followed by a 1-h incubation period. Samples were assayed colorometrically at 640 nm according to Coseteng and Lee, (1987) and compared to tannic acid standards ra nging from 0 to 100 gmL-1. The chlorogenic acid assay involved combining 0.5 mL of the sample with 0.5 mL 5% sodium-molybdate and 2 mL DI-water. Samples were assayed colorometrically at 370 nm according to Coseteng and Lee, (1987) and compared to chlorogeni c acid standards ranging from 0 to 25 gmL-1. Peel tissue samples stored at -80C were us ed to determine if an ethanol or aqueous based polyphenol oxidase (PPO) extraction (C oseteng and Lee 1987) were viable options for measuring enzyme activity in banana peel. PPO from ethylene-treated fruit peel from Day 4 was extracted in a 5.0 mM cystie ne hydrochloride, 0.2 M phosphate buffer solution (pH 7.2). A parallel extraction utili zed ethanol during ho mogenization and the subsequent pellet as the source of PPO. The pellet was re-suspended in the buffer described above and assayed along side the bu ffer enzyme extract. Both extracts were assayed at 420 nm with readings taken at 5 min. intervals over a 60 min. period to determine the change in absorbance values ( Abs.) over time. All assays were

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65 conducted using a Shimadzu UV-1201 Spectrophotometer (Shimadzu Scientific Instruments Inc., Colombia, Maryland) and disposable plastic cuvettes. PPO from ethylene-treated fruit peel from Day 8 was extracted in a 5.0 mM cystiene hydrochloride, 0.2 M phosphate buffer solution (pH 7.2) and th en separately in an identical buffer containing 360,000 MW polvinylpolypyrrolidone (PVP) to scavenge free phenolics. PPO from these extractions was assayed at 420 nm every 3 min. for a total of 21 min. to determine the window of ti me where most activity occurred and if the PVP component added any benefit to the assay. Abs. were compared between all samples with activity and with boiled samples assumed to have no activity due to protein denaturation. Total protein was quantified using the BCA assay according to Smith et al. (1985). Statistical Analysis No statistical analysis was conducted in th is experiment as the primary purpose was to test the specified assays’ suit ability for banana tissue analysis. Results and Discussion Peel Color Fruit color score varied between contro l and ethylene treatm ents upon arrival at the Postharvest Horticulture Laboratory at the University of Florida with control fruit initially at color score 2.0 while ethylene fruit was at color score 3.0 (Table 4-2, Figure 4-1A, 1B). By Day 5, the treatment gr oups were significantly different (P < 0.05) with control fruit score remaining near scor e 2.0 while ethylene fr uit score approached score 6.0. The difference lasted for the durat ion of the experiment with C fruit score between 4 and 5 while all ethylene fruit were at score 7.0 and had been since Day 10.

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66 Variability in ripening rates was also obs erved in hands within the control group on Day 10 with fruit ranging from score 2 to sc ore 5 (Figure 4-1C). Many of the control hands never passed score 4.0 and began showi ng signs of decay by Day 14. Observations of delayed ripening and the variability in ri pening rates of non-ethylene treated control bananas lead to the decision that all subse quent experiments will use fruit commercially treated with ethylene (300 LL-1, 24 h, 15C, 90% RH). Incidence of Chilling Injury During the first 24 h of low temperature stor age (5C) both control and ethylene hands showed minimal signs of chilling injury (CI). Upon removal from low temperature storage (5C) all hands showed visible CI discoloration. 24 h after being transferred to 20C fruit showed severe discoloration a nd browning. Discoloration in control and ethylene-treated fruit was of two types, gray ing of all fingers of the hand and blotchy browning on individual fingers (discussed in Browning Section). Analysis of banana fruit chilling injury has allowed the developm ent of a chilling injury rating scale based on a modification of that described by Nguyen et al., (2004). The scale ranges from 1 to 5 with descriptions of each numerical rating as follows: 1, no chilling injury; 2, mild CI, grayish tint and first sign of browning; 3, moderate CI, development of browning up to 25% fruit surface covered; 4, severe CI, in creased browning approximately 25 to 50% fruit surface covered; 5, very severe CI, increased browning, greater than 50% fruit surface covered. The graying observed was different than the graying observed in the first experiment (Chapter 3) as it appeared within 24 h of fruit being returned to 20C, was opaque (not as dark) and uniformly present on every finger of the hand (Figure 4-2A); while graying in the first experiment (Cha pter 3) appeared at random times during

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67 ripening, was darker in the proximal and distal areas of affected fruit than in the center and not always present on all fingers of a given hand (Figure 4-2C). In addition to the discoloration, the chilled fruit seemed to ha ve dryer peduncle area s indicating increased water loss in this region of the fruit. Peel Browning Browning was observed in both control and ethylene-treated fruit due to three factors: 1) senescent spotting, 2) chilling injury and 3) repetitive impact compression, which occurred while placing fruit on and off trays (for grading, color score etc.). Senescent spotting occurred in ethylene-treated fruit while control fruit were not ripe enough by the termination of the experiment to exhibit senescent spotting. Chilling injury browning symptoms were expressed in both control and ethyl ene-treated fruit as blotchy brown areas similar to those observe d in the first experiment (Chapter 3). Increased browning observed on the bottoms of ethylene-treated and ethylene-treated fruit at 5C, 48 h, bananas due to repetitiv e compression impacts was another source of browning observed (Figure 4-3). For this reas on all future sampling of fruit for soluble sugars, PPO and total phenolics assays were taken from fingers on the top of the hand. Total Soluble Sugars Pulp tissue samples stored at -30C and assayed according to the phenol-sulfuric method (Dubios et al., 1956) showed incr easing soluble sugar levels as ripening progressed (data not shown). Peel Soluble Phenolic Compounds Peel tissue samples stored at -80C and assayed for total phenolics and chlorogenic acid content according (Coseteng and Lee, 1987) showed increasing levels of each as ripening progressed (data not shown).

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68 Peel Polyphenol Oxidase Activity The ethanol extract of Day 4 peel yielde d no PPO activity (data not shown). The phosphate buffer extraction procedure yielded a Abs. of 0.712 over a 60-min. period with more than 50% of the Abs. (0.0 to 0.415) occurring in the first 15 min. Based on these results it was concluded that the alc ohol extraction was not a viable method to measure PPO activity. The phosphate buffer PPO extraction of Day 8 banana peel yielded a Abs. of 0.706, similar to that of Day 4 phosphate buffe r extraction described previously. The only difference between the Day 4 and Day 8 assay was the maximum absorbance values of 0.712 and 1.083, respectively and the time required to reach the maximum absorbance levels, 60 and 21 min., respectively. These diffe rences can be attributed to the color score of the peel on each day (Day 4 vs. Day 8) which can be direc tly correlated with fruit ripeness. Day 4 peel requir ed a 60-min period to achieve a 0.712 Abs. because less ripe fruit usually have lower levels of PPO protein. Consequently, Day 8 peel only required a 21-min period to achieve a 1.083 Abs. because the sample, derived from fruit at a more advanced stage of ripeni ng, would have higher le vels of PPO protein synthesized (Choehom et al., 2004). Samples extracted with the PVP phosphate buffer became brown within minutes of completing the extraction procedure. Th e color was manifest in high absorbance readings (> 2.5) and therefore no Abs. was observed during th e assays. The results of the assay indicate that the aqueous phosphate buffer protocol without PVP is a suitable extraction procedure for banana PPO.

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69 Conclusions The results of this experiment lead to the conclusion that commercial ethylene application (300 LL-1, 24 h, 15C, 90% RH) promotes uni form fruit ripening. Fruit not treated with ethylene do not ripen uniformly enough and were not used in further experiments. The non-uniform ripening also precluded the possibility of 1-MCP application prior to ethylene application. The low temperature storage treatment il lustrated that 24 h at 5C was enough chilling time to induce minimal CI signs and facilitated the developm ent of a rating scale of 1 to 5 for quantifying the degree of CI. The CI also revealed that fruit graying related to low temperature storage can be visibly di fferentiated from fruit graying observed in commercial shipments. The CI and browning observations showed that the areas of the fruit most likely to be bruised in handling was the fruit bottoms and therefore these fruit would not serve as accurate representations of the hand for the PPO assays. Sampling therefore occured from the top layer of fruit. The results from the soluble sugars, tota l phenolics and chlor ogenic acid assays verify that the respective assays produce useab le results. The PPO assays showed that neither the ethanol extraction nor the aqueous extraction with PVP yielded any activity and therefore the aqueous phosphate buffer extraction outlined by Coseteng and Lee (1987) was utilized in all future assays.

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70 Table 4-1. Control and ethylene treatmen t 5C storage regime s beginning Day 3. Treatment Day 3 Day 5 Treatment label Control 15C storage (no chi ll) 15C storage (no chill) C Control 48 h, 5C 20C storage C48 Ethylene 15C storage (no chill) 15C storage (no chill) E Ethylene 48 h, 5C 20C storage E48 Table 4-2. Color score of control and ethylen e-treated fruit ripened at 15C for 14 days. Treatment Day 0 Day 5 Day 10 Day 14 Control 2.0 0 2.28 0.31 3.025 .85 4.23 0.96 Ethylene 3.0 0.35 5.96 0.13 7.0 0 7.0 0 Table 4-3. Absorbance readings of banana peel PPO extracted in phosphate buffer according to Coseteng and Lee (1987). Values are means of 4 replicates standard error. Time (minutes) Peel sample 1 Peel sample 1 boiled Peel sample 1 PVP 0 (initial) 0.376 0.0150 0.145 0.0057 2.613 3 0.617 0.0022 0.147 0.0056 2.613 6 0.740 0.0089 0.152 0.0036 2.613 9 0.841 0.0089 0.151 0.0075 2.613 12 0.916 0.0109 0.147 0.0055 2.613 15 0.978 0.0202 0.149 0.0034 2.613 18 1.045 0.0140 0.148 0.0057 21 (final) 1.083 0.0143 0.149 0.0038 absorbance 0.706 0.004 0.000

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71 A B C Figure 4-1. Variation in fruit ripening resulting from ethylene treatment. A) Variation between ethylene (top) and contro l (bottom) treatments on Day 6. B) Variation between ethylene (top) and control (bottom) treatments on Day 10. C) Variation within control treatment group on Day 10. A B C Figure 4-2. Graying of ethylene-treated fruit 24 h after removal from chilling injury temperatures. A) Ethylene-treated fru it (top) compared to ethylene-treated fruit at 5C, 48 h. B) Et hylene-treated fruit (left) compared to CI-induced graying on ethylene-treated fruit at 5C, 48 h, (right). C) Graying of fruit in the first experiment (Chapter 3) for comparison purposes.

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72 A B Figure 4-3. Banana fruit brow ning as a result of repetitive impact compression. A) The top surfaces ethylene-treated fruit (top) and ethylene-treated fruit at 5C, 48 h, fruit (bottom). B) Identical fruit fli pped showing ethylene-treated fruit (top) and ethylene-treated fruit at 5C, 48 h, fruit (bottom) bottoms exhibiting more browning than top surfaces.

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73 CHAPTER 5 EFFECTS OF 1-MCP CONCENTRAT ION ON BANANA RIPENING Introduction Suppression of ethylene action via 1-me thylcyclopropene (1-MCP) treatment has been shown to delay ripening and possibly prolong the marketable shelf-life of many tropical climacteric fruits such as papa ya, avocado and mango (Ergun and Huber 2004; Jeong et al., 2003; Jiang and Joy ce 2000). Studies monitoring banana ripening responses to ethylene suppression have been conducted in a variety of postharve st settings ranging from rural (Jiang et al., 1999b) to commercial (Harris et al ., 2000). Specifically, it has been shown that 1-MCP treatment of pr eclimacteric banana fruit retards color development, delays the onset of the resp iratory and ethylene climacterics, decreases eventual total volatiles production (Golding et al., 1998, Jiang et al., 1999a) and slows whole fruit and fresh-cut fruit softening (J iang et al., 1999a, VilasBoas and Kader 2006). Banana peel color and fruit firmness are key indicators of fr uit edibility and quality (Kerbel 2004). Peel color devel opment due to chloro phyll catabolism and carotenoid synthesis occurs concomitantly w ith fruit softening in ripening banana (Drury et al., 1999, Seymour, 1993, Gross and Fl ugel, 1982). Fruit soft ening is the result of cell wall and starch degr ading enzymes (Asif and Nath 2005; DoNascimento et al., 2006). Softening is also related to favorab le changes in flavor such as increased sweetness, decreased astr ingency (Pesis 2005, Matuso and Ito 1982, Esguerra et al., 1992) and increased acidity (Rhodes 1980, Wyman and Palmer 1964), therefore the effects of ethylene suppression on fruit soften ing may affect eventual fruit flavor.

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74 Soluble sugar accumulation is an important contributor to fruit flavor (Baldwin 2004). The pulp of unripe, green bananas consis t of approximately 20 to 25% starch that is eventually converted to soluble sugars as fruit ripen (Prabha and Bhagyalakshmi 1998, DoNascimento et al., 2006). Soluble sugar content in the fruit remains below 1% until ripening and starch-sugar conversions begin whic h eventually leads to final soluble sugar values between 18 to 25% (Prabha and Bh agyalakshmi, 1998; Israeli et al., 1986). DoNascimento et al., (2006) noted non-ethyle ne-treated fruit and 1-MCP-treated fruit initiate starch degradation approximately 10 d after the ethylene-treated fruit begin starch degradation. This delay in starch degradat ion should correspond to a delay in soluble sugar accumulation, therefore the following e xperiment explored the effects of 1-MCP on soluble sugar accumulation in ethylene-treated fruit. Banana peel browning in the form of senescent spotting is an inevitable consequence of fruit ripening and is also manifest during fruit bruising (Israeli et al., 1986). Browning can be considered a postharve st disorder if it pr events consumers from purchasing fruit (Choehom et al. 2004, Kerb el, 2004). Browning is related to the interaction of phenolic compounds with polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO), the enzyme responsible for catalyzing enzymatic browning in plants and fruits (Yoruk et al., 2003). Both ba nana pulp and peel PPO have been characterized and partially purified (G ooding et al., 2001; Yang et al., 2001). While many studies monitoring the response of bana na peel PPO to temperature and MAP have been conducted (Choehom et al., 2004, N guyen et al., 2004, Nguyen et al., 2003), few have addressed how PPO activity is a ffected by inhibition of ethylene action (Moradinezhad et al., 2006).

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75 In regards to the timing of 1-MCP app lication, treatment during the 12-h period after propyleneor ethylene-i nduced ripening has been most effective at creating ripening delays (Golding et al., 1998). Experiments examining 1-MCP applic ation after ethyleneinduced ripening have concluded that 1MCP application 36 to 48 h after ethylene treatment promotes inconsistent ripening of fr uit (Pelayo et al., 2003) and application 3 to 5 d after ethylene treatment is ineffective at suppressing ethylene action even at 1-MCP concentrations of up to 10,000 nLL-1 (Jiang et al., 1999a) . These results have lead researchers to focus on 1-MCP application with in the 8to 12-h period after ethylene gassing. The purpose of this experiment was to determine how inhibiting ethylene action using two 1-MCP concentrations affected measurable ripening parameters of commercially harvested, transported and ethylen e-gassed banana fruit. The effect of 1-MCP on peel color change was monitored beca use peel color is directly correlated with fruit ripeness and used by retailers as an indi cator of when to display fruit. Whole fruit firmness measurements are required to dete rmine which 1-MCP concentration allows adequate fruit softening in ripening fruit wh ile eventually maintaining firmness of fully ripe fruit. Pulp soluble solids measuremen ts determine if 1-MCP application allows sufficient starch-sugar conversion to permit acce ptable ripe fruit sweetness and flavor. The effect of 1-MCP on peel total phenolic compounds, ch lorogenic acid content and PPO serve to expose any benefici al effects on fruit browning. Materials and Methods Plant Material and 1-MCP Treatments In the first experi ment involving 500 nLL-1 1-MCP application, banana fruit ( Musa acuminata , Cavendish subgroup cv. Williams) were obtained from Chiquita ripening

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76 facilities in Bradenton, FL with in 8 h of ethyl ene gassing (300 LL-1, 24 h, 15C, 90% RH) while fruit were green (color score 2). Upon arrival in Ga inesville fruit were sorted for uniformity of size and shape. Fruit mass was meas ured in kg and converted to a volume of airspace (L) occupi ed in the 174 L chambers us ed for 1-MCP treatment. This volume was used to determine the mass of 1-MCP powder requir ed to achieve the 500 nLL-1. The source of 1-MCP was a comm ercial powder formulation (0.14%) (SmartFresh AgroFresh, Inc., a division of Rohm and Hass Co., Philadelphia, PA), and was prepared by dissolving 197.74, 196.93, 203.86 and 225.34 mg powder in four separate 125 mL Erlynmeyer fl asks containing 40 mL deionized water. The control flask (DI water only) and 1-MCP containing flask we re placed immediately in their respective chambers along with twenty hands of fruit per chamber. The chambers were then sealed for the fi rst 12-h period. Sixty hands of fruit were gassed with 500 nLL-1 1-MCP for the first of two 12-h periods at 18C while the other sixty hands were maintained without 1-MCP fo r an equal time period at 18C to serve as Controls. The process was then repeated for a second 12-h period. The two 12-h periods were utilized to avoid carbon dioxide (CO2) build-up as it is known that elevated CO2 levels can result in pulp softening and unus ual fruit texture (Wei et al., 1993) and in general, high CO2 levels lead to improper fr uit ripening (Seymour, 1993). In the second experiment involving 250 nLL-1 1-MCP application banana fruit were delivered to U.F. from Bradenton, FL by Chiquita (15C) within 8 h of ethylene gassing (300 LL-1, 24 h, 15C, 90% RH) while fruit were green (color score 2). Fruit were unloaded, sorted and wei ghed as described above to determine the mass of 1-MCP powder required to achieve the 250 nLL-1 1-MCP. The source of 1-MCP was a

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77 commercial powder formulation (0.14%) (Sma rtFresh AgroFresh, Inc., a division of Rohm and Hass Co., Philadelphia, PA), and was prepared by dissolving 98.27, 99.55, 99.88 and 99.35 mg powder in four separate 12 5 mL Erlynmeyer flas ks containing 40 mL deionized water. The chambers were then seal ed for the first 12-h pe riod. Sixty hands of fruit were gassed with 250 nLL-1 1-MCP for the first of two 12-h periods at 18C while the other sixty hands were maintained without 1-MCP for an equal time period at 18C to serve as Controls. The process was then repe ated for a second 12-h period as described above. Fruit Ripening Parameters In both the 500 nLL-1 1-MCP and the 250 nLL-1 1-MCP experiments, fruit were allowed to ripen (18C, 80 to 90% RH) ove r a 14-d period and were examined and digitally photographed every da y on black velvet cloth under food-grade evaluation lights simulating the full visible spectrum. Data were measured and recorded on whole fruit ripening parameters for color score, inci dence of graying and fungal appearance upon arrival (Day 0) and every ot her day thereafter acc ording to procedures outlined in Chapter 3. Whole fruit firmness was determin ed on four individual fruit per treatment using an Instron Universal Testing Instru ment (Model 4411, Instron Instruments Inc. Norwood, MA). A 50-kg load ce ll fitted with a 10-mm flat, stainless steel probe was positioned at zero-force contact on the equatorial region of each banana fruit positioned horizontally on its side on a solid platform. Fruit was compressed to a depth of 6 mm at a rate of 10 mmmin-1. The maximum force encountered ov er the distance of probe travel was recorded and expressed in Newtons (N). Fruit were discar ded after destructive firmness tests. Fruit firmness measurements were taken within 1 h after removal from 18C. Fruit were discarded af ter destructive firmness tests.

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78 Total Soluble Sugars, Peel Soluble Phen olic Compounds and Peel Polyphenol Oxidase Activity In the first experi ment involving 500 nLL-1 1-MCP application, three individual fingers were sampled from the Control and 1-MCP treatments beginning Day 0 and every other day thereafter thereafter for determination of pulp total soluble sugars content, peel soluble phenolic compounds, peel chlorogeni c acid content and peel polyphenol oxidase (PPO) activity. A three-inch length of pulp a nd peel tissue from the center, equatorial region of the fruit was cut using a knife (proxi mal and distal ends discarded). Pulp and peel tissue were separated by peel ing, frozen in liquid nitrogen (N2) and stored in plastic bags (Ziploc, SC Johnson Inc., Racine, Wisconsin) at -30C and -80C, respectively. For total sugar determination, 2 g of bana na pulp tissue were ho mogenized in 8 mL 95% ethanol using a Polytron homogeniz er (Brinkman, PT 10-35 Lenz Kruenz, Switzerland) (1 min., speed 4) after which sa mples were placed at -20C for 2 hours. The homogenate (10 mL) was centrifuged (17,600xg2, 5 min., 20C), the supernatant decanted and saved, the pellet washed and resuspended with 10 mL 80% ethanol and centrifuged again (17,600xg, 5 min.) finally comb ining supernatants and adjusting to 20 ml final volume with 80% ethanol. Samp les were diluted 1:100 with 80% ethanol, combined with 0.5 mL 5% phenol and 2.5 ml H2SO4 and allowed to cool as the color developed (Dubios et al., ( 1956). Samples were assayed colorometrically according to Dubios et al., (1956).at 490 nm. Glucose was used as a standard. The refractometric procedure involved homogenizing 2 g banana pulp in 8 mL 95% ethanol and comparing 2 G-force units (xg) refer to the average g-force at the midpoint of the centrifuge tube and were calculated using the following equation: RCF = 1.12 r (RPM/1000)2 where RCF = relative centrifugal field (xg units), RPM = revolutions per minute, and r = radius (in mm) or the distance from the center of the centrifuge rotor to the center of the centrifuge tube.

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79 degree Brix (Brix) readings of these samples to sucrose standards prepared in ethanol. Sucrose standards were prepared by dissolving 4, 8, 12, 16 and 20 g sucrose, in 20 mL H2O and bringing each to 100 mL with 95% ethanol. Preparation for the peel total phenolics and chlorogenic acid assays based on Coseteng and Lee, (1987) involved homogeni zing 5 g peel in 20 mL 95% ethanol (2 min., speed 9) using a Waring blender (W aring Science Inc., Torrington, CT). The homogenate was boiled for 10 min., centr ifuged (5020xg, 10 min., 20C), and filtered through Miracloth (Calbiochem, EMD Bioscien ces, Inc., San Diego, CA) into storage tubes. The pellet was re-suspended in 20 mL 80% ethanol, boiled, centrifuged and filtered again, finally combining supernatants and adjusting to 50 mL final volume with 80% ethanol. The total phenolics assay involved sample d ilution with water (1:5) and combining 0.5 mL of this dilution with 1 mL DI-wat er, 2.5 mL 0.2 N Folin-Ciocalteu reagent and 2 mL 0.7 M NaCO3 followed by a 1-h incubation period. Samples were assayed colorometrically at 640 nm according to Coseteng and Lee, (1987) and compared to tannic acid standards ra nging from 0 to 100 gmL-1. The chlorogenic acid assay involved combining 0.5 mL of the extracts w ith 0.5 mL 5% sodium-m olybdate and 2 mL DI-water. Samples were assayed colorometrically at 370 nm according to Coseteng and Lee, (1987) and compared to chlorogeni c acid standards ranging from 0 to 25 gmL-1. Polyphenol oxidase (PPO) activity in peel tissue was measured using a modification of Coseteng and Lee, (1987). 10 g peel tissue were homogenized in 20 mL 5mM cystiene hydrochloride / 0.2 M phosphate buffer (pH 7.2) for 1 min. using a Waring blender (Waring Science Inc., Torrington, CT ).surrounded by ice. The homogenate was

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80 centrifuged (17,600xg, 15 min., 4C) and filtere d through Miracloth (Calbiochem, EMD Biosciences, Inc., San Diego, CA) saving both the filtrate and residue. The residue was re-suspended in 20 mL 1% potassium chlori de (-5C) and sti rred for 30 min. The centrifugation (17,600xg, 15 min., 4C) and Miracloth filtration process were repeated with the filtrate from this step, combined w ith the previous filtrate. The final volume was then brought to 50 mL with 1% potassium chloride. The filtrate was used as source of the PPO assay which involved combining 0.5 mL enzyme extract, 2.0 mL 0.1 M sodium citr ate (pH 5.0) and 0.5 mL 0.5 M catechol and assaying at 420 nm over a 3-min time period according to Coseteng and Lee, (1987). PPO activity is expressed in units where one unit of enzyme activity is defined as the change in absorbance at 420 nm of 1.0 over a 3 min. period, per milligram protein (units mg protein-1). Activity of these samples were compared to the following controls: boiled sample, (-) substrate control and (-) enzyme control. Total protein was measured using the BCA Assay (Smith, 1985; Pierce Chemical Co., Rockford, IL). The original extraction of Coseteng and Lee, (1987) wa s modified to optimize activity by testing various buffer pH, two substrates (catechol and dopamine) and substrate molarities as described in Table 5-1. All laboratory procedures from 500 nLL-1 treatment were repeated in the 250 nLL-1 treatment with changes made to the following: the refract ometric procedure for measuring soluble sugars in pulp tissue was forgone in favor of the phenol-sulfuric procedure due to unusually high readings recorded in 500 nLL-1 treatment; the aqueous PPO extraction utilizing the phosphate buffer was replaced with a procedure utilizing acetone powders based on modifications of N guyen et al., (2003), Chang et al., (2000)

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81 and Yoruk et al., (2003a). Brie fly, 25 g peel were homogenize d (2:1 v/w) in 50 mL cold acetone (-20C) for 1 min. using a Waring bl ender (Waring Laboratory Science Inc., Torrington, CT). The residue was vacuum filtered thru Miracloth (Calbiochem, EMD Biosciences, Inc., San Diego, CA) and the hom ogenization/filtration process repeated two more times with the final vacuum filtration lasting for 15 min. to facilitate drying of the residue. The powder residue was placed on al uminum foil and allowed to air dry for 15 min. The acetone powders were sealed in freezer bags and stored at -20C. PPO activity was extracted from 500 mg of the acetone powders by suspending in 10 mL 0.1 M sodium citrate (pH 6.2) and stirri ng while in an ice bucket for 20 min. The suspension was centrifuged (11,670xg, 10 min., 5C) and filtered thr ough miracloth. The filtrate was used as source of the PPO en zyme which involved combining 1.0 mL 0.1 M sodium citrate (pH 6.2), 1.5 mL 0.3 M catechol and 1.0 mL enzyme extract and assaying at 420 nm over a 3-min time period. One unit of enzyme activity is defined as the change in absorbance at 420 nm of 1.0 over a 3 mi n. incubation period, per milligram protein (units mg protein-1). Activity of these samples were compared to the following controls: boiled sample, (-) substrate control and (-) enzy me control. Total protein was measured using the BCA Assay (Smith, 1985; Pierce Ch emical Co., Rockford, IL) using bovine serum albumin as a standard. Statistical Analysis Data were analyzed using the Statisti cal Analysis Software version 9.1.3 (SAS Institute Inc. Cary, NC) “Proc Mixed” procedur e. In applicable cas es a Tukey adjustment was utilized for the Day x Treatment interacti on to adjust the experi ment-wise error rate for multiple comparisons within a treatment group.

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82 Results and Discussion Peel Color In the 500 nLL-1 treatment, Control and 1-MCP fru it began at color score 2.0 when measured approximately 8 h after ethylene treatment on Day 0 (Figure 5-1). By Day 2 both treatment groups began to diverge signi ficantly (P < 0.05) as the Control fruit approached 2.5 and the 1-MCP-treated fruit re mained near 2.0. By Day 9 the Control approached color score 7 (past marketabilit y) while the 1-MCP remained below 4.0. The 1-MCP group surpassed color score 4 on Day 14 yet did not approach color score 5 before beginning to deteriorate due to patchy peel discoloration and fungal proliferation. Uneven peel degreening of fruit treated w ith 1-MCP at a color score 2 resulting in unacceptable peel discoloration has also been observed in a number of previous studies (Jiang et al. 2004; Harris et al . 2000; Jiang et al. 1999b). Th is lack of adequate color development is not commercially desirable due to consumer preference for purchasing and consuming banana fruit with surface color score 5 and 6 (Chiquita, 2000). Therefore, the 500 nLL-1 1-MCP concentration may have exceed ed the fruits’ ability to recover from the treatment. In the 250 nLL-1 treatment, fruit arrived in a slig htly riper condition (compared to the fruit monitored in the 500 nLL-1 treatment) as indicated by the initial color score of 2.5 when measured approximately 8 h afte r ethylene treatment on Day 0 (minor variability in degree of ripening is normal in commercial shipments). Regardless of the higher color score, both Contro l and 1-MCP groups followed a similar pattern of ripening as observed in the 500 nLL-1 treatment (Figure 5-1, Figure 52) where color score values began to diverge by Day 2 and by Day 3 th e values were significantly different (P < 0.05). On Day 8 the Control approached color score 7 (past marketability) while the

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83 1-MCP remained between 4.0 and 4.5. By Day 10 the 1-MCP group eventually reached color score 5.0, a color score considered adequa te for purchase and consumption. It must be noted that in 1-MCP fruit, color scores of 4.0 and above did not appear as yellow as the Control fruit at similar ripening stag es, indicating that either the chlorophyll breakdown process (Drury et al., 1999; Jana ve et al., 2004) or carotenoid synthesis (Gross and Flugel, 1982) is affect ed by 1-MCP application. The color development data confirms the findings of Sisler et al. (1997) where higher 1-MCP concentrations have increased efficacy in suppressing ethylene perception and associated ripening changes. The results al so revealed that treating fruit at a slightly later ripening stage (higher color score) may decrease treatment efficacy because after autocatalytic ethylene production is initiated, various ripe ning processes become free of further ethylene requirements (Golding et al ., 1998). Comparisons of 1-MCP treatment effects on ‘Khai’ banana fruit color by Jansas ithorn and Kanlavanar at (2006) revealed no significant difference between the 50, 100 and 250 nLL-1 treatments which suggest 1-MCP concentrations below 250 nLL-1 are not as effective as higher concentrations in delaying fruit color change. Bagnato et al. (2003) showed banana fruit treated with 300 nLL-1 1-MCP required 3.9 d to progress from co lor score 2 to 4 and another 6.8 d for fruit to ripen from color score 4 to 7. Alt hough this experiment treated fruit with a lower 1-MCP concentration, fruit required 6 d to re ach color score 4 and never reached color score 7. In an experiment analyzing the effect of ethylene pretreatment (varying concentrations and durations) on 1-MCP e fficacy, Moradinezhad et al.,(2006) observed the degree of discoloration (measured by a discoloration index) of 300 nLL-1 1-MCP-treated fruit varied based on the seas on fruit were harvested in addition to the

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84 ethylene treatments. Acknowledgment of this variation is important when comparing studies as banana fruit are produced and experimented with year-round. Overall, the 250 nLL-1 concentration was more effective for extending fruit color at the yellow stage (between color score 4 and 6) while at the same time allowing adequate ripening of the pulp tissue. A dditionally this concentration grants the commercial applicator the bene fits of reduced costs associated with treating at lower 1-MCP concentrations. Peel Graying Graying was observed first in 1-MC P-treated fruit in both the 500 nLL-1 and the 250 nLL-1 treatments. In the 500 nLL-1 treatment graying began in 1-MCP fruit on Day 4 while in the 250 nLL-1 treatment graying began in 1-MCP fruit two days earlier (Day 2) which coincides with its more advanced stage of ripening (Figure 5-3, Figure 5-4). Control fruit bega n graying at later color scores than the 1-MCP fruit in 500 nLL-1 treatment and the 250 nLL-1 treatment. This suggests that the 1-MCP may exacerbate graying but does not cause it. By the end of each experiment all treatment groups exhibited greater than 30% fruit affect ed by graying (Figure 5-3, Figure 5-4). The only difference observed between treatments was at 500 nLL-1 1-MCP graying peaked above 30% and at 250 nLL-1 1-MCP graying peaked above 45%. Thus far the only noted occurrence of gray ing has been related to low temperature storage and the resulting peel chilling injury symptoms which were referred to as graying (Morrelli et al. 2003). The lack of informa tion and the data outlined above suggest that some unknown preor postharvest treatment or environmental factor plays a role in graying, as the 500 nLL-1 treatment 1-MCP concentrati on did not result in higher percentages of fruit graying. The temporal association of graying with 1-MCP-treated

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85 fruit at ripening color score 3 suggests either the discoloration is masked by chlorophyll or it develops only at certain physiological stages of ripe ning. Further research is necessary to elucidat e the genesis and physiological m echanisms behind banana peel graying as this phenomenon has been observed in retail displays as well as experimental settings. Incidence of Fungal Infection Fungal decay in the 250 nLL-1 treatment appeared on both Control and 1-MCP fruit by Day 4 and continued to progress to le vels near 40% fruit affected by the end of the experiment (Figure 5-5). In the 500 nLL-1 treatment infections began 2 days earlier (Day 2) which may be related to more adva nced fruit ripeness upon arrival and therefore decreased defense mechanisms (Figure 5-6) (Kader, 2002b). Infections began on the stem end of the fruit and were limited to th ese regions in intact fruit. As ripening progressed, fruit began to exhibit infections on the distal tip and in some cases the infections spread to bruised or damaged tissue over time. The degree of infection varied among fruit with the earliest infected e xhibiting the most mycelia and the latest exhibiting the least while overa ll percentages of infecti on between Control and 1-MCP fruit did not differ throughout the experiment. Studies analyzing i nhibition of ethylene action using 1-MCP acknowledged fungal prolif eration on plant material before the organs regained sensitivity to ethylene (Sis ler and Serek 1997; Bagnato et al. 2003). This experiment shows that even when banana fruit are semi-responsive to ethylene, as manifested by their continued ripening, they are still susceptible to colonization by fungus.

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86 Whole Fruit Firmness In the 500 nLL-1 treatment mean whole fruit firmness began at 140 N and within 2 days softened to 40 N at which point the Control and 1-MCP treatment groups began to diverge (Figure 5-7). After Day 4 the 1-MCP group maintained firmness levels between 30 and 35 N while the Control group continued so ftening at a rate of 2 N per day finally reaching 15 N at the termination of the experiment. Between Day 0 and Day 4 firmness values did not differ significantly yet by Day 6 firmness values differed significantly (P < 0.05) between Control and 1-MCP groups. Within the Control group the firmness value of 40 N recorded on Day 2 was significantly different (P < 0.05) than the final firmness value of 15 N, while within the 1-MCP group the firmness value of approximately 40 N recorded on Day 2 was not significantly different (P < 0.05) than the final firmness valu e of 30 N. The data show that adequate firmness after 1-MCP treatment is maintained for the duration of fruit shelf-life. Similar results occu rred in the 250 nLL-1 treatment between Day 0 and Day 2 where fruit firmness began at 55 N and decreased to the 30 to 35 N range by Day 2. On Day 6, Control and 1-MCP firmness values of 24 N and 35 N, respectively, differed significantly (P < 0.05) and remained so for th e duration of the experiment. Control fruit continued to soften at a rate of 2 to 4 N per day eventually reaching 17 N. The 1-MCP group maintained firmness levels between 30 and 35 N for the duration of the experiment, as compared to firmness values in the 500 nLL-1 treatment with final firmness values not significantly different (P < 0.05) from firmness values on Day 2. In both the 250 nLL-1 and 500 nLL-1 treatments, firmness of Control fruit reached values below 20 N while 1-MCP fruit remained near 30 N. Fruit firmness values under 20 N were associated with over-ripe, color sc ore 7 fruit exhibiting senescent spotting and

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87 therefore maintenance of firmness levels above this 20 N mark was desirable. Comparisons between final firmness values of 1-MCP-treated fruit from the two concentrations tested (500 nLL-1 and 250 nLL-1) showed no significant difference between the two. The data indicate that 1-MCP had desi rable effects on firmness retention as banana fruit softening has been shown to be dependent on ethylene (Zhang et al., 2006; Jiang et al., 1999a). Firmness results from Zhang et al., 2006 showed no significant difference (P>0.05) in firmness measurements of Control and 1-MCP fruit before Day 4, a result coherent with the firmness tests from both concentrations tested in this experiment. Firmness results from Bagnato et al. (2003) showed Control and 300 nLL-1 1-MCP treated fruit at color score 6 to be 116 and 148 kPa, respectively, which translates into the Control being 22% softer than the 1-MCP fruit. Firmness of Control fruit at color score 6 versus the final firmness meas urement of 1-MCP fruit (1-MCP fruit never reached color score 6) in the 500 and 250 nLL-1 treatment showed Control fruit being 40% and 18% softer, respectively, than the 1-MCP fruit. Jiang et al., (1999a) expressed banana softening as a rela tive response (%) based on the difference in firmness between control and 1-MCP-treated fruit divided by firmness of control fruit. Their tests revealed that bana na fruit treated with 100 LL-1 ethylene followed by treatment with 100 or 1000 nLL-1 1-MCP resulted in a 35% and 50% relative response, respectively, on Day 8 (the fi nal measurement). These results show the higher 1-MCP concentration resulted in a hi gher relative response i ndicative of firmer fruit. When data from Day 8 of this e xperiment are expressed using this type of calculation the 500 nLL-1 1-MCP concentration yielded a 64% relative response while

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88 the 250 nLL-1 1-MCP yielded a 51% relative response. Although these values are higher, they correlate with the findings of Jiang et al., (1999a) as the highe r 1-MCP concentration resulted in a higher relative response. Using fruit that was 1-MCP treated at various concentr ations, then treated with 100 LL-1 ethylene, Macnish et al., (2000) measur ed fruit firmness subjectively using a 1 to 5 rating scale and object ively using a digital meter according to (Macnish et al., 1997). These values were reported as shel f-life measurements where shelf-life was considered the time in days (from day 0) for fruit to reach “eating ripe condition of a firmness color score of 4, ‘eating soft’” (Mac nish et al., 2000). Fr uit treated with higher 1-MCP concentrations were afforded greater protection from the subsequent ethylene treatment which resulted in the following average shelf-life durations: no 1-MCP + 100 LL-1 ethylene = < 10 d; no 1-MCP, no ethylene = 30 d; 15 nLL-1 1-MCP + no ethylene = 40 d; 15 nLL-1 1-MCP + 100 LL-1 ethylene = 40 d (Mac nish et al., 2000). These results reveal the inhibitory effects of 1-MCP in preventing the effects of both exogenous and endogenous ethylene. Although the order of treatments utilized by Macnish et al., (2000) was 1-MCP + ethylene and this experiment performed ethylene + 1-MCP, a similar shelf-life comparison can be made between Control and 1-MCP-treated fruit in this experiment based on the edibility of fruit at color scor e 4 and firmness measurements approaching or below 30 N. In the 500 nLL-1 treatment Control and 1-MCP fruit shelf-life would have been 5 d and 11 d, respectively. In the 250 nLL-1 treatment Control and 1-MCP fruit shelf-life would have been 3 d and 6 d, respectively. These shelf-life measurements

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89 reveal shelf-life may be doubled when fru it are prevented from perceiving ethylene during initial ripening using either the 500 or 250 nLL-1 1-MCP concentration. Total Soluble Sugars Changes in external frui t characteristics occur simulta neously with carbohydrate catabolism activities in the pul p (Prabha and Bhagyalakshmi, 1998). These events lead to increased soluble sugar levels and decreased fi rmness indicating edibility of the fruit as a dessert banana. In the 500 nLL-1 treatment both Control and 1MCP fruit began at 2.5% sugar content (25 mggfw-1) on Day 0 (Figure 5-9). By Day 4 values began to diverge with the Control surpassing 14% and the 1-MCP remaining near 10% soluble sugar content. Sugar levels remained significan tly different (P < 0.05) between Day 6 and Day 10 reflecting an approximate 3to 6-d delay in ripeni ng caused by lack of ethylene perception in the 1-MCP group. By Day 12 (a nd for the duration of the experiment) the sugar content of both groups (near 16%) was not signifi cantly different (P < 0.05) indicating the eventual accumu lation of equivalent sugar levels in both Control and 1-MCP groups. More variation was obser ved in the 1-MCP gr oup indicating that individual hands differed in their response to 1-MCP. This phenomenon was attributable to physiological differences in receptivity to 1-MCP based on within-bunch and between-bunch harvest maturity (Harris et al., 2000). Variati on was also manifest in the color development data but less apparent due to the large numb er of replicates. In the 250 nLL-1 treatment fruit began at 4.5% soluble sugars (45 mggfw-1) on Day 0. On Day 2 a significant difference (P < 0.05) was observed between Control and 1-MCP fruit (8% vs. 10%, respectively) yet th e delay did not continue as observed in the 500 nLL-1 treatment (Figure 5-9, Figure 5-10). Between Day 4 and Day 14 Control and

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90 1-MCP fruit did not significantly differ (P < 0.05) in sugar content and by Day 14 both treatment groups surpassed 14% (140 mggfw-1) soluble sugars. The data indicate that 250 nLL-1 1-MCP application did not cause the 3to 4-d delay in sugar accumulation observed in the 500 nLL-1 1-MCP experiment. Total sugar accumulation was greater in the 500 nLL-1 treatment (~16%) than in the 250 nLL-1 treatment (~14%) yet it is assumed the fina l values were independent of the 1-MCP application because the Control groups in the respective treatmen ts achieved levels similar to those of the 1-MCP groups. Jiang, et al., (2004) noted that applying 200 nLL-1 1-MCP to non-ethylene treated fruit (cv. Zhonggang) caused Control and 1MCP-treated fruit to require 8 and 21 d, respectively, to surpass the 10% total soluble sugar level considered necessary to achieve what the authors called the “bes t taste of banana fruit used in the study.” The data from the 500 nLL-1 1-MCP treatment also showed a dela y in accumulation of total soluble sugar between Control and 1-MCP fruit, fo llowed by eventual recovery of the 1-MCP fruit. Specifically, the Control required 3 d to surpass the 10% total soluble sugar level while the 1-MCP-treated fruit required 4 d. In the 250 nLL-1 1-MCP treatment a 1-d delay was observed as fruit crossed the 10% so luble sugar mark, yet the delay did not last for the duration of the experiment. Although Jiang et al., (2004) treated fruit with a slightly lower 1-MCP concentratio n than used here (200 vs. 250 nLL-1 1-MCP) they observed a greater delay in to tal soluble sugar accumulation. A possible explanation is related to conclusions drawn by Golding et al., (1998) that stated “once autocatalytic ethylene production is in itiated certain processes become independent of further ethylene action.” The fruit used in both the 500 and 250 nLL-1 1-MCP treatments were ethylene

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91 treated prior to 1-MCP treatment (appr oximately 6 to 8 h before) and possibly, autocatalytic ethylene production was partially underway during 1-MCP application. The total soluble sugar accumulation process ma y therefore only be dependent on a minimal threshold amount of ethylene action allowed by the 250 nLL-1 concentration. In addition, the longer ripening time span (>24 d) reported by Jiang et al., (2004) was due to the fact that non-ethylene trea ted fruit were used while utili zing ethylene trea ted fruit in the 500 vs. 250 nLL-1 experiment resulted in a decreased ripening time span (~14 d). Another possible explanation for why a slight delay was observed in the 250 nLL-1 treatment (besides random variation) may be that ethylene pre-treatment makes the fruit more receptive to ethylene even after an inhibitor such as 1-MCP is applied. This line of reasoning is based on th e observation of Goldi ng et al., (1998) that unbound 1-MCP continued to diffuse from fruit for days after initial treatment which indicated a “binding saturation point” for 1-MCP and the active ethylene receptors. It is assumed that the proportion of ethylene recep tors occupied is highest upon initial treatment and decreases during the days after application as it has been suggested that new ethylene receptors are generated as ri pening progresses (Pathak et al., 2003, Jiang et al., 1999a). Therefore the suspected delay in sugar accumulation in the 250 nLL-1 1-MCP treatment was most apparent when th e inhibitor was most severely affecting signal transduction and the resulting ripening effects of ethylene perception. Banana fruit total soluble sugars measured refractometrically yielded high readings with little increase over time (Table 5-2) while the refractometric readings (Brix) of sucrose standards showed a linear increase of approximately 2.1 Brix per 4 g sucrose interval (Table 5-3). The B rix readings of all samples a nd standards indicated elevated

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92 background readings due to unknown optical prop erties of the 80% alcohol extracts. Increasing readings from fruit samples w ould be expected due to soluble sugar accumulation during fruit ripening as shown using the phenol-sulfuric method (Figure 5-9, Figure 5-10). For example, Control Day 12 samp les using the phenolsulfuric method showed 16 to18% soluble solid s (Figure 5-9). Ther efore, Day 12 Control Brix readings should be greater than 30.0 corre sponding to the 16 g mL-1 sucrose standard (16%). This was not the case as Day 12 Contro l Brix readings were 20.7. Therefore all soluble sugar assays employe d the phenol-sulfuric method rather than the refractometric method. Peel Soluble Phenolic Compounds Peel soluble phenolic compounds play an im portant role in banana fruit ripening, functioningas antimicrobial agents preventing pa thogen proliferation and as substrates for enzymatic browning when associated with polyp henol oxidase (Marshal l et al., 2000). In the 500 nLL-1 treatment total phenolic compounds were initially in the range of 1.0 to 1.5 mggfw-1 (Figure 5-11). No significant diffe rence (P < 0.05) between Control and 1-MCP samples was observed until Day 12 wh en a delay in accumulation of 1-MCP phenolics past 3 mggfw-1 became evident. The final values in the range of 4 mggfw-1 were achieved in Control peel by Day 12 while 1-MCP-treated fruit required 2 more days (Day 14) to achieve equivale nt levels. In the 250 nLL-1 treatment total phenolic compounds began at slightly higher levels of 2.0 mggfw-1 (Figure 5-12). Less of a delay occurred as both Control and 1-MCP-trea ted fruit reached levels between 3.5 to 4.0 mggfw-1 by Day 14 with no significant differe nce (P < 0.05) between Control and 1-MCP fruit observed .

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93 Studies have reported how peel tota l phenolic compound levels respond to modified atmosphere packaging (Nguyen et al., 2004 Choehom et al., 2004) and low temperature storage (Nguyen et al., 2004) yet no studies have reported how peel total phenolic levels react to inhibition of et hylene action via 1-MCP application. Nguyen et al., (2003) observed differences in the initial leve ls of total phenolic s in cv. ‘Sucrier’ and cv. ‘Kluai Hom Thong’ bana nas and attributed this to cu ltivar-related variation. Both cultivars showed slightly d ecreasing levels of total phe nolic compounds during ripening at 10C and a more rapid d ecrease of total phenolic com pounds during ripeni ng at 6C. Although Choehom et al., (2004) used the same cv. ‘Sucrier’, they observed higher initial total phenolic levels along with increases as ripening proceeded, possible due to the 29 to 30C storage temperature utilized during ri pening. The trends of increasing total phenolics as fruit ripen coincide with the data presented here for cv. ‘Williams’ where total phenolic levels of both Control and 1-MCP fruit steadily increased as ripening proceeded at 18C. Chlorogenic acid is a subset of total phenolics (Marshall et al., 2000). In the 500 nLL-1 treatment chlorogenic acid began at 0.18 mggfw-1 and accumulated to levels of 0.5 mggfw-1 in Control fruit by Day 10 while 1-MCP-treated fruit reached similar levels by Day 16 (Figure 5-13). Significant difference (P < 0.05) between Control and 1-MCP fruit were observed on Day 2, 4 and 10. In the 250 nLL-1 treatment, chlorogenic acid levels began at 0.38 mggfw-1 and became significantly different (P < 0.05) be tween Day 4 and Day 12. Final chlorogenic acid levels were measured above 0.5 mggfw-1 in Control peel by Day 14 while 1-MCP-treated fruit were at levels of 0.45 mggfw-1 on the same day (Figure 5-14).

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94 Peel Polyphenol Oxidase Activity The aqueous PPO extract yielded substantia l activity (Figure 5-15) as was observed in the trial tests from the non-ethylene vs. ethylene treated ripening experiment (Chapter 4, Table 4-2). The peaks displayed in the solid lin e replicate were due to an unstable cuvette (caused by corrosion of th e spectrophotometer cuvette holder) rather than jumps in activity (i.e. a physical event rather than a physiological one). When the same extraction and assay procedures were re peated, no activity occu rred (Figure 5-16). The most likely reason for the lack of activit y was protein denaturation due to extraction at room temperature. For this reason the alternative extraction and assay procedures described in Table 5-1 were util ized to derive maximum activity. PPO activity is expressed in units where one unit of enzyme activity is defined as the change in absorbance at 420 nm of 1.0 over a 3 min. period, per milligram protein (units mg protein-1). In the 500 nLL-1 treatment PPO activity began at 2 units and diverged significantly (P < 0.05) between Control and 1-MCP samples by Day 4 (Figure 5-17). Control and 1-MCP values remained significantly different for the duration of the experiment and were reco rded near 16 and 12 units respectively by Day 16. PPO activity from the 250 nLL-1 treatment began slightly below 2 units and diverged significantly (P < 0.05) between Control and 1-MCP samples by Day 4 as was observed in the 500 nLL-1 treatment (Figure 5-18). Control activity was similar to activity observed in the 500 nLL-1 treatment where final values surpassed 14 units by Day 14 while 1-MCP values surpassed 10 units by Day 14. In both Control and 1-MCP th e final activity units were approximately 2 units less in the 250 nLL-1 treatment than in the 500 nLL-1 treatment. This difference may be attributed to intrinsic differences in the bana na fruit used in each experiment as it has

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95 been shown that ripening responses to 1MCP application vary based on the season fruit are harvested (Moradinezhad et al., 2006) in ad dition to maturity differences within fruit bunches (due to developmental factors) and between bunches harvested at different days after bunch emergence (Harris et al. 2000). Choehom et al., (2004) monitored PPO activit y in relation to senescent spotting of control and modified atmosphere packaged fruit (MAP, covered with “Sun wrap” polyvinyl chloride film) during ripening at 30C over a 6-d pe riod. The experiment noted MAP fruit exhibited higher PPO act ivity levels (20 unitsmg protein-1 vs. 10 unitsmg protein-1 in the control) increased levels of total phenolics (6.0 mggfw-1 vs. 4.5 mggfw-1) and less senescent spotting during ripening. The inverse relationship between decreased in vivo spotting of MAP fruit and increased in vitro activity was assumed to be due to “a feedback mechanism whereby more active protein is produced in the face of PPO inhibition” by decreased oxyge n concentrations resulting from the MAP (Choehom et al., 2004). While Choehom et al., (2004) showed MAP treatment caused increased PPO activity and decreased total phenolics, it appears 1-MCP treatment caused decreased PPO activity and allowed normal accumulation of total phenolics. In both the 500 nLL-1 treatment and the 250 nLL-1 treatment, total phenolic levels steadily increased over the 14-d period with no significant difference (P < 0.05) between Control and 1-MCP fruit observed in the 500 nLL-1 treatment until Day 12 and none in the 250 nLL-1 treatment (Figure 5-11, Figure 5-12). This data reve als accumulation of pheno lic substrates is not as severely affected by decreased ethylene perception as is the accumulation of the PPO protein itself.

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96 Gooding et al. (2001) measured peel PPO activity (defined as the uptake of 1.0 mol oxygen min-1 rather than units mg protein-1 as used in this experiment) and noted only slight increases in activity as fruit ripened, compared to significant increases observed in both the 500 and 250 nLL-1 1-MCP treatments from this experiment. Perhaps defining activity based on oxygen uptak e (using an oxygen elec trode) results in small increases in activity while defining a unit based on photometric measurements of catechol’s conversion (the substrate used here and in Gooding et al . (2001)) to melanin pigments results in marked increases in activ ity. Both assays woul d have to be conducted side by side to verify this notion. Other nondestructive treatments such as spraying with 1.0 mM n-propyl dihydrojasmonate (PDJ) and 0.25 mM abscisic acid (ABA) have resulted in lower peel PPO activity levels as compared to control fr uit (Pongprasert et al., 2006 ). The results of Pongprasert et al., (2006) along w ith those presented here sugg est the need to test the PDJ/ABA and 1-MCP treatments side by side to determine their relative effects on PPO activity and furthermore, testi ng varying concentrations of th ese treatments in tandem to determine the possibility of utilization as an ti-browning agents. The overall results from the 500 vs. 250 nLL-1 1-MCP treatments indicate that decreased PPO activity was the result of delayed ripening a nd inhibition of ethylene action. Conclusions The results of the 500 nLL-1 treatment and the 250 nLL-1 treatment confirmed that 1-MCP application for two consecutive 12h periods at both concentrations had significant effects on delaying fruit ripeni ng over the 14-d measurement period (18C, 80 to 90% RH). The 250 nLL-1 concentration allowed adequa te fruit ripening and peel

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97 color change while maintaining fruit firmness as compared with the 500 nLL-1 concentration which did not allow suffici ent ripening and peel color change. The data revealed that graying is not caused by 1-MCP application and the degree of fruit affected does not dependent on 1-MCP concentration. The exact cause of graying, however, remains unknown. A similar result was seen with fungal appearance where approximately equal levels of infec tion occurred in Contro l and 1-MCP groups in both treatments. 1-MCP had desirable eff ects on whole fruit firmness retention yet treating fruit with higher concen trations did not significantly affect final firmness values. Results demonstrated that total soluble sugar accumulation of Control and 1-MCP in both the 500 nLL-1 and the 250 nLL-1 treatment achieved acceptable levels (14 to 16%) and therefore it was concluded that fi nal values were independent of the 1-MCP application. Application of 250 nLL-1 1-MCP did not cause the 3to 4-d delay in sugar accumulation observed in the 500 nLL-1 1-MCP experiment yet th is result was likely due to the fruit’s ripeness at treatment in addition to the decreased effects of ethylene inhibition from the lower 1-MCP concentration. The results from the total phe nolics assays in the 500 nLL-1 treatment and the 250 nLL-1 treatment showed less of a delay than obs erved in the chlor ogenic acid assay. Although accumulation of total phenolic levels were not substantially delayed by 1-MCP application, accumulation of individual phenolic compounds may be and therefore assays of the phenolic compounds in questi on must be done individually to determine levels during ripening. In both the 500 nLL-1 and the 250 nLL-1 treatment PPO activity was delayed by a minimum of 4 d which indicates 1-MC P has significant effects on delaying in vitro PPO

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98 activity. Delayed activity was most likely due to delayed accumulation of total PPO protein or decreased accumulation of specific is ozymes rather than a direct effect of 1-MCP on the enzyme. In all commercially relevant ripening para meters measured (color score, graying appearance, fungal incidence and soluble sugar accumula tion) there were no added benefits from treating at the hi gher 1-MCP concentration of 500 nLL-1. It was therefore concluded the 250 nLL-1 concentration was more desirable for further experiments.

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99 Table 5-1. Extraction buffers and substrates used to assa y PPO. Assays involving the sodium phosphate buffer were modificatio ns of Coseteng and Lee, (1987); the assay involving the sodium citrate buffe r was a modification of Nguyen et al., (2003). Buffer component 1 Buffer component 2Salt pH Substrate 0.2 M sodium phosphate 5 mM cystiene hydrochloride 1% potassium chloride 5.5 0.3 M catechol 0.2 M sodium phosphate 5 mM cystiene hydrochloride 1% potassium chloride 6.2 0.3 M catechol 0.2 M sodium phosphate 5 mM cystiene hydrochloride 1% potassium chloride 7.2 0.3 M catechol 0.2 M sodium phosphate 5 mM cystiene hydrochloride 1% potassium chloride 7.2 0.3 M dopamine 0.2 M sodium phosphate 5 mM cystiene hydrochloride 1% potassium chloride 7.2 1.0 M dopamine 0.1 M sodium citrate none none 6.2 0.3 M catechol Table 5-2. Fruit pulp samples soluble so lids content (Brix) measured using the refractometric procedure. Values sh own are the mean of 3 replicates. Sample Day 0 Day 2 Day 4 Day 8 Day 12 Day 16 Control Brix 19.5 19.8 20.2 20.3 20.7 1-MCP Brix 19.8 20.3 20.5 20.6 20.7 Table 5-3. Sucrose standards soluble so lids content (Brix) measured using the refractometric procedure. Values sh own are the mean of 3 replicates. Standards (g mL-1) 0 g 4 g 8 g 12 g 16 g 20 g Brix 19.2 22.1 24.9 27.4 30.0 32.6

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100 1 2 3 4 5 6 7 1234567891011121314 Days in Storage (18C, 80 to 90% RH)Color Score Commercial Ripening Chart Control 1-MCP 500 nL/L Figure 5-1. Peel color of Control and 1-MCP-treated fruit (500 nLL-1) during storage (18C, 80 to 90% RH). (n=60). Vert ical bars represent standard error.

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101 1 2 3 4 5 6 7 1234567891011121314 Days in Storage (18C, 80 to 90% RH)Color Score Commercial Ripening Chart Control 1-MCP 250 nL/L Figure 5-2. Peel color of Control and 1-MCP-treated fruit (250 nLL-1) during storage (18C, 80 to 90% RH). (n= 60). Ver tical bars represent standard error.

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102 0 5 10 15 20 25 30 35 234567891011121314 Days in Storage (18C, 80 to 90% RH)Fruit Exhibiting Graying (%) Control 1-MCP 500 nL/L Figure 5-3. Degree of graying in Cont rol and 1-MCP-trea ted fruit (500 nLL-1) during storage (18C, 80 to 90% RH). Each poi nt represents the percentage of the total hands exhibiting graying. (n = 60). Standard error bars are not present because the “present” or “absent” me thod of measurement does not produce this type of statistic.

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103 0 5 10 15 20 25 30 35 40 45 50 1234567891011121314 Days in Storage (18C, 80 to 90% RH)Fruit Exhibiting Graying (%) Control 1-MCP 250 nL/L Figure 5-4. Degree of graying in Cont rol and 1-MCP-trea ted fruit (250 nLL-1) during storage (18C, 80 to 90% RH). Each poi nt represents the percentage of the total hands exhibiting graying. (n = 60). Standard error bars are not present because the “present” or “absent” me thod of measurement does not produce this type of statistic.

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104 0 5 10 15 20 25 30 35 40 45 234567891011121314 Days in Storage (18C, 80 to 90% RH)Fruit Exhibiting Fungal Infection (%) Control 1-MCP 500 nL/L Figure 5-5. Incidence of funga l infection in Control and 1-MCP-treated fruit (500 nLL-1) during storage (18C, 80 to 90% RH). Each poi nt represents the perc entage of the total hands exhibiting mycelia. (n = 60). Standard error bars are not present because the “present” or “absent” method of measuremen t does not produce this type of statistic.

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105 Figure 5-6. Incidence of funga l infection in Control and 1-MCP-treated fruit (250 nLL-1) during storage (18C, 80 to 90% RH). E ach point represents the percentage of the total hands exhibiting mycelia. (n = 60). Standard error bars are not present because the “pre sent” or “absent” method of measurement does not produce this type of statistic. Control 1-MCP 250 nL/L70 50 60 0 40 30 20 10 Control 1-MCP 500 nL/L123456789101112 Days in Storage (18 C, 80-90% RH) Fruit Exhibiting F ungal Infection (%)

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106 0 20 40 60 80 100 120 140 160 0246810121416 Days in Storage (18C, 80 to 90% RH)Firmness @ Max Load (Newtons) Control 1-MCP 500 nL/L Figure 5-7. Firmness of Control a nd 1-MCP-treated fruit (500 nLL-1) during storage (18C, 80 to 90% RH). (n = 4). Vert ical bars represent standard error.

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107 0 10 20 30 40 50 60 02468101214 Days in Storage (18C, 80 to 90% RH)Firmness @ Max Load (Newtons) Control 1-MCP 250 nL/L Figure 5-8. Firmness of Control a nd 1-MCP-treated fruit (250 nLL-1) during storage (18C, 80 to 90% RH). (n=4). Ver tical bars represent standard error.

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108 0 20 40 60 80 100 120 140 160 180 200 0246810121416 Days in Storage (18C, 80 to 90% RH)Soluble Sugars (mg/gFW) Control 1-MCP 500 nL/L Figure 5-9. Soluble sugar content of C ontrol and 1-MCP-treated fruit (500 nLL-1) during storage (18C, 80 to 90% RH). (n=3). Vertical bars represent standard error.

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109 20 40 60 80 100 120 140 160 024681214 Days in Storage (18C, 80 to 90% RH)Soluble Sugars (mg/gFW) Control 1-MCP 250 nL/L Figure 5-10. Soluble sugar content of Control and 1MCP-treated fruit (250 nLL-1) during storage (18C, 80 to 90% RH). (n=3 ). Vertical bars represent standard error.

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110 1 1.5 2 2.5 3 3.5 4 4.5 0246810121416 Days in Storage (18C, 80 to 90% RH)Total Phenolics (mg/gFW) Control 1-MCP 500 nL/L Figure 5-11. Peel soluble phenolic cont ent of Control and 1-MCP-treated fruit (500 nLL-1) during storage (18C, 80 to 90% RH). (n=3). Vertical bars represent standard error.

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111 1.5 2 2.5 3 3.5 4 0481214 Days in Storage (18C, 80 to 90% RH)Total Phenolics (mg/gFW) Control 1-MCP 250 nL/L Figure 5-12. Peel soluble phenolic cont ent of Control and 1-MCP-treated fruit (250 nLL-1) during storage (18C, 80 to 90% RH). (n=3). Vertical bars represent standard error.

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112 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0246810121416 Days in Storage (18C, 80 to 90% RH)Chlorogenic Acid (mg/gFW) Control 1-MCP 500 nL/L Figure 5-13. Chlorogenic acid content of Control and 1-MCP-treated fruit (500 nLL-1) during storage (18C, 80 to 90% RH). (n=3 ). Vertical bars represent standard error.

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113 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0481214 Days in Storage (18C, 80 to 90% RH)Chlorogenic Acid (mg/gFW) Control 1-MCP 250 nL/L Figure 5-14. Chlorogenic acid content of Control and 1-MCP-treated fruit (250 nLL-1) during storage (18C, 80 to 90% RH). (n=3 ). Vertical bars represent standard error.

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114 Figure 5-15. Increasing polyphe nol oxidase (PPO) activity of color score 7+ Control fruit. Each line represents one replicate of the sample. Figure 5-16. Non-increasing polyphenol oxi dase (PPO) activity of color score 7+ Control fruit. Each line represen ts one replicate of the sample.

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115 0 2 4 6 8 10 12 14 16 0481216 Days in Storage (18C, 80 to 90% RH)Total Activity (Units/mg protien) Control 1-MCP 500 nL/L Figure 5-17. Total PPO activity of C ontrol and 1-MCP-treated fruit (500 nLL-1) during storage (18C, 80 to 90% RH). (n=3). Vertical bars represent standard error.

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116 0 2 4 6 8 10 12 14 16 0481216 Days in Storage (18C, 80 to 90% RH)Total Activity (Units/mg protien) Control 1-MCP 250 nL/L Figure 5-18. Total PPO activity of C ontrol and 1-MCP-treated fruit (250 nLL-1) during storage (18C, 80 to 90% RH). (n=3). Vertical bars represent standard error.

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117 CHAPTER 6 EFFECTS OF ETHYLENE-ACTION I NHIBITION AND LOW TEMPERATURE STORAGE ON BANANA RIPENING Introduction Low temperature storage is an effectiv e way to delay fruit ripening and prolong whole and fresh-cut fruit quality and shelf-lif e (Kader, 2002b). Storage of sensitive crops below their chilling thresholds , however, may result in a vari ety of chilling injury (CI) symptoms that may be detrimental to fruit sa les as external appearance is an important parameter by which consumers judge fruit quality. CI symptoms in banana are usually manifested 18 to 24 hours after fruit are rem oved from temperatures below their chilling threshold (storage below 13C, durations of more than a few hours) or when fruit are stored continuously at CI temperatures for extended periods of time (Kerbel, 2004; Wang 2004). In ripe bananas, CI is evident as peel and pulp tissue disc oloration (e.g. browning, water-soaking) and increased sensitivity to mechanical injury (Turner, 1997; Wang 2004). In unripe green bananas, CI may not be visible initi ally, but becomes evident as disrupted ripening leads to decreased soften ing, altered volatile profiles and a general inability of fruit to properl y ripen (Turner, 1997; Wang 2004). The exact mechanism of how low temperatur es affect fruits is currently being studied. Marangoni et al., (1996) has presented evidence of low temperature’s affect on cellular membranes while Matsui et al., (2003) suggested that low-temperature-induced free radicals contribute to cell death. Jiang et al., (2004a) de scribed increased electrolyte leakage from peel tissue of bananas stor ed at temperatures below 8C and this

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118 phenomenon may be related to the increased membrane permeability noted by Marangoni et al., (1996). Electrolyte leakage may also pl ay a role in the app earance of peel water soaking symptoms (Jiang et al., 2004a) while peel browning is assumed to be caused by rupture of cellular components, specifical ly the vacuole, and exposure of this compartment’s phenolic substrates to e ndogenous polyphenol oxidase (PPO) (Marshall et al., 2000). Nguyen et al., ( 2003) also noted that as CI developed over time, peel PPO activity increased while total free phenolics decreased. Banana fruit exhibit decreased ethylene binding when stored below 13C indicating that temperature and duration of storage play a role in ethylene perception and ripening (Jiang et al., 2004). The authors assumed lo w temperature storage caused disruption of membrane-bound ethylene receptors that in turn lead to decreased ethylene perception and inadequate fruit ripening. Receptor prot ein disruption by low temperature was also noted by Macnish et al., (2000) where 1-met hylcyclopropene, an inhibitor of ethylene action, was applied at 10 nLL-1 (12 h) and binding to the ethylene receptors was not achieved at temperatures of 2C comp ared to the contro l fruit at 20C. Experiments addressing amelioration of CI symptoms in banana fruit have involved treatments ranging from modified atmosphere packaging (MAP) (Nguyen et al., 2004) to the application of jasmonat e derivatives (Kondo et al., 2005 ; Chaiprasart et al., 2002; Pongprasert et al., 2006). N guyen et al., (2004) noted th at fruit stored in MAP (polyethylene bags, et hylene absorbers, CO2 scrubber) began exhi biting CI symptoms 12 days after control fruit and the extent of the CI symptoms were less. Kondo et al., (2005) reported that CI in bananas (quantif ied using a peel browning scale) decreased

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119 when bananas were treated with the jasmonate derivative n -propyl dihydrojasmonate before storage at 6C versus contro l fruit held at 6C and 12C. The purpose of the present experiment wa s to determine the effects of ethylene suppression (through 1-MCP treatment) and storage at 5C on various ripening parameters of commercially harvested, transported and ethylene-gassed banana fruit. Whole-fruit ripening parameters of color development, CI development, senescent spotting, pulp firmness, total soluble suga rs, total phenolics an d PPO activity were measured to determine the possibility of utilizing 1-MCP treatment and low temperature storage in the food service or processing industry where peel appearance is not a determining factor in long-term fruit quality. Materials and Methods Plant Material and 1-MCP Treatments Banana fruit ( Musa acuminata , Cavendish subgroup cv. Williams) were obtained from Chiquita ripening facilities in Bradenton, FL within 8 h of ethylene gassing (300 LL-1, 24 h, 15C, 90% RH) while fruit were gr een (color score 2). Upon arrival in Gainesville fruit were sorted for uniformity of size and shape. Fruit mass was measured in kg and converted to a volume of airspace (L ) occupied in the 174 L chambers used for 1-MCP treatment. This volume was used to determine the mass of 1-MCP powder required to achieve the 250 nLL-1. The source of 1-MCP was a commercial powder formulation (0.14%) (SmartFresh AgroFresh, Inc., a division of Rohm and Hass Co., Philadelphia, PA), and was prepared by di ssolving 98.08 mg powder in 40 mL deionized water in a 125 mL Erlynmeyer flask contai ning 40 mL deionized water. The control flask (DI water only) and 1-MCP containing flask were placed immediately in their respective chambers along with twenty hands of fruit per chamber.

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120 The chambers were then sealed for the first 12-h period. Twenty hands of fruit were gassed with 250 nLL-1 1-MCP for the first of two 12h periods at 18C while the other twenty hands were maintained without 1-MCP for an equal time period at 18C to serve as Controls. The process was then re peated for a second 12-h period. The two 12-h periods were utilized to avoid carbon dioxide (CO2) build-up as it is known that elevated CO2 levels can result in pulp softening a nd unusual fruit texture (Wei et al., 1993) and in general, high CO2 levels lead to improper fr uit ripening (Seymour, 1993). Fruit Ripening Parameters Fruit were allowed to ripen over a 15-d period while data were measured and recorded on whole fruit ripeni ng parameters for color score, incidence of graying and fungal appearance upon arrival (Day 0) and every third day thereafter according to procedures outlined in Chapter 3. Changes in pulp firmness were measured as follows: a 2-cm thick disc was sliced from the equa torial portion of the fruit and peel was removed; two measurements were taken from the outer mesocarp tissue using an Instron Universal Testing Instrument (Model 4411, Instron Instruments Inc. Norwood, MA) equipped with a 5.0 kg load cell, 10.00 mmmin-1 travel rate, 3.0 mm probe travel distance and 5 mm diameter convex probe. The maximum fo rce encountered over the distance of probe travel was recorded a nd expressed in Newtons (N). Firmness measurements of both non-chilled and chilled fruit were taken with in 1 h after removal from their respective storage temperatures3 of 18C and 5C (see subsequent section on Storage Under Conditions that Induce Chilling Injury for detail ed storage regimes). Fruit 3 Pulp temperature of non-chilled and chilled fruit wa s not measured prior to firmness tests, hence it is unknown weather pulp temperature played any role in firmness measurements. Pulp firmness could have been measured using a pulp temperat ure probe similar to the one shown in The Appendix, Figure A-14.

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121 were discarded after destructive firmness tests. Chilling injury development was quantified using the modification of the N guyen et al., (2004) chilling injury index ranging from 1 to 5: 1, no chilling injury; 2, mild CI, grayish tint and first sign of browning; 3, moderate CI, development of browning up to 25% fruit surface covered; 4, severe CI, increased browning approximately 25 to 50% fruit surface covered; 5, very severe CI, increased browning, greater than 50% fruit surf ace covered. Severity of senescent spotting was measured using the Horsfall-Barrett Scal e (Table 6-1). Storage Under Conditions that Induce Chilling Injury At the start of Day 6, twelve hands from the Control and twelve hands from the 1-MCP treatment groups4 were transferred from 18C to 5C for 24 h. After the 24-h period, six hands from the Control group a nd six hands from the 1-MCP group were returned to 18C where they remained fo r the duration of the experiment and were labeled Control 5C, 24 h and 1-MCP 5C, 24 h, respectively. The remaining six Control and six 1-MCP fruit hands were kept at 5C for the 9-d remainder of the experiment and were labeled Control 5C, 9 d and 1-MCP 5C, 9 d, respectively. The original six Control and six 1-MCP hands not exposed to low temp erature storage were used as “No Chill” controls in the experiment and were labeled No Chill Control and No Chill 1-MCP, respectively (Table 6-2). Total Soluble Sugars, Peel Soluble Phen olic Compounds and Peel Polyphenol Oxidase Activity Three individual fingers were sampled fr om the Control and 1-MCP treatments beginning Day 0 and every third day thereafter for determination of pulp total soluble 4 At this transfer point, Control fruit were above color score 5.5 while 1-MCP-treated fruit were above color score 4.

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122 sugars content, peel soluble phenolic compounds , peel chlorogenic ac id content and peel polyphenol oxidase (PPO) activity. A three-inch length of peel and pulp tissue from the center, equatorial region of the fruit was cu t using a knife (proximal and distal ends discarded). Pulp and peel tissue were separated by peeli ng, frozen in liquid nitrogen (N2) and stored in plastic bags (Ziploc, SC Johnson Inc., Racine, Wisconsin) at -30C and -80C, respectively. For total sugar determination, 2 g of bana na pulp tissue were ho mogenized in 8 mL 95% ethanol using a Polytron homogeniz er (Brinkman, PT 10-35 Lenz Kruenz, Switzerland) (1 min., speed 4) after which sa mples were placed at -20C for 2 hours. The homogenate (10 mL) was centrifuged (17,600xg5, 5 min., 20C), the supernatant decanted and saved, the pellet washed and resuspended with 10 mL 80% ethanol and centrifuged again (17,600xg, 5 min.) finally co mbining supernatants and adjusting to 20 ml final volume with 80% ethanol. Samp les were diluted 1:100 with 80% ethanol, combined with 0.5 mL 5% phenol and 2.5 ml H2SO4 and allowed to cool as the color developed (Dubios et al., ( 1956). Samples were assayed colorometrically according to Dubios et al., (1956).at 490 nm. Glucose was used as a standard. Preparation for the peel total phenolics and chlorogenic acid assays based on Coseteng and Lee, (1987) involved homogeni zing 5 g peel in 20 mL 95% ethanol (2 min., speed 9) using a Waring blender (W aring Laboratory Science Inc., Torrington, CT). The homogenate was boiled for 10 min., centrifuged (5020xg, 10 min., 20C), and filtered through Miracloth (Calbiochem, EM D Biosciences, Inc., San Diego, CA) into 5 G-force units (xg) refer to the average g-force at the midpoint of the centrifuge tube and were calculated using the following equation: RCF = 1.12 r (RPM/1000)2 where RCF = relative centrifugal field (xg units), RPM = revolutions per minute, and r = radius (in mm) or the distance from the center of the centrifuge rotor to the center of the centrifuge tube.

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123 storage tubes. The pellet was re-suspende d in 20 mL 80% etha nol, boiled, centrifuged and filtered again, finally combining supernatants and adjusting to 50 mL final volume with 80% ethanol. The total phenolics assay involved sample d ilution with water (1:5) and combining 0.5 mL of this dilution with 1 mL DI-wat er, 2.5 mL 0.2 N Folin-Ciocalteu reagent and 2 mL 0.7 M NaCO3 followed by a 1-h incubation period. Samples were assayed colorometrically at 640 nm according to Coseteng and Lee, (1987) and compared to tannic acid standards ra nging from 0 to 100 gmL-1. The chlorogenic acid assay involved combining 0.5 mL of the extracts w ith 0.5 mL 5% sodium-m olybdate and 2 mL DI-water. Samples were assayed colorometrically at 370 nm according to Coseteng and Lee, (1987) and compared to chlorogeni c acid standards ranging from 0 to 25 gmL-1. The PPO extraction was based on a modifi cation of Nguyen et al., (2003), Chang et al., (2000) and Yoruk et al., (2003a). Briefly, 25 g peel we re homogenized (2:1 v/w) in 50 mL cold acetone (-20C) for 1 min. using a Waring blender (Waring Laboratory Science Inc., Torrington, CT). The residue was vacuum filtered thru Miracloth (Calbiochem, EMD Biosciences, Inc., San Diego, CA) and the homogenization/filtration process repeated two more times with the final vacuum filtration lasting for 15 min. to facilitate drying of the residue. The powde r residue was placed on aluminum foil and allowed to air dry for 15 min. The acetone powders were sealed in freezer bags and stored at -20C. PPO activity was extracted from 500 mg of the acetone powders by suspending in 10 mL 0.1 M sodium citrate (pH 6.2) and stirri ng while in an ice bucket for 20 min. The suspension was centrifuged (11,670xg, 10 min., 5C) and filtered thr ough miracloth. The

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124 filtrate was used as source of the PPO en zyme which involved combining 1.0 mL 0.1 M sodium citrate (pH 6.2), 1.5 mL 0.3 M catechol and 1.0 mL enzyme extract and assaying at 420 nm over a 3-min time period. One unit of enzyme activity is defined as the change in absorbance at 420 nm of 1.0 over a 3 mi n. incubation period, per milligram protein (units mg protein-1). Activity of these samples were compared to the following controls: boiled sample, (-) substrate control and (-) enzy me control. Total protein was measured using the BCA Assay (Smith, 1985; Pierce Ch emical Co., Rockford, IL) using bovine serum albumin as a standard. Statistical Analysis Data were analyzed using the Statisti cal Analysis Software version 9.1.3 (SAS Institute Inc. Cary, NC) “Proc Mixed” procedur e. In applicable cas es a Tukey adjustment was utilized for the Day x Treatment interacti on to adjust the experi ment-wise error rate for multiple comparisons within a treatm ent group. Comparisons for statistical significance (P < 0.05) were made within ea ch Control and 1-MCP treatment group and then made between the Control and 1-MCP-tr eated fruit for each chilling treatment. Results displayed in table format pertain to Day 6, Day 9 (two days after chilling) and Day 15 (final measurements). Results and Discussion Peel Color Banana fruit had initial peel color scor e of 2 when measured approximately 8 h after ethylene treatment on Day 0 (Figure 6-1). No Chill Control and No Chill 1-MCP fruit began to diverge significantly (P < 0.05) by Day 3, with No Chill Control at color score 4 and the No Chill 1-MCP fruit above color score 3 (Table 6-1). After Day 3,

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125 No Chill Control and No Chill 1-MCP fru it color score remained significantly different (P < 0.05). Within the Control group, significant di fferences (P < 0.05) in color score were observed between No Chill Control and Contro l 5C 9 d treatments from Day 9 through Day 15. Significant differences (P < 0.05) between No Chill Control and Control 5C 24 h occurred on Day 12 and Day 15 as No Chill Control approached color score 7 (past marketability) while the Cont rol 5C 24 h remained near 6.5. By Day 12, significant differences (P < 0.05) were al so present between the color score of Control 5C 24 h and Control 5C 9 d at 6.3 and 5.9, respectively, a nd these differences remained for the duration of the experiment. No Chill 1-MCP fruit exhibited color development and ripening patterns similar to those of 1-MCP-treated fruit observed in previous experiments involving 250 nLL-1 1-MCP treatment (Chapter 5). From Day 9 through Day 15 significant differences (P < 0.05) in color score were observed be tween No Chill 1-MCP and 1-MCP 5C 24 h as well as between No Chill 1-MCP and 1-MCP 5 C 9 d (Table 6-1). However, color score comparisons of 1-MCP 5C 24 h and 1-MCP 5 C 9 d showed no signi ficant differences by Day 9. The approximately equivalent co lor scores of 1-MCP 5C 24 h and 1-MCP 5C 9 d on Day 9 suggested that the 3-d period after chilling was not sufficient time for differentiating the effects of 24-h and exte nded 5C storage on color development in 1-MCP-treated fruit although CI symptoms of 1-MCP 5C 9 d were markedly greater than those of 1-MCP 5C 24 h by this time (Figure 6-2). While No Chill 1-MCP and 1-MCP 5C 24 h fruit eventually surpassed co lor score 5, the peel did not exhibit the

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126 uniformly yellow pigmentation observed in either No Chill Control or Control 5C 24 h fruit. Uneven peel coloration of 1-MCP-treated fr uit has also been noted by Harris et al., (2000) in fruit treated at levels of 500 nLL-1 (24 h, 20C). Uneven peel coloration, (amongst other ripening delays discussed late r) can be generally attributed to 1-MCP functioning as an ethylene antagonist by bi nding to the ethylene receptor preventing the signal transduction that regulates ethylene action and ripening (Sisler and Serek 1997). Specifically, Golding et al., (1998) has shown that proce sses such as peel color development and volatile production require ethylene perception throughout the early stages of ripening to achieve levels characteristic of nor mal fruit ripening. On the molecular level, Gupta et al., (2006) has shown that “treatme nt [of banana fruit] with 1-methylcyclopropene inhibits ripening and represses the expression of most of the up-regulated genes [identified in the study] indicating that thei r expression is directly or indirectly governed by ethylene.” The decr eased color developmen t of 1-MCP-treated fruit observed in the present experiment conf irmed the notions that color development is regulated by ethylene (Golding et al., 1998; Gupta et al., 2006). While decreased ethylene perception due to 1-MCP treatment plays a role in delayed and/or uneven peel color developm ent, it may not totally account for the decreased ripening observed in fruit stored at 5C. Golding et al., (1998) reported that other ripening processes including the ethyl ene and respiratory climacterics become independent of ethylene once autocatalytic et hylene production begins. Banana fruit stored at 5C may also experience decreased ethylene perception as Jiang et al., (2004a) observed that low temperature storage of banana fruit (3C and 8C) resulted in decreased

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127 ethylene binding manifest by a 14C-ethylene release assay. Jiang et al., (2004a) also reported that lower temperatures and longe r durations of chil ling both resulted in decreased ethylene binding. Similarly, M acnish et al., (2000) showed that 1-MCP binding in banana fruit is di srupted at low temperatures possibly due to conformational changes of the ethylene receptor. The aut hors suggested this because 1-MCP treatment (10 nLL-1, 12 h) at 2 C did not in hibit ethylene-induced ripening6 to the extent that identical 1-MCP and ethylene treatments conducted at 20C did. The data presented by Macnish et al., ( 2000) and Jiang et al., (2004a) offer insight as to why fruit from both Control and 1-MCP gr oups stored for either 24 h or 9 d at 5C were unable to achieve color scores equivalent to their No Chill counterparts. It is likely that the 24 h and 9 d storage at 5C resulte d in conformational changes to the ethylene receptor and disrupted ethylene binding enough to delay and/or prevent full color development and ripening. Just as Jiang et al., (2004a) noted that lo wer temperatures and longer durations of chilling resulted in decr eased ethylene binding, similar patterns were observed in the color score data in both Control and 1-MCP groups where the lower temperature and the longest durations at 5C resulted in the lowest color score. Inadequate peel color development in the 1-MCP 5C 24 h and 1-MCP 5C 9 d may have been a result of subjecting the fru it to low temperatur e storage too early7 in their ripening (color score 4 for the 1-MCP group ve rsus color score 5.5 fo r the Control group) 6 Ethylene was applied at 100 LL-1, 24 h, 20C. Ripening was quantified as “shelf life” which was the time in days for fruit to reach “eating ripe” condition based on a color score of 6, a hue angle of 90 and firmness score of 4,“eating soft”. 7 Fruit were place in low temperature storage on the same day (Day 6) to ensure identical treatment time after commercial ethylene and 1-MCP treatment. Conversely, fruit could have been placed in low temperature storage at equivalent color scores, yet th is would have required transfer to low temperature storage on separate days.

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128 to have become fully independent of their ethylene perception requirements. To avoid this, fruit should be transferred to low temper ature storage after they have reached color score 5. This recommendation is based on th e data from the Control group where fruit ripened past color score 5 and subsequently subjected to 5C storage eventually ripened past color score 6. Incidence of Chilling Injury In the present experiment, CI symptoms were characterized primarily by peel discoloration in all fruit exposed to short or long-term storage at 5C. By Day 7 (after 24 h at 5C) Control 5C 24 h, Control 5C 9 d, 1-MCP 5C 24 h and 1-MCP 5C 9 d fruit were at a CI rating of 2 (Fi gure 6-2) with mild CI symp toms consisting of an opaque grayish tint uniformly present on every finger of the hand accompanied by minor browning (limited to small areas where fruit had mechanical damage from shipping and handling). While all fruit exhibited a CI ra ting of 2, CI symptoms in Control 5C 24 h and Control 5C 9 d appeared to have more browning present than 1-MCP 5C 24 h and 1-MCP 5C 9 d. Decreased incidence of brow ning in 1-MCP-treated fruit may be related to delayed ripening and differences in colo r score between Control and 1-MCP-treated fruit. By Day 9, both Control 5C 24 and 1MCP 5C 24 h exhibited a CI rating of 2.6, a value significantly different (P < 0.05) fr om the ratings of Control 5C 9 d and 1-MCP 5C 9 d at 3.5 and 4.0, respectively (Table 6-4). The CI ratings of Control 5C 9 d and 1-MCP 5C 9 d at 3.5 and 4.0, respectively, were also significantly different (P < 0.05) from each other on Da y 9, but became equivalent with further storage. No significant differences in CI ratings (P < 0.05) were observed between Control 5C 24 h and 1-MCP 5C 24 h on any day. This occurrence may lead to the conclusion

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129 that 1-MCP had no effect in preventing or inducing CI symptoms during or after 24 h, 5C storage. Yet the 1 to 5 scale used in this experiment may not be sensitive enough to detect smaller changes in peel discolorati on that are characteristic of CI symptom development. The 1 to 5 rating scale may have to be replaced with a more precise scale or an objective reading from an instrument such as a colorimeter to track more minute differences. It is worth noting that subjectiv e visual analysis of the 1-MCP 5C 24 h fruit pulp (compared to Control 5C 24 h fruit pu lp) revealed a whiter color, decreased browning, and decreased water so aking (data not shown). The data also revealed that extended 5C storage initially increased the appearance of peel CI symptoms in 1-MC P 5C 9 d fruit as compared to Control 5C 9 d fruit during the 3-d period after low temperature storage be gan. However, any benefits of increased ethylene perception present in the Control 5 C 9 d fruit decreased over time resulting in no significant difference (P < 0.05) between Control 5C 9 d and 1-MCP 5C 9 d CI ratings on Day 12 or Day 15. This convergen ce of Control 5C 9 d and 1-MCP 5C 9 d CI ratings over time may be a result of d ecreased ethylene action in the Control 5C 9 d over time due to a low-temperature-induced decline in ethylene binding as noted by Jiang et al., (2004a) and discussed earlier. Peel discoloration during low temperature storage observed in Control 5C 24 h and Control 5C 9 d has also been observed by Nguyen et al., (2003) in non-1-MCP-treated banana fruit stored at 6 and 10C. Low-te mperature storage may cause changes in the “physical and chemical characte ristics of membranes which in turn lead to alterations in cellular metabolism and accelerated death” (M arangoni et al., 1996; Levitt, 1980). The peel CI observed in all fruit stored at 5C may be symptomatic of these cellular

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130 membrane changes. In intact fruit cells, phenolic compounds are st ored in the vacuole (Vamos-Vigyazo, 1981; Walker and Ferrar, 1998) while polyphenol oxidase (PPO), the enzyme responsible for browning reactions is located primarily in the plastids and associated with the thylakoid membrane (V aughn and Duke, 1984; Gooding et al., 2001). As normal senescence progresses or ce llular damage occurs through membrane disintegration, as in the case of CI, PPO-mediated oxidati on of phenolic compounds into insoluble melanin pigments results in ch illing-induced browning (Nguyen et al., 2003; Marshall et al., 2000). In addition to the proposed peel discoloration resulting from the browning processes described above, ot her detrimental processes asso ciated with CI may include decreased peel “coloration” resulting from the disruption of carot enoid synthesis that takes place primarily after fruit reach color sc ore 4 (Gross and Flugel, 1982). It is also assumed that the altered metabolic processe s resulting from extende d 5C storage disrupts defense mechanisms, allowing pathogen prolifer ation to further degrade cellular integrity and function (Wang 2004). These processes of cellular disintegration therefore account for the CI symptoms and peel discolor ation observed in Control 5C 24 h and Control 5C 9 d. Data from the present experiment also s howed that 1-MCP treatment prior to lowtemperature exposure slightly in creased the incidence of peel CI. Increased incidence of CI in 1-MCP-treated fruit may be relate d to subjecting these fruit to injurious temperatures at color score 4 (as compared to color score 5.5 for Control fruit) as it has been reported that less ripe fruit are more susceptible to CI (Kerbel, 2004). The observation of increased CI in 1-MCPtreated fruit concurs with those of

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131 Jiang et al., (2004a), who repor ted that 1-MCP treatment of banana fruit increased the incidence of CI, membrane permeability and el ectrolyte leakage. While Jiang et al. (2004a) reported that decreased ethylene pe rception (1-MCP treatment) resulted in increased CI symptoms8, Wang et al., (2006) reported th at propylene-induced ripening prior to low-temperature storage decreased CI symptoms9. These findings, along with the data presented here, suggest that preventi on of CI is an ethylene-dependent process while development of CI is not. Theref ore, utilizing 1-MCP to decrease ethylene perception combined with the decreased ethylene binding and increased cellular disintegration that occur at low temperatures resulted in increased peel CI symptoms and delays in ripening greater than observe d with either 1-MCP or chilling alone. While 1-MCP application resulted in incr eased CI, other experiments have shown that application of chemicals such as n -propyl dihydrojasmonate (PDJ) and abscisic acid (ABA) to banana fruit before low temper ature storage (various temperatures and durations) resulted in a d ecreased rate of CI development during ripening (Chaiprasart et al., 2002; Kondo et al., 2005; Pongprasert et al. 2006). Kondo et al., 2005 reported that endogenous levels of PD J and ABA (among other phytochemicals) increased as CI developed. The authors reas oned that these phytochemicals serve as part of a defense mechanism used to ameliorate CI, hence pretreatments with synthetic analogs may provide a readily available supply of the CI ameliorating substances. Future 8 CI symptoms were defined as “the relative extent of darkened areas” on the peel and rated using a scale of 1 to 5 which was convert ed into a CI severity index (Jiang et al., 2004a). 9 CI symptoms were defined as “pitting a nd brown patches” on the peel, yet no rating scale or CI index were used (Wang et al., 2006).

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132 experiments exploring the interaction of 1MCP, PDJ and ABA should be carried out to determine the effect of these chemicals on CI, fruit ripening and fruit quality Senescent Spotting Between Day 0 and Day 6, the No Chill Control fruit developed senescent spotting severity ratings between 1 and 1.6 units, values significantly different (P < 0.05) than the No Chill 1-MCP fruit that ranged from 0 to 1 (Figure 6-3, Table 6-4). The difference of 1 to 2 Horsfall-Barrett Scale units 10 between the No Chill Control and No Chill 1-MCP treatments continued for the duration of th e experiment. Within the Control group, no significant difference (P < 0.05) occurred between any of the treatments for the duration of the experiment. An identical result was observed within the 1-MCP group where no significant difference (P < 0.05) occurred between any of the treatments for the duration of the experiment. It is assumed, however, that different physiol ogical processes set in motion by 1-MCP treatment and low temperature storage resulted in equivalent senescent spotting ratings of the No Chill 1-MCP, 1-MCP 5C 24 h and 1-MCP 5C 9 treatments. In general, senescent spo tting levels of 1-MCP 5C 24 h and 1-MCP 5C 9 trailed Control 5C 24 h and Control 5C 9 d by a pproximately 1 to 2 Horsfall-Barrett Scale units resulting in significant differences (P < 0.05) from Day 9 through Day 15. During the latter days of the experiment, spotti ng measurements in Control 5C 9 d and 1-MCP 5C 9 d sometimes became confounded by CI symptom proliferation that may have resulted in readings that did not accurate ly reflect actual levels of senescent spotting present. 10 The Horsfall-Barrett Scale units change percentage-w ise as the Scale reached its midpoint, therefore lower numbers (0 to 3) represent less percentage surf ace area than mid-range numbers (4 to 6). Refer to Table 6-1 for exact percentage ranges corresponding to each numerical unit value.

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133 Decreased senescent spotting in ripe fr uit is a desirable trait as consumer purchasing and consumption preferences decrease by 33% and 52%, respectively, as spotting becomes apparent (Chiquita, 2000). The lower senescent spotting ratings observed in the 1-MCP group is assumed to be a result of the delayed ripening caused by decreased ethylene perception (Zhang, et al ., 2006; Jiang et al., 1999; Golding et al., 1998) and chilling temperature storage (J iang et al., 2004a; Macn ish et al., 2000). Ripening banana fruit at modera tely low temperatures (12C) has also been reported to affect senescent spotting development (Tra kulnaleumsai et al., 2006). Banana fruit cv. Sucrier11 treated with ethephon (dippe d for 1 to 2 min. in 500 mgL-1) and ripened at 12C did not develop senescent spotting and their ripening and color development was slower than fruit at 18C or 27C that did develop senes cent spotting. Trakulnaleumsai et al., (2006) also noted that ‘S ucrier’ bananas held at 27C for 2 d and then transferred to 12C exhibited a delay, but not a complete prevention of senescent spotting. Trakulnaleumsai et al., (2006)’s pattern of initial ripening at a higher temperature followed by transfer to a lower temperat ure not preventing senescent spotting was consistently observed in Cont rol 5C 9 d and 1-MCP 5C 9 d in the present experiment. The pattern was evident in both Control 5C 9 d and 1-MCP 5C 9 d treatments as these fruit were held at 18C for 6 d then tran sferred to 5C and still developed senescent spotting. It can therefore be concluded that temperatures below 12 C have an effect on slowing the rate of senescent spotting occu rrence during banana fruit ripening, yet the 11 Banana fruit cv. Sucrier is an Asian bana na known to develop senescent spotting early in fruit ripening near score 4, as fruit become more yellow than green. Banana fruit cv. Williams used in this experiment develop senescent spotting only after fruit reach score 6 or full yellow.

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134 timing of this treatment in regard to color development and ripening needs to be further explored. Other treatments shown to inhibit the development of senescent spotting in ‘Sucrier’ bananas involved ut ilizing polyvinyl chloride film in modified atmosphere packaging (MAP) which resulted in increa sed levels of ethylene, carbon dioxide, and relative humidity while oxygen levels decrea sed compared to the control fruit (Choehom et al. 2004). The authors attributed the de velopment of senescent spotting under MAP to lowered oxygen concentrations. While Choeho m et al. (2004) demonstrated that low oxygen levels resulted in decreased spotting, th e present experiment showed that ambient levels of oxygen during ripening combined w ith decreased ethylene perception (due to 1-MCP application and chilling) also resulted in decreased spotting. Overall, senescent spotting never surpa ssed 6 units on the Horsfall-Barrett Scale (50.1 to 75% of the fruits’ surface area covere d by senescent spotting) in any Control or 1-MCP treatment. Edible bananas are rarely used for consumption when they develop more than 60% senescent spotting and thos e that are kept beyond this point become highly susceptible to secondary fungal pa thogen colonization that renders them unmarketable for fresh-cut processing. In a ddition, this stage of fruit ripening is usually associated with less desirable quality paramete rs related to fruit texture (Kerbel, 2004; Abbot 1999) and volatile profiles (Pesis, 2005; Hyodo et al., 1983). Peel Graying Peel graying is a disorder of unknown or igin and is characterized by grayish discoloration appearing near th e neck and/or distal ends of individual finge rs with the discoloration eventually spread ing toward the center of the finger as ripening progresses. In the present experiment, in some cases all fingers of the hand developed graying

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135 simultaneously and in other cases graying deve loped only on selected fingers of the hand with no apparent pattern by which finger it appeared. Fruit peel graying was also observed in previous experiments in both Cont rol and 1-MCP-treated fruit (Chapter 5). Data for graying were recorded only on No Chill Control and No Chill 1-MCP fruit and not recorded on chilled fruit because low temperature storage also caused peel discoloration that rendered the standard gr aying scores of “present” or “absent” unreliable. Graying was observed in No Chill Control and No Chill 1-MCP fruit by Day 3 with approximately 10% of No Chill Control fruit exhibiting graying while No Chill 1-MCP fruit remained near 5% (Figure 6-4). No Chill Control and No Chill 1-MCP fruit exhibited similar pe rcentages of fruit graying throughout the experiment as has been observed in expe riments involving 1-MCP treatment at 500 and 250 nLL-1 (Chapter 5). However, the present experiment showed No Chill Control and No Chill 1-MCP fruit with more than 60% of fruit graying by Day 15, a considerably larger percentage than the 48% maximum observed in previous experiments. The observation of similar incidence of graying in non-chilled Contro l and 1-MCP fruit in three separate experiments suggests that gr aying is not caused by 1-MCP application. Thus far the latest mention of banana fruit “graying” has been related to low temperature storage, wherein pe el CI symptoms were collectively referred to as graying (Morrelli et al., 2003). The lack of informa tion on graying and the data presented here suggest that some inherent factor present in the entire sample of fruit arriving from the distributor (or grower) in conjunction with commercial postharvest treatments and environmental conditions induced peel grayi ng development. A possible cause may be the change in relative humidity (RH) from extremely humid field conditions to less

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136 humid postharvest shipping conditions. This RH change may initiate individual cell death from dehydration, resulting in a compro mised ability to fully degrade chlorophyll and synthesize carotenoids. In addition, the repeated observation that graying appeared in fruit only after atta ining color score 3 sugge sts that the discolora tion is already present and masked by chlorophyll or it develops only at certain physiological stages of ripening. Further research focusing specifically on peel graying is necessary to test these notions and definitively elucidate the genesis and physiological mechanisms contributing to banana peel graying. Incidence of Fungal Infection Data for incidence of fungal infections were recorded only on No Chill Control and No Chill 1-MCP fruit. The reason for this was based on the random sampling procedures employed in selecting fruit for 1-MCP treatm ent and chilling. Upon arrival (before treatment with 250 nLL-1 1-MCP), all fruit were check ed for incidence of fungal infection12 and divided into two groups, infected and non-infected. Half of the hands from the non-infected fruit were randomly sele cted to become part of the Control group while the other half became part of the 1-MCP group. This type of random selection procedure was also employed for the infected fruit where half were randomly selected to become part of the Control group while the other half became part of the 1-MCP group thereby ensuring an equal distribution of non-infected and infected fruit in each treatment. A random selection process was also employed for chilling, yet fungal appearance was not considered during the select ion process because of the artificially low 12 Initial infections were characterized by small am ounts of white mycelia, usually limited to a 2 to 3 mm area. Nevertheless, once visible, these infections we re recorded no matter how small. The appearance of this type of minor pathogen infec tion on fruit received from ripening facilities is considered normal and does not adversely affect fruit quality. For this reason, infected fruit were not removed from the experiment.

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137 or high percentages of fruit infected this would have produced13. It might therefore have been assumed that these non-representative re adings were a byproduct of the treatments, when in actuality they were due to the sampling procedures. Between Day 3 and Day 9 levels of infection were initially greater in No Chill Control than No Chill 1-MCP with ranges of 28 to 40% and 17 to 35% fruit infected, respectively (Figure 6-5). Between Day 12 and Day 15, both No Chill Control and No Chill 1-MCP fruit exhibited approximately equal percentage of infected fruit. The data lead to the conclusion that f ungal appearance is related to decreases in plant defenses with ripening (Kader, 2002b) rather than any prophylactic effect 1-MCP may have in preventing fruit from developing fungal inf ections. Confirmation of this idea would require separate tests of et hylene and 1-MCP on fungal organisms sampled from banana tissue to determine if these organisms respond directly to a lack of ethyl ene perception. Pulp Firmness Previous experiments (Chapter 5) have shown that whole fruit firmness of non1-MCP-treated fruit decreased more than 50% within 3 d of commercial ethylene gassing. In the present experiment, firmness of pulp tissue was initially 9 N and dropped below 5 N within 3 d in both No Chill Control and No Chill 1-MCP pulp (Figure 6-6). By Day 3, No Chill Control and No Chill 1MCP pulp exhibited a significant difference (P < 0.05) in pulp firmness of nearly 1 N (3.8 N and 4.7 N, respectively). This 1 N difference persisted for the duration of storage with final values of No Chill Control near 2.4 N and No Chill 1-MCP near 3.5 N. 13 For example, if 2 of the randomly selected 6 hand s designated for the 1-MCP 5C 24 h treatment were infected, this would have resulted in 33% of fruit affected. At the same time if all 6 hands randomly selected for Control 5C 24 h treatment were infected this would have resulted in 100% fruit affected.

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138 No significant difference in pulp firmne ss was observed between any treatment within the Control group until Day 12, when Control 5C 24 h and Control 5C 9 d differed significantly (P < 0.05) (Table 6-6). The firmness difference between the pulp of Control 5C 24 h and Control 5C 9 d may be attributable to the 24-h chilling period eventually causing a more rapid softening of Control 5C 24 h pulp upon return to 18C than observed in the No Chill Control (held at 18C) or Control 5C 9 d. Results from the 1-MCP group showed that no significant diffe rence (P < 0.05) in pulp firmness occurred between any treatments on any day, suggesti ng that the lack of ethylene perception affected the correlation between decreased ripening temperatures and decreased softening. The results also showed that stor age of 1-MCP-treated fr uit at 5C for 9 d did not confer any additional be nefits to pulp firmness maintenance as compared with 1-MCP 5C 24 h and No Chill 1-MCP fruit pulp. Comparisons between Control 5C 24 h and 1-MCP 5C 24 h showed significant differences (P < 0.05) in pulp firmness from Day 9 through Day 15. From Day 12 through Day 15, significant differences (P < 0.05) also occurred between No Chill Control and No Chill 1-MCP, as well as between Control 5C 9 d and 1-MCP 5C 9 d. During the latter part of the experiment, C ontrol 5C 24 h pulp was the softest of all treatments (approaching 2 N) while No Chill 1-MCP and 1-MCP 5C 24 h pulp remained firmest at values near 3.4 N. The pulp firm ness data suggest that 1-MCP has a beneficial effect upon pulp firmness retention both during and after 5C storage as compared to non1-MCP-treated fruit. Studies by Trakulnaleumsai et al., (2006) ha ve shown that ripening banana fruit at 12C resulted in decreased fruit softening as compared with fruit ripened at 18C. This

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139 pattern of ripening at low temp eratures resulting in decreased softening was consistently observed in the Control group ri pened at injurious temperatur es. Trakulnaleumsai et al., (2006) also performed low temper ature storage of fruit (12C, 4 d) followed by a return to room temperature (26 to 27C) for the durat ion of the experiment yet no firmness data were presented for these fruit for comparisons to Control 5C 24 h. The firmness data from the 1-MCP group correspond with the findings of Zhang et al., (2006) where the authors reported that 1-MCP application inhibited fruit softening of banana fruit cv. Zhonggang. The only differe nce lies in the patter ns of softening as 1-MCP-treated fruit in the present experiment exhibited more than 50% softening within the first 3 d of storage whereas fruit from Zhang et al., (2006) showed a rapid decline only after 4 d in storage. These differences may be attributed to the fact that fruit in the present experiment were pre-treate d with ethylene before 250 nLL-1 1-MCP application whereas fruit in Zhang et al., (2006) were not pre-treated with ethylene before 200 nLL-1 1-MCP application. Ethylene pre-treatment in the present experiment initiated the fruits’ climacteric period, which would have been underway during the 3 d period during which > 50% of the pulp softening occurred while fruit from Zhang et al., (2006) exhibited delayed climacteric initiation due to decrea sed ethylene perception. Data on respiration and ethylene production rates from Zhang et al., (2006) showed that the climacteric ethylene production and respir ation peaks occurred during the rapid period of fruit softening after 4 d in storage. In addition, cultivar-related differences may also contribute to the slight vari ation in firmness results obser ved in Zhang et al., (2006) compared with the data reported herein.

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140 Total Soluble Sugars Total soluble sugar content of fruit pulp is expressed as a percentage of fresh weight or in milligrams per gram fresh weight (mgg FW-1). Soluble sugar content was initially above 3% (30 mggFW-1) on Day 0. By Day 3, soluble sugar values in the No Chill Control and No Chill 1-MCP fruit were significantly different (P < 0.05) and remained so for the duration of the experi ment (Figure 6-7, Table 6-8). Although No Chill 1-MCP soluble sugar values were significantly lower throughout, they eventually achieved levels above 14% which is near the lo wer range of soluble sugars in ripe banana fruit pulp (Prabha and Bhagyalakshmi, 1998). Between Day 0 and Day 6, a 1-d delay14 in soluble sugar accumulation occurred between the No Chill Control and No Chill 1-MC P fruit. However, by Day 9 (and for the duration of storage), a 3-d delay in soluble sugar accumulation occurred between the No Chill Control and No Chill 1-MCP fruit. The 3-d delay in soluble sugar accumulation seems to be an intermediate delay when compared with the maximum delays15 observed between Control and 1-MCP-trea ted fruit in the 500 and 250 nLL-1 1-MCP treatments previously analyzed (Chapter 5). The experiment utilizing 500 nLL-1 1-MCP (Chapter 5) showed a maximum delay of 6 d between Control and 1-MCP treatments while the experiment utilizing 250 nLL-1 1-MCP (Chapter 5) showed no delay (0 d) in soluble sugar accumulation between Control a nd 1-MCP treatments (Table 6-7). 14 Delays between two treatments were determined by approximating the difference in days required for the treatments to achieve equivalent soluble sugar levels. For example, the No Chill Control fruit required 2 d to achieve 60 mggFW-1 soluble sugars while the No Chill 1-MCP fruit required 3 d to achieve 60 mggFW1. Therefore, the No Chill 1-MCP treatment trailed the No Chill Control treatment by 1 d, hence a 1-d delay in soluble sugar accumulation. This method was used to calculate all delays in soluble sugar accumulation comparisons between any set of treatments. 15 Maximum delays were defined as the largest delay observed between two specified treatments during the experiment.

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141 The intermediate delay in soluble sugar accumulation observed in the present experiment involving 250 nLL-1 1-MCP application may have been related to the fruit’s ripeness upon arrival (as measured by color sc ore). Color score upon arrival is related to the time elapsed between commercial ethylene application and the time of arrival and color score measurement. Fruit from the 500 nLL-1 treatment had an average color score upon arrival of 2.0; fruit from the first 250 nLL-1 treatment had an average color score of 2.6; and fruit from the present 250 nLL-1 treatment had an average color score of 2.2. The data reveal that fruit w ith the lowest color score upon arrival exhibited the greatest delays in sugar accumulation between Cont rol and 1-MCP treatments, fruit with intermediate color score upon a rrival exhibited intermediate delays and fruit with the highest color score upon arrival exhibited the shortest delays (Table 6-7). These results support the notion that as fruit ripen they b ecome less receptive to the effects of 1-MCP and ethylene inhibition (G olding et al., 1998). Comparisons of soluble sugar accumula tion within the Control group showed no significant difference (P < 0.05) between No Chill Control and Control 5C 24 h from Day 9 through Day 15 (Figure 6-7, Table 6-8) . On Day 9, a significant difference in soluble sugar accumulation (P < 0.05) was observed between No Chill Control and Control 5C 9 d with values at 12.8% (128 mggFW-1) and 11.5% (115 mggFW-1), respectively. The significant difference in soluble sugar accumulation (P < 0.05) between No Chill Control and Control 5C 9 d lasted fo r the duration of the experiment with final values on Day 15 at 15.7% (157 mggFW-1) and 13.4% (134 mggFW-1), respectively.

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142 On Day 12 and Day 15, the soluble sugar values of Control 5C 24 h were 14.4% (144 mggFW-1) and 15.5% (155 mggFW-1), respectively, and were significantly different (P < 0.05) from C ontrol 5C 9 d soluble sugar values on those days. The above data illustrate delays in st arch-sugar conversion caused by extended 5C storage. The reduction of tota l soluble sugar levels resul ting from delayed ripening at injurious temperatures observed in the Control group in the present experiment have also been observed in ‘Sucrier’ banana fruit ripe ned at 12C (Trakulnaleu msai et al., 2006). ‘Sucrier’ bananas ripened at 12C exhibited a 2-d dela y in total soluble sugar accumulation when compared with fruit ripe ned at 18C (Trakulnaleumsai et al., 2006) while Control 5C 9 d fruit in the present expe riment showed a 3-d delay in total soluble sugar accumulation when compared with No Chill Control fruit ripened at 18C. Other reports have noted extended that 5C storag e of banana fruit cv. Berangan resulted in increased soluble solids conten t as measured refractometri cally (Ratule et al., 2006). However, increases in soluble solids content may not accurately estimate the soluble sugar content of the fruit because other solu ble compounds such as ascorbic acid, organic acids, amino acids, pectins, pigments and phe nolics are intrinsically incorporated into refractometer readings (Kader et al., 2003). Comparisons of sugar accumulation with in the 1-MCP group s howed significant differences (P < 0.05) occurred between all treatments on Day 9 with the No Chill 1-MCP and 1-MCP 5C 24 h showing a 1.2% (12 mggFW-1) difference, 1-MCP 5C 24 h and 1-MCP 5C 9 d showing a 0.8% (8 mggFW-1) difference and No Chill 1-MCP and 1-MCP 5C 9 d showing a 2.0% (20 mggFW-1) difference. Both No Chill 1-MCP and 1-MCP 5C 24 h continue d along the upward trend and there was no

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143 difference between their soluble sugar levels of 15.7% (145 mggFW-1) and 15.5% (140 mggFW-1), respectively, by Day 15. Soluble sugar values of 1-MCP 5C 9 d on Day 12 and Day 15 were 11.0% (110 mggFW-1) and 11.8% (118 mggFW-1), respectively, and were significantly differe nt (P < 0.05) from both No Chill 1-MCP and 1-MCP 5C 24 h on those days. Jiang et al., (2004b) reported that inhibition of ethylen e action through application of 200 nLL-1 1-MCP (24 h, 20C) had significant effects at delaying soluble sugar accumulation of ‘Zhonggang’ banana fruit. Th ese 1-MCP-treated fruit were ripened at 20C and exhibited a 5-d delay in soluble sugar accumulation as compared to control fruit (Jiang et al., 2004b). This 5-d delay in so luble sugar accumulation was greater than the 3-d delay observed between No Chill Control and No Chill 1-MCP fruit in the present experiment,; however, final soluble sugar leve ls of Control and 1-MCP-treated fruit from both Jiang et al., (2004b) and the present expe riment reached levels above 14%. Jiang et al., (2004b) also reported th at 1-MCP-treated fruit ripene d at 13C resulted in more than a 10-d delay in soluble sugar accumulation as compared with 1-MCP-treated fruit ripened at 20C. The results from the present experiment support the findings that 1-MCP treatment and extended low temper ature storage sign ificantly delayed accumulation of total soluble sugars and may be an effective means for extending postharvest shelf life of banana fruit. Peel Soluble Phenolic Compounds Soluble phenolic compounds in the p eel tissue were initially 1.3 mggFW-1 on Day 0 (Figure 6-8). Significant differen ces (P < 0.05) between No Chill Control and No Chill 1-MCP fruit began on Day 6 where soluble phenolic levels were 2.0 and 1.7 mggFW-1, respectively. This significan t difference (P < 0.05) between

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144 No Chill Control and No Chill 1-MCP fruit pheno lic levels lasted for the duration of the experiment and the difference ranged from 0.53 mggFW-1 on Day 9 to 0.77 mggFW-1 on Day 15 (Table 6-9). Comparisons within the Control group on Day 9 showed significant differences (P < 0.05) in soluble phenolic levels betw een No Chill Control and Control 5C 24 h of 0.34 mggFW-1, while No Chill Control and Control 5C 9 d showed a difference of 0.22 mggFW-1. No significant difference (P < 0.05) in soluble phenolic levels was observed between Control 5C 24 h and Cont rol 5C 9 d on Day 9 or any other day thereafter. By Day 15 a significan t difference (P < 0.05) of 0.39 mggFW-1 was observed between No Chill Control and Control 5 C 24 h soluble phenolic levels, while a significant difference (P < 0.05) of 0.48 mggFW-1 was observed between No Chill Control and Control 5C 9 d. The only significant difference (P < 0.05) in soluble phenolic levels observed within the 1-MCP group on Day 9 was between No Chill 1-MCP and 1-MCP 5C 9 d that showed a difference of 0.22 mggFW-1. This 0.22 mggFW-1 difference in soluble phenolic levels was equivalent to the di fference observed on Day 9 between No Chill Control and Control 5C 9 d indicating that the effects of chilling on soluble phenolic compound accumulation were expressed equall y in both the Control and 1-MCP groups during the 3-d period after 5C storage. By Day 15, all treatments within the 1-MCP group showed significantly differences (P < 0.05) in soluble phenolic levels with No Chill 1-MCP reaching levels of 3.13 mggFW-1, 1-MCP 5C 24 h reaching levels of 2.54 mggFW-1, and 1-MCP 5C 9 d reaching levels of 2.34 mggFW-1.

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145 Trakulnaleumsai et al., (2006) observed a f our-fold increase in soluble phenolic levels in the peel of control fruit ripened at 26C over an 8-d peri od while Choehom et al., (2004) observed both a twoand a four-fold increa se in soluble phenolic levels in peel of control fruit ripened at 30C over a 6-d period. In general agreement with the studies of these authors, the present experiment show ed a four-fold increase in total soluble phenolics in the peel of No Chill Control fr uit ripened at 18C over a 15-d period. The higher ripening temperatures utilized in Trakulnaleumsai et al., (2006) and Choehom et al., (2004) are the most likely cause of th e accelerated rate of peel soluble phenolics accumulation as it is known that higher temper atures result in more rapid fruit ripening (Kader 2002b, Kerbel 2004). The decreased levels of soluble phenolics observed in No Chill 1-MCP as compared with No Chill C ontrol was likely due to delayed ripening caused by lack of ethylene perception, as wa s observed for other ripening parameters including soluble sugar accumulation and peel color development. This phenomenon of decreased ethylene perception causing re duced rates of total soluble phenolics accumulation was also observed in the two previous experiments involving 500 and 250 nLL-1 1-MCP application (Chapter 5). The main effect of low temperature stor age on Control 5C 24 h, Control 5C 9 d, 1-MCP 5C 24 h and 1-MCP 5C 9 d was to decrease the rate of soluble phenolic accumulation as compared to the No Chill Control and No Chill 1-MCP counterparts. The cause of the reduced solubl e phenolic levels in fruit st ored at 5C is most likely related to the utiliz ation of phenolic compounds by pol yphenol oxidase (PPO) in peel browning reactions caused by membrane disint egrations during low temperature storage (Maragoni et al., 1996; Yoruk et al., 2003b; Nguyen et al., 2003). While the present

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146 experiment showed that 5C storage reduced the rate of soluble phenolic accumulation, there was still an overall upward trend over ti me (Figure 6-8). An increase in soluble phenolic accumulation was not observed in Nguye n et al., (2003) where ‘Sucrier’ banana fruit ripened at 10C showed decreasing levels of soluble phenolics over time with a more rapid decrease in fruit ripened16 at 6C. Nguyen et al., (2004) also noted a downward trend of soluble phenolic levels in ‘Sucrier’ banana fruit ripened at 10C, both with and without modified atmosphere p ackaging (MAP). Experiments conducted by Choehom et al., (2004) invol ving ‘Sucrier’ banana fruit and MAP reported overall increases (upward trends) in soluble pheno lic levels in fru it ripened at 30C. The upward trends in soluble phenolic leve ls observed in the present experiment and by Choehom et al., (2004) were possibly due to the rate of soluble phenolic substrate depletion (by PPO in the CI-induced browni ng reactions) being less than the rate of soluble phenolics synthesis associated w ith normal ripening. Conversely, the downward trends observed by Nguyen et al., (2003) woul d indicate that low temperature storage caused faster substrate depletion than accumulation. Regardless of trends, the inverse relationship between decreasing soluble phe nolic levels corresponding to increasing incidence of peel CI symptoms noted by N guyen et al., (2003) was consistently observed in the present experiment. This inverse re lationship between CI occurrence and phenolic levels was most apparent in Control 5C 9 d and 1-MCP 5C 9 d fruit as these treatments exhibited the highest CI rati ngs (Figure 6-2) and the lowe st soluble phenolic levels (Figure 6-8) when compared with their No Chill counterparts. It is worth noting that 16 The experiment focused on CI effects on phenolic compounds, hence ripening in the sense of edibility was not reported. It is assumed, however that fruit held at 6C for extended periods of time did not exhibit characteristic peel color change and ripening patte rns of fruit ripened at ambient temperatures.

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147 while the CI ratings of Control 5C 9 d and 1-MCP 5C 9 d were not significantly different on Day 12 and 15, their soluble pheno lic levels were signi ficantly different (P < 0.05). This observation i ndicates that there were physio logical effects not manifest completely in the CI ratings and therefore there was a discrepancy between visual CI assessment and the data derive d from laboratory analysis. Peel chlorogenic acid cont ent was initially 0.2 mggFW-1 on Day 0 (Figure 6-9). A significant difference (P < 0.05) in chlorogenic acid content of 0.092 mggFW-1 was observed between the No Chill Control and No Chill 1-MCP fruit on Day 3, yet the magnitude of the differences may not be accurate as the difference decreased to 0.04 mggFW-1 by Day 6 indicating no signi ficant difference (P < 0.05) (Table 6-10). On Day 9 and Day 12, however, the No Chill Control and No Chill 1-MCP fruit showed a significant difference (P < 0.05) in ch lorogenic acid content of 0.06 mggFW-1 which increased to 0.08 mggFW-1 by Day 15. Comparisons of chlorogenic acid cont ent within the Cont rol group showed no significant differences (P < 0.05) on Day 9 or Day 12. By Day 15, however, all treatments were significantly different (P < 0.05) with the No Chill Control reaching levels of 0.64 mggFW-1, Control 5C 24 h reaching levels of 0.61 mggFW-1, and Control 5C 9 d reaching levels of 0.56 mggFW-1. Within the 1-MCP group, significant differences (P < 0.05) in chlorogenic acid content ranging from 0.05 to 0.03 mggFW-1 were observed between No Chill 1-MCP a nd 1-MCP 5C 9 d on Day 9 through Day 15. Comparisons of chlorogenic acid content between No Chill 1-MCP and 1-MCP 5C 24 h showed a significant difference (P < 0.05) of 0.04 mggFW-1 only on Day 12.

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148 Final chlorogenic acid levels ranged between 0.5 and 0.65 mggFW-1 in all treatments, values slightly higher than those observed in previous experiments (Chapter 5). Higher final chlorogenic acid levels are likely due to physiological differences in fruit shipments as banana fru it arrive from a variety of Central and South American countries that vary in envi ronmental conditions (precipitation, soils, temperature etc.) at similar times of year and within each country based on the growing season. This variation leads to physiological differences in the fruits that are most apparent in comparisons between fruit from di fferent shipments at different times of year (Harris et al. 2000, Moradi nezhad et al., 2006). Little has been mentioned regarding ch lorogenic acid content in banana peel outside of Palmer (1963) and Kondo et al., (2005 ). Utilizing HPLC, Kondo et al., (2005) did not find detectable leve ls of cholorgenic acid in the peel of banana fruit17 harvested 84 days after full bloom. Data from the pres ent experiment showed extremely low levels of cholorgenic acid measured at less than 0.2 mggFW-1 in banana peel at a color score slightly above 2. The upward trends in chol orgenic acid content indicated that this phenolic compound like others may be synthesize d as ripening proceeds. The differences between samples from the Control and 1MCP groups suggest that synthesis or accumulation may be disrupted by inhibition of ethylene action and low-temperature storage. Peel Polyphenol Oxidase Activity Peel polyphenol oxidase (PPO ) activity was expressed in units where one unit was defined as the amount of enzy me activity that caused a cha nge in absorbance at 420 nm 17 It is assumed these fruit were green and at a color score no greater than 2.

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149 of 1.0 over a 3 min. period, per milligram protein (units mg protein-1). PPO activity was less than 1 activity unit (AU) on Day 0. PPO activity of No Chill Control and No Chill 1-MCP peel samples diverged significantly (P < 0.05) by Day 3 with values at 5.7 and 3.6 AU, respectively (Figure 6-10, Table 611). PPO AU of No Chill Control and No Chill 1-MCP peel samples remained significantly different for the duration of the experiment with the greatest difference of 4.6 AU occurring on Day 9 and the least difference of 2.6 AU occurring on Day 12. Final PPO AU from No Chill Control and No Chill 1-MCP peel samples were near 13.7 and 10.7 units, respectively, on Day 15. On Day 9, PPO AU from the Control group peel samples showed No Chill Control at 10.7 AU, a value significantly lower (P < 0.05) than both Control 5C 24 h and Control 5C 9 d PPO activity measured at 13.0 and 12.2 AU, respectively. The 1.5 to 2.3 AU increase exhibited by Control 5C 24 h a nd Control 5C 9 d sugg est that 24 h, 5C and extended 5C exposure cont ributed to the increased PPO activity measured. By Day 15 No Chill Control PPO activity was m easured at 13.7 AU, a value significantly different (P < 0.05) from C ontrol 5C 9 d PPO activity at 14.8 AU, but not significantly different (P < 0.05) than Control 5C 24 h PPO activity at 14.1 AU. Day 15 also showed a significant difference (P < 0.05) between PPO activity of Control 5C 24 h and Control 5C 9 d. Results from the 1-MCP group on Day 9 showed PPO activity of No Chill 1-MCP at 6.1 AU. This 6.1 AU was significantly diffe rent (P < 0.05) from both 1-MCP 5C 24 h and 1-MCP 5C 9 d PPO activities measur ed at 7.4 and 8.4 AU, respectively. The significant differences (P < 0.05) be tween No Chill 1-MCP and 1-MCP 5C 24 h on Day 9 lasted for the duration of the experiment with final PPO AU of No Chill 1-MCP

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150 and 1-MCP 5C 24 h d at 10.7 and 12.1 AU, re spectively. The significant differences (P < 0.05) observed between No Chill 1-MCP and 1-MCP 5C 9 d on Day 9 also lasted for the duration of the experiment with fi nal PPO AU at 10.7 and 12.4 AU, respectively. From Day 9 through Day 15 no significant differences (P < 0.05) occurred between 1-MCP 5C 24 h and 1-MCP 5C 9 d. The effects of inhibiting ethylene perception were manifest in the PPO data as 1-MCP-treated fruit showed the lowest PPO activity when compared to their Control counterparts. Decreased PPO activity in re sponse to inhibition of ethylene perception was observed in the previous experiments involving 500 and 250 nLL-1 1-MCP treatment (Chapter 5) yet little has been men tioned in the literature in regards to 1-MCP effects on banana peel PPO activity. However, 1-MCP application (5 LL-1, 12 h, 20C) to loquat fruit18 ( Eriobotrya japonica Lindl. cv. Luoyangqing) resulted in significantly lower PPO activity in pulp tissue samples from fruit ripened at 20C fo r 8 d (Cai et al., 2006). Similarly, 1-MCP application (300 nLL-1, 18 h, 20C) to three different avocado varieties was shown to signi ficantly decrease mesocarp PPO activity after 3.5 weeks at 5C as well as after this chilling period was followed by 1 week at 20C (Hershkovitz et al., 2005). While the data from the previous experiments involving 500 and 250 nLL-1 1-MCP treatment (Chapter 5) revealed that inhi bition of ethylene acti on caused decreased PPO activity, the findings of the pr esent experiment showed that low temperature storage increased PPO activity as the highest PPO ac tivity (within the Cont rol or 1-MCP group, 18 Loquat fruit has been classified by some as a non-climacteric tropical fruit (Kader 2002c; Blumenfeld 1980) while othe r reports classify it as a low to medium climacteric tropical fruit (Amoros et al ., 2006; Hamauzu et al., 1997).

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151 respectively) was measured in fruit stored at 5C, 9 d followed by fru it stored at 5C, 24 h and lastly No Chill fruit. These results support the findings of Nguyen et al., (2003) where peel from fruit stored at 6C showed higher levels of PPO activity as ripening proceeded than did peel from fruit stored at 10C. It is known that low temperature storag e causes cellular membrane disruptions (Maragoni et al., 1996) and abundant PPO-phe nolic compound interactions resulting in peel browning amongst other CI symptoms (N guyen et al., 2003). While these cellular disruptions represent disintegrative processes, it is likely that low temperature also results in coinciding synthesis processes such as increased transcription and/or translation involved in protein synthesis (Gooding et al., 2001; Nguyen et al., 2003) as this has been suggested to be occurring in the case of PAL induction du ring low temperature storage (Wang et al., 2007). These synthesis proces ses may play a role in the increased in-vivo PPO activity observed in the CI inde x (Figure 6-2) and the increased in-vitro PPO activity measured photometrically (Figure 6-10 ). Both PAL and PPO are involved in the browning pathway utilized by fruits to produ ce melanin pigments from the amino acid phenylalanine (Marshall et al., 2000), theref ore synthesis of these enzymes may be regulated by similar environmental factors in cluding low temperature storage. Further studies must be conducted to specifically identify such processes as related to PPO induction and activity as has been done with PAL. On a larger scale, synthesis processes induced by low temperature storage may be part of natural plant defense systems involved in inhibiting and/or ameliorating CI and other detrimental low temperature effects (Kondo et al., 2005).

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152 Conclusions The results of the present experiment s showed that application of 250 nLL-1 1-MCP in conjunction with 5C storage for 24 h or 9 d caused greater delays in banana fruit ripening than observed with low temper ature storage or 1-MCP treatment alone. While peel color development of No Chill 1-MCP and 1-MCP 5C 24 h surpassed color score 5, these fruit still lacked the distinct yellow color characteristic of their Control counterparts. However, beyond color sc ore 5, pulp of No Chill 1-MCP and 1-MCP 5C 24 h softened less than their C ontrol counterparts. Therefore diminishing ethylene perception may be desirable for main taining pulp firmness in situations where short-term low temperature storage is require d to delay ripening and where peel color is of no concern. Color development of 1-MCP 5C 9 d never surpassed color score 5 and the firmness results from this treatment show ed more rapid softening (as compared to No Chill 1-MCP and 1-MCP 5C 24 h). Conseque ntly, extended 5C storage may not be desirable for 1-MCP-treated fruit. The data confirmed that 1-MCP treatment did not increase the incidence of peel graying or increase the degree of fungal a ppearance during ripening at 18C. Peel graying, however, became blurred with chillin g-induced peel discoloration in all fruit stored at 5C. It was therefore concluded th at there are two separa te graying disorders, chilling-induced graying and nonchilling-induced graying, wh ich occur independently of each other and originate from different preand postharvest circumstances. Closer scrutiny of production and postharvest marketi ng conditions are required to determine the cause of non-chilling-induced graying. At that point a clear distinction could be made in identifying and quantifying non-chilling-indu ced graying from chilling-induced graying.

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153 Soluble sugars achieved acceptable levels (14 to 16%) in all Control and 1-MCP treatments (excluding 1-MCP 5C 9 d which r eached 12%), therefore the combination of 250 nLL-1 1-MCP treatment with 24 h, 5C storage is presumed acceptable for inducing slight delays in soluble sugar accumulation and fruit ripening. This combination of decreased ethylene perception and low temperatur e storage also resulted in reduced total phenolic levels while chlorogenic acid levels ap peared to be less affected. In regards to the PPO data, it was concluded that PPO activ ity exhibited a negativ e relationship with storage temperature and a positive relationship w ith duration of storage at 5C. The fact that peel CI symptoms were manifest re gardless of 1-MCP-trea ted fruits’ decreased ability to perceive ethylene indicated that CI development may not be an ethylenedependent process. While 24 h, 5C storage negatively affected fruit external appearance in all treatments, subjective visual analysis reveal ed 1-MCP-treated fruit exhibited superior pulp appearance after 24 h, 5C storage as comp ared to Control fruit held in identical conditions . Maintained firmne ss, adequate soluble sugar le vels and accepta ble internal appearance of 1-MCP-treat ed fruit lead to the conclusion that 250 nLL-11-MCP application in conjunction with 24 h, 5C st orage could be utilized as a postharvest treatment to delay fruit ripeni ng in the food service, freshcut or processing industries where peel appearance and external CI symp toms are not a determin ing factor in fruit quality. Furthermore, extended 5C storage could be utilized after fruit have surpassed an acceptable ripeness stage (i.e. Day 9 when fru it are clearly past color score 4 and/or soluble sugars are above 10%). In all cases, further analysis of fruit flavor is warranted as this quality parameter was not explored in this study. Taste tests such as those

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154 conducted by Salvador et al., (2006) could be utilized to determine the effects of decreased ethylene perception and low temperat ure storage on banana fruit flavor as subjective taste tests revealed specific preferences and dislikes for 1-MCP-treated fruit.

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155 Table 6-1. Horsfall-Barrett scale units used to quantify senescent spotting development. Units Percent of fruit surface affected 0 0% 1 0.1 to 3.0% 2 3.1 to 6.0% 3 6.1 to 12% 4 12.1 to 25% 5 25.1 to 50% 6 50.1 to 75% 7 75.1 to 88% 8 88.1 to 94% 9 94.1 to 97% 10 97.1 to 99.9% 11 100% Table 6-2. Control and 1-MCP treatment groups 5C storage re gimes beginning Day 6. Group label 1-MCP treatment 5C st orage duration Treatment label Control none no chill No Chill Control Control none 24 h Control 5C, 24 h Control none 9 d Control 5C, 9 d 1-MCP 250 nLL-1 1-MCP no chill No Chill 1-MCP 1-MCP 250 nLL-1 1-MCP 24 h 1-MCP 5C, 24 h 1-MCP 250 nLL-1 1-MCP 9 d 1-MCP 5C, 9 d

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156 Table 6-3. Peel color comp arisons between specified tr eatments before CI storage (Day 6) and after removal from CI storage (Day 9, Day 15). Peel color comparison* Day 6 Day 9 Day 15 No Chill Control vs. Control 5C 24 h nd ns ns Control 5C 24 h vs. Control 5C 9 d nd ns s No Chill Control vs. Control 5C 9 d nd s s No Chill 1-MCP vs. 1-MCP 5C 24 h nd s s 1-MCP 5C 24 h vs. 1MCP 5C 9 d nd ns s No Chill 1-MCP vs. 1-MCP 5C 9 d nd s s No Chill Control vs. No Chill 1-MCP s s s Control 5C 24 h vs. 1-MCP 5C 24 h nd s s Control 5C 9 d vs. 1-MCP 5C 9 d nd s s *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6. Table 6-4. CI development co mparisons between specified treatments after removal from 5C, 80 to 90% RH, storage (Day 7, Day 9, Day 15). CI development comparison* Day 7 Day 9 Day 15 No Chill Control vs. Control 5C 24 h ns s s Control 5C 24 h vs. Control 5C 9 d ns s s No Chill Control vs. Control 5C 9 d ns s s No Chill 1-MCP vs. 1-MCP 5C 24 h ns s s 1-MCP 5C 24 h vs. 1-MCP 5C 9 d ns s s No Chill 1-MCP vs. 1-MCP 5C 9 d ns s s ns No Chill Control vs. No Chill 1-MCP ns ns ns Control 5C 24 h vs. 1-MCP 5C 24 h ns ns ns Control 5C 9 d vs. 1-MCP 5C 9 d ns s ns *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6.

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157 Table 6-5. Senescent spotting development comparisons between specified treatments before CI storage (Day 6) and after rem oval from CI storage (Day 9, Day 15). Senescent spotting comparison* Day 6 Day 9 Day 15 No Chill Control vs. Control 5C 24 h nd ns ns Control 5C 24 h vs. Control 5C 9 d nd ns ns No Chill Control vs. Control 5C 9 d nd ns ns nd ns ns No Chill 1-MCP vs. 1-MCP 5C 24 h nd ns ns 1-MCP 5C 24 h vs. 1-MC P 5C 9 d nd ns ns No Chill 1-MCP vs. 1-MCP 5C 9 d nd ns ns No Chill Control vs. No Chill 1-MCP s s s Control 5C 24 h vs. 1-MCP 5C 24 h nd s s Control 5C 9 d vs. 1-MCP 5C 9 d nd s s *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6. Table 6-6. Fruit firmness comparisons between specified treatments before CI storage (Day 6) and after removal from CI storage (Day 9, Day 15). Fruit firmness comparison* Day 6 Day 9 Day 15 No Chill Control vs. Control 5C 24 h nd ns ns Control 5C 24 h vs. Control 5C 9 d nd ns S No Chill Control vs. Control 5C 9 d nd ns ns ns ns No Chill 1-MCP vs. 1-MCP 5C 24 h nd ns ns 1-MCP 5C 24 h vs. 1-MC P 5C 9 d nd ns ns No Chill 1-MCP vs. 1-MCP 5C 9 d nd ns ns No Chill Control vs. No Chill 1-MCP s s s Control 5C 24 h vs. 1-MCP 5C 24 h nd s s Control 5C 9 d vs. 1-MCP 5C 9 d nd ns s *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6. Table 6-7. Maximum delays observed in so luble sugar accumulation between Control and 1-MCP-treated fruit corre lated with fruit color scor e upon arrival (Day 0). Chapter indicates where the data were presented. Experiment Chapter Color score Maximum delay Control vs. 500 nLL-1 1-MCP 5 2.0 6 d Control vs. 250 nLL-1 1-MCP 5 2.6 0 d No Chill Control vs. 250 nLL-1 1-MCP 6 2.2 3 d

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158 Table 6-8. Soluble sugar content comparisons between specified treatments before CI storage (Day 6) and after removal from CI storage (Day 9, Day 15). Soluble sugar content comparison* Day 6 Day 9 Day 15 No Chill Control vs. Control 5C 24 h nd ns NSns Control 5C 24 h vs. Control 5C 9 d nd ns s No Chill Control vs. Control 5C 9 d nd s s No Chill 1-MCP vs. 1-MCP 5C 24 h nd s ns 1-MCP 5C 24 h vs. 1-MCP 5C 9 d nd s s No Chill 1-MCP vs. 1-MCP 5C 9 d nd s s No Chill Control vs. No Chill 1-MCP s s s Control 5C 24 h vs. 1-MCP 5C 24 h nd s s Control 5C 9 d vs. 1-MCP 5C 9 d nd s s *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6. Table 6-9. Peel soluble phenolics content comparisons between specified treatments before CI storage (Day 6) and after re moval from CI storage (Day 9, Day 15). Peel phenolics content comparison* Day 6 Day 9 Day 15 No Chill Control vs. Control 5C 24 h nd s s Control 5C 24 h vs. Control 5C 9 d nd ns ns No Chill Control vs. Control 5C 9 d nd s s No Chill 1-MCP vs. 1-MCP 5C 24 h nd ns s 1-MCP 5C 24 h vs. 1MCP 5C 9 d nd ns s No Chill 1-MCP vs. 1-MCP 5C 9 d nd s s No Chill Control vs. No Chill 1-MCP s s s Control 5C 24 h vs. 1-MCP 5C 24 h nd s s Control 5C 9 d vs. 1-MCP 5C 9 d nd s s *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6.

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159 Table 6-10. Chlorogenic acid content comparis ons between specified treatments before CI storage (Day 6) and after remova l from CI storage (Day 9, Day 15). Chlorogenic acid content comparison* Day 6 Day 9 Day 15 No Chill Control vs. Control 5C 24 h nd ns s Control 5C 24 h vs. Control 5C 9 d nd ns s No Chill Control vs. Control 5C 9 d nd ns s No Chill 1-MCP vs. 1-MCP 5C 24 h nd ns ns 1-MCP 5C 24 h vs. 1-MCP 5C 9 d nd s s No Chill 1-MCP vs. 1-MCP 5C 9 d nd s s No Chill Control vs. No Chill 1-MCP ns s s Control 5C 24 h vs. 1-MCP 5C 24 h nd s s Control 5C 9 d vs. 1-MCP 5C 9 d nd s s *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6. Table 6-11. Polyphenol oxidase (PPO) ac tivity comparisons between specified treatments before CI storage (Day 6) and after removal from CI storage (Day 9, Day 15). PPO activity comparison* Day 6 Day 9 Day 15 No Chill Control vs. Control 5C 24 h nd s ns Control 5C 24 h vs. Control 5C 9 d nd ns s No Chill Control vs. Control 5C 9 d nd s s No Chill 1-MCP vs. 1-MCP 5C 24 h nd s s 1-MCP 5C 24 h vs. 1-MC P 5C 9 d nd ns ns No Chill 1-MCP vs. 1-MCP 5C 9 d nd s s No Chill Control vs. No Chill 1-MCP s s s Control 5C 24 h vs. 1-MCP 5C 24 h nd s s Control 5C 9 d vs. 1-MCP 5C 9 d nd s s *s = significant difference (P < 0.05); ns = not significant; nd = no data as the 5C treatments occurred after Day 6.

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160 2 3 4 5 6 7 03691215 Da y s in Stora g e Color Score Commercial Ripening Chart No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-1. Peel color in res ponse to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH. At Day 6, two-thirds of the Control and 1-MCP-treated fruit were transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C were returned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. For Day 0, 3 and 6 n=18; Day 9, 12 and 15 n=6. Vertical bars represent standard error.

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161 1 2 3 4 5 036791215 Days in StorageChilling Injury Index No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-2. Peel chilling injury in response to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH. At Day 6, two-thirds of the Control and 1-MCP-treated fruit were transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C were returned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. For Day 0, 3 and 6 n=18; Day 9, 12 and 15 n=6. Vertical bars represent standard error.

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162 0 1 2 3 4 5 6 03691215 Da y s in Stora g e Senescent Spotting Horsfall-Barrett Scale No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-3. Peel senescent spot ting in response to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH . At Day 6, two-thirds of the Control and 1-MCP-treated fruit we re transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C were retu rned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. For Day 0, 3 and 6 n=18; Day 9, 12 and 15 n=6. Vertical bars represent standard error.

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163 0 10 20 30 40 50 60 70 03691215 Days in StorageFruit Exhibiting Graying (%) No Chill Control No Chill 1-MCP Figure 6-4. Non-chilling-induced peel grayi ng of Control and 1-MCP-treated fruit during storage (18C, 80 to 90% RH). Each poi nt represents the percentage of the total hands exhibiting graying. (n=18). Standard error bars are not present in the graying graph because the “present” or “absent” method of measurement does not produce this type of statistic.

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164 0 10 20 30 40 50 60 70 03691215 Da y s in Stora g e Fruit Exhibiting Fungal Infection (%) No Chill Control No Chill 1-MCP Figure 6-5. Incidence of funga l infection of intact Control and 1-MCP-treated fruit during storage (18C, 80 to 90% RH). E ach point represents the percentage of the total hands exhibiting mycelia. (n =18). Standard error bars are not present in the fungal incidence graphs because the “present” or “absent” method of measurement does not pro duce this type of statistic.

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165 2 4 6 8 10 03691215 Days in StorageFirmness @ Max Load (Newtons) No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-6. Fruit mesocarp firmness in response to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH . At Day 6, two-thirds of the Control and 1-MCP-treated fruit we re transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C were retu rned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. (n=3). Vertical bars represent standard error.

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166 20 40 60 80 100 120 140 160 03691215 Days in StorageSoluble Sugar Content (mg/gFW) No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-7. Soluble sugar content in response to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH . At Day 6, two-thirds of the Control and 1-MCP-treated fruit we re transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C were retu rned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. (n=3). Vertical bars represent standard error.

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167 1 1.5 2 2.5 3 3.5 4 03691215 Days in StorageTotal Phenolics Content (mg/gFW) No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-8. Peel soluble phenolic s content in response to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH. At Day 6, two-thirds of the Control and 1-MCP-treated fruit were transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C we re returned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. (n=3). Vertical ba rs represent standard error.

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168 0.1 0.2 0.3 0.4 0.5 0.6 0.7 03691215 Days in StorageChlorogenic Acid Content (mg/gFW) No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-9. Peel chlorogenic acid content in response to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH. At Day 6, two-thirds of the Control and 1-MCP-treated fruit were transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C we re returned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. (n=3). Vertical ba rs represent standard error.

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169 0 2 4 6 8 10 12 14 16 03691215 Days in StorageTotal Activity (Units/mg protien) No Chill Control Control 5C, 24 h Control 5C, 9 d No Chill 1-MCP 1-MCP 5C, 24 h 1-MCP 5C, 9 d Figure 6-10. Total PPO activity in response to low-temperature. For the first 6 d, all fruit were stored at 18C, 80 to 90% RH. At Day 6, two-thirds of the Control and 1-MCP-treated fruit were transferred to 5C, 80 to 90% RH. After 24 h, half of the fruit at 5C were returned to 18C, 80 to 90% RH and the remaining fruit maintained at 5C, 80 to 90% RH for the remainder of the experiment. (n=3). Vertical bars represent standard error.

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170 CHAPTER 7 FINAL CONCLUSIONS Conclusions Suppressing ethylene action of commerci ally marketed banana fruit through 1-MCP application caused in delays in all ripe ning parameters measured. Visible delays in peel color change and senescent spotting occurrence are beneficial yet peel color of 1-MCP-treated fruit never achieved the charac teristic yellow color consumers use for determining ripeness and fruit quality. Mainte nance of textural prope rties such as wholeand fresh-cut fruit firmness are desirable qua lities for processing industries where peel color is not an issue. Delayed accumulation of intrinsic pulp and peel components such as phenolic compounds and soluble sugars also reveal the distinct effects 1-MCP has on fruit ripening. Implications for Experimental and Research Settings 1-MCP remains an excellent tool for understanding how ethylene affects fruit ripening in climacteric and non-climacteric frui t. Experimental an alysis of individual ripening parameters has reinfor ced previous studies’ findings and has also contributed to the current body of knowledge pertaining to the physiological mech anisms of banana fruit ripening affected by 1-MCP application under controlled conditions. The data has also opened the door to furthe r exploration of the physiological mechanisms behind the appearance of non-chilling-indu ced peel graying and how ri pening-related events are affected by low temperature storage.

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171 Implications for the Postharvest Industry The approval of 1-MCP for selected commercial uses by the Food and Drug Administration has allowed fresh fruit producer s the opportunity to extend fruit shelf-life while incurring minimal costs to do so. L onger fruit shelf-life implies decreased losses due to quality degradation and decay. In econom ic terms, this potentially translates into increased sales and revenue from an identical quantity of fruit produced which equals larger profit margins. The escalating demand for 1-MCP also presents benefits for the manufacturer/patent holder of 1-MCP as they may profit from increased sales of the chemical. Implications for Consumers In the future, 1-MCP may allow consum ers access to fruit with longer at-home shelf-life. Extended at-home shelf-life also means extended commer cial shelf-life, the idea of which may erode consumer s’ perception that the fruit is “fresh.” Consideration must also be given to labeling as consumer s have the right to know what chemicals are applied to their food supply. Finally, alt hough subjective instrument/laboratory analysis is important for determining individual qua lity components, there is no substitution for human perception of overall qual ity (all attributes combined) as this is the final deciding factor in all repurchas ing decisions. The next step in determining the practicality of applying 1-MCP to commercially handled ba nana fruit would be to conduct objective taste tests to determine how overall fruit flavor and quality are affected by 1-MCP application.

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172 APPENDIX POSTHARVEST SUPPLY CHAIN: PLANT PHYSIOLOGY, ECONOMICS AND FOOD SCIENCE Introduction Postharvest physiology is the study of biologi cal processes associated with ripening and senescence of harvested plants and plant organs during handling a nd storage. Proper management of the postharvest marketing chai n is of utmost impor tance to agricultural producers, postharvest handlers and final cons umers because it is estimated that nearly two-thirds of the retail price of fresh produ ce is due to postharvest handling and storage costs (Kader 2002). These costs are associat ed with harvesting field/greenhouse grown agricultural commodities, cleaning and removi ng farm by-products, sorting for defective units, grading for size, shape, degree of ripeness and/or color, packaging for shipping, shipping, wholesale storage, and eventual re tail display until the product is purchased by the final consumer. In some cases, value adde d steps are incorporated into the procedure to provide consumers with a product that’s conveniently ready to eat. Commercial Postharvest Handling of Banana Fruit Banana production areas are geographical ly isolated from many of the major consumer markets in temperate regions. Th e negative correlation between ripening and shipping endurance requires harv est of banana fruit in a gr een, pre-ripe, pre-climacteric stage to facilitate pos tharvest handling and shipping. Photos of banana plantation and processing were taken with permission in Siqu erres, Costa Rica, while photos of banana ripening and shipping facilities were taken with permission in St. Croix, U.S.V.I. and Bradenton, FL by Daniel A. Stanley. Field Harvest Bagging of the bunch is done two weeks afte r fruit emergence to protect the fruit from cosmetic damage due to wind scar a nd insect herbivory (F igure A-1). During harvest the blue bags are tied above the bunc h and foam pads are placed between bands to prevent bruising and abrasi on (Object 1). Fruit are hand harvested at a physiologically and horticulturally mature green stage 90-120 days after anthesis. Fru it bunch is cut from the stem end and carried to the banana ra il system where it is mounted (Figure A-2, Object 1). After bunch harvest the plant is cu t down to allow the small shoot to grow and eventually produce fruit. When full, the bana na rail is manually pulled from the field into the processing area where foam pads are removed (Figure A-3, Object 1).

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173 Banana Processing Plant Activities No pre-cooling is done to harvested fruit before processing pro cedures. Processing begins with the removal of distal hands de stined for local markets and progresses up the bunch to proximal hands destined for export ma rkets (Figure A-4A, Object 1). Hands are immediately placed in fungicide dips which function to decrease pathogen inoculum and as a wash to remove latex and field debris (Figure A-4B). Si ngle fruit and visibly damaged hands are eliminated while hands ar e and transferred to the second fungicide dip (Figure A-5). Hands are removed fr om the second fungicide dip and placed on drying trays mounted on roller bars for ease of transport (Figure A-6) . Trays are rolled down the line to the location where the stem wound sealant is applied to prevent pathogen attack on wounded tissue and prevent fr uit desiccation (Figur e A-7). Fruit are then weighed and boxed in corrugated cardboard boxes with paper padding for protection from physical damage and polyethylene pl astic liners to re duce fruit desiccation (Figure A-8). Boxed fruit are then manually palletized for shipping (Figure A-9). The boxes have holes designed to allow airflow through the boxes when palletized. Pallets are then loaded onto refrigerated trucks (15 to 18C) for transport to embarkation ports. At this point the cold chain begins and is maintained throughout the postharvest handling chain until retail display. Storage and Transportation Palletized boxes are loaded onto trucks then transported to seaports to be loaded onto boats for international shipping. Boats ma y hold entire trailers in on their deck or individual pallets may be loaded into the cargo hold of the ships where they may be subject to modified atmosphere conditions. Fruit cargo are sea-sh ipped to destination ports such as Miami and Tampa in Florida and others throughout the United States and European Union. Upon arrival in destination ports trailers are unl oaded and trucked to ripening facilities (Figure A-10, Figure A-11). Upon arrival at ripening facilities, fruit pallets are unloaded from trailers and me rchants take random samples of fruit from various boxes to measure pulp temperature to ensure proper ha ndling has occurred (Figure A-12). Postharvest Ripening Facilities and Procedures Fruit pallets are unloaded into ripeni ng rooms where they will undergo ethylene treatment and storage until shipped to retail locations. Ripening rooms are designed to maximize airflow and ethylene circulation when pallets are properly placed (Figure A-13). Ethylene generators are either stationary or portable for in trailer ripening (Figure A-14). Stationary un its are controlled by external control panels while portable units have all controls on the unit. Both utilize an ethanol based liquid to generate ethylene. Fruit are usually gassed at concentrations between above 300 LL-1 for 24 h at 15C and 90% relative humidity (RH) (Figur e A-15). Pallets are then reloaded into refrigerated trailers at 15C (59F) and shipped to retailers within 4 to 6 days of ethylene gassing (Figure A-16). Fruit arri ve at retail locati ons between stage 3 to 4 and are retail displayed at ambient temperatures at stag e 5 due to increased consumer purchasing preferences for this stage (Figure A-17).

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174 A B Figure A-1. Perforated polyeth ylene bag covering banana fruit. A) Fruit with bag during development. B) Removal of bag during harvest. A B Figure A-2. Fruit harvest. A) Foam pads placed between hands to prevent bruising and abrasion during transport. B) Transpor tation to on-site pr ocessing plant via banana railway system.

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175 Figure A-3. Railway arrival at processing plant functions as temporary storage before processing. Object 1. Video of banana harvest, manual railway transportation from the field to the processing plant, removal of proximal to distal hands, placing in, and removal from fungicide dips (4.7 MB, Banana_ha rvest.wmv, 1:41 total running time). A B Figure A-4. Primary processing activities. A) Removal of proximal to distal hands. B) Hands placed in first fungicide dip.

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176 A B Figure A-5. Secondary processing activities. A) Damaged and single fruit elimination and transfer to second fungicide di p. B) Hands floating through second fungicide dip. Figure A-6. Hands removed from second fungicide dip and placed on drying tray.

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177 Figure A-7. Stem woun d sealant application. A B Figure A-8. Fruit weighed and boxed for shi pping. A) Large (proximal) fruit destined for export markets. B) Small (distal) fruit destined for local markets.

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178 A B Figure A-9. Boxed fruit palletized for sh ipping. A) Initial stacking. B) Pallet stabilization. A B Figure A-10. Fruit unloaded at ports and sh ipped by refrigerated tr uck 15C (59F) to ripening facility. A) Bradenton, FL. B) St. Croix, U.S.V.I. A B Figure A-11. Interior of fully loaded trailers capable of transporting 960, 40 pound (18 kg) boxes. A) Bradenton, FL. B) St. Croix, U.S.V.I.

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179 Figure A-12. Temperature probe meter used to sample fruit pulp temperature upon arrival. A B Figure A-13. Empty ripening room. A) Ai r flows down between tarp and wall (green arrows) while air is removed (red arro ws) through ceiling fans. Ethylene generator (yellow circle). B) Ceiling fans used to remove air (at the top of red the arrows).

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180 A B C Figure A-14. Ethylene generato rs. A) Fixed ethylene ge nerator unit inside ripening chamber. B) External control unit for adjusting air flow rates, temperature, relative humidity (RH) and ethylene concentration. C) Portable ethylene generator and ethanol for in trailer ripening. A B Figure A-15. Ethylene gassing of pall etized and boxed fruit at 300 LL-1 C2H4 gas, 24 h, 15C, 90% RH. A) Fruit pallet s unloaded into ripening chambers. B) Fruit ripened in trailer for immediate shipment.

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181 Figure A-16. Fruit pallets reloaded into refrig erated trailers at 15C (59F) and shipped to retailers.

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182 Figure A-17. Chiquita peel color score (s tage) chart used to monitor fruit ripening detailing consumer purchase and consumptio n preferences correlated with fruit stage of ripeness. [Reprint ed with permission from Wanner, J. 2000. Chiquita ripening chart . Chiquita, N.A.]

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183 LIST OF REFERENCES Abbott, J.A., 1999. Quality measurement of fr uits and vegetables. Postharvest Biology and Technology 15, 207-225. Abeles, F.B., Morgan, P.W., Saltveit, M.E., 1992. Ethylene in Plant Biology, 2nd Ed. Academic Press, Inc., U.S.A. p. 1. Adams, D.O., Yang, S.F., 1979. Ethylen e biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc . Natl. Acad. Sci. USA 76, 170. Amors, A., Zapata, P., Pretel, M.T., Bo tella, M.A., Almansa M.S., Serrano M., 2005. Ripening physiology of five loquat ( Eriobotrya japonica Lindl.) cultivars. Dept. Biologa Aplicada, Universidad Miguel He rnndez. Orihuela, Alicante, Spain. Arthey, V.O., 1975. Quality of horticultural products. John Wiley & Sons. New York. 228. 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 1-methylcyclopropene. International J ournal of Food Science and Technology 38, 745. Bagnato, N., Barrett, R., Sedgley, M., K lieber, A., 2002. Optimising Ripening temperatures of Cavendish bananas var. ‘Williams’ harvested throughout the year in Queensland, Australia. Australian J ournal of Experimental Agriculture 42, 1017. Baldwin, E.A., 2004. Flavor. In: USDA ARS Agriculture Handbook Number 66: The Commercial Storage of Fruits, Vegeta bles, and Florist and Nursery Stocks. http://www.ba.ars.usda .gov/hb66/contents.html . USDA ARS, Washington D.C.. Last accessed December 2006. Bishop, D., 1990. Controlled atmosphere stor age. In: C.J.V. Dellino, (Ed.), Cold and Chilled Storage Technology. Blackie, London. p. 66-98. Blankenship, S.M., Dole, J.M., 2003. 1-Me thylcyclopropene: a review. Postharvest Biology and Technology 28, 1-25. Brady, C.J., O’Connell, P.B.H., 1976. The pectin esterase of the pulp of the banana fruit. Australian Journal of Plant Physiology. 3, 301-310.

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184 Burg, S.P., Burg, E.A., 1962. Role of et hylene in fruit ripening. Plant Physiology. 37, 179-189. Burg, S.P., Burg, E.A., 1965. Relationship between ethylene production and ripening in bananas. Botanical Gazzette 126, 200-204. Buttery, R.G., 1993. Quantitative and sensory aspects of flavor of tomato and other vegetable and fruits. In: T.E. Acree and R. Teranishi, (Ed.), Flavor Science: Sensible Principles and Tec hniques, ACS, Wash. DC, p. 259-286. Cai, C., Chen, K., Xu, W., Zhang, W., Li X., Ferguson, I., 2006. Effect of 1-MCP on postharvest quality of loquat fruit . Postharvest Biology and Technology 40, 155-162. Carr, G., 2005. Musaceae. University of Hawaii Botany Department. www.botany.hawaii.edu/faculty/carr/mus.htm . University of Hawaii at Manoa Honolulu, HI. Last accessed December 2006. Chaiprasart, P., Gemma, H., Iwahori, S., 2002. Reduction of chilling injury in stored banana fruits by jasmonic acid derivatives a nd abscisic acid treatment. Acta Hort. 575, 89-696. Chiquita 2000. Consumer Color Preference Ripening Chart. Chiquita Solutions. Chiquita, N.A.. Choehom , R., Ketsa, S. , van Doorn, W., 2004. Senescent spotting of banana peel is inhibited by modified atmosphere pack aging. Postharvest Biology and Technology 31, 167. Coseteng M.Y., Lee C.Y., 1987. Changes in apple polyphenoloxidase and polyphenol concentrations in relation to degree of browning. J. Food Science 52, 985-989. Dominguez, M., Vendrell, M., 1993. Ethylene biosynthesis in banana fruit evolution of EFE activity and ACC levels in peel and pulp during ripening. J. Hortic. Sci. 60, 63. DoNascimento, J., Jnior, A., Bassinello, P., Cordenunsi, B., 2006. Beta-amylase expression and starch degradation during banana ripening Po stharvest Biology and Technology 40, 41. Drury, R., Hortensteiner, S., Donnison, I ., Bird, C, Seymour, G., 1999. Chlorophyll catabolism and gene expression in the peel of ripening banana fruits. Physiologia Plantarum 107, 32-38. Dubios, M., Gilles, K., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chemistry 28, 350-356.

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186 Hyodo, H., Ikeda, N., Nagatani, A., Tana ka, K., 1983. The increase in alcohol dehydrogenase activity and ethanol content du ring ripening of banana fruit. J. Jpn. Soc. Hortic. Sci. 52, 196. Inaba, A., Nakamura, R., 1986. Effect of exogenous ethylene concentration and fruit temperature on the minimum treatment time necessary to induce ripening in banana fruit. J. Jpn. Soc. Hortic. Sci. 55, 348. International Network for the Improvement of Banana and Plantain (INIBAP), 2005. Bananas. http://bananas.bioversityinter national.org/files/files/pdf /publications/brochure_bana nas.pdf . Bioversity International. Montpel lier, France. Last accessed December 2006. Israeli, Y., Lahav, E., 1986. Banana. In: Monselise, S.P. (Ed.), Handbook of Fruit Set and Development. CRC Press. Boca Raton, Florida. p. 45-73. Janave M., Sharma A., 2004. Partial purificatio n of chlorophyll degrading enzymes from Cavendish banana ( Musa cavendishi ). Indian Journal of Biochemistry & Biophysics, 41, 154-161. Jansasithorn, R., Kanlavanarat, S., 2006. E ffect of 1-MCP on physiological changes in banana ‘Khai’. Acta Hort. (ISHS) 712,723-728. Jeong, J., Huber, D.J., Sargent, S.A., 2003. Delay of avocado ( Persea americana ) fruit ripening by 1-methylcycloprope ne and wax treatments. Postharvest Biology and Technology 28, 247. Jiang, Y., Joyce, D.C., 2000. Effects of 1methylcyclopropene alone and in combination with polyethylene bags on the postharvest life of mango fruit. Annals of Applied Biology 137, 321-327. Jiang Y, Joyce, D.C., Macnish, A.J., 1999a. Re sponses of banana fruit to treatment with 1-methylcyclopropene. Plan t Growth Regulation 28, 78. Jiang Y, Joyce, D.C., Macnish, A.J., 1999b. Ex tension of the shelf life of banana fruit by 1-methylcyclopropene in combination wi th polyethylene bags. Postharvest Biology and Technology 16, 187-193. Jiang Y, Joyce, D.C., Macnish, A.J., 2002. Softening response of banana fruit treated with 1-methylcyclopropene to high temperature exposure. Plant Growth Regulation 36, 7-11. Jiang Y, Joyce, D.C., Jiang, W., Lu W., 2004a. Effects of chilling temperatures on ethylene binding by banana fruit. Plant Growth Regulation 43, 109.

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187 Jiang, W., Zhang, M., He, J. Zhou, L., 2004b. Regulation of 1-MCP-treated banana fruit quality by exogenous ethylene and temper ature. Food Science and Technology International 1, 15-20. Kader, A.A., 1985. Quality Factors: Defi nition and evolution for fresh horticultural crops. Publication 3311, Univer sity of California, Davis. Kader, A.A., 1993. Modified and controlled atmosphere storage of tropical fruits. In: Postharvest handling of tropical fruits. Proc. Intl. Conf., Chiang Mai, Thailand, December 1993, p. 239-249. Kader, A.A., 1997. A summary of CA recommendations for fru its other than apples and pears. In: 7th Intl. Contr. Atmos. Res. Conf., Univ. of Califor nia, Davis. p 1-34. Kader, A.A., 2002a. Banana. In: Postha rvest Technology Research & Information Center. http://rics.ucdavis.edu/postharvest2/produce/producefacts/fruit/banana.shtml . Univ. of California, Davis, CA. Last accessed December 2006. Kader, A.A., 2002b. Postharvest Technology of Horticultural Crops, Third Edition. Univ. of California Agriculture and Natural Resources Publication 3311. p.41. Kader, A.A., 2002c. Loquat. In: Postha rvest Technology Research & Information Center. http://postharvest.ucdavis.edu/Produc e/ProduceFacts/Fruit/loquat.shtml . Univ. of California, Davis, CA. Last accessed December 2006. Kader, A.A., 2004. Controlled Atmosphe re Storage. In: USDA ARS Agriculture Handbook Number 66: The Comme rcial Storage of Fruits, Vegetables, and Florist and Nursery Stocks. http://www.ba.ars.usda.gov/hb66/contents.html . USDA ARS, Washington D.C.. Last accessed December 2006. Kader, A.A., Hess-Pierce, B., Almenar, E ., 2003. Relative cont ributions of fruit constituents to soluble solids content meas ured by a refractometer. HortScience 38, 833. Kader A.A., Perkins-Veazie, P., Lester, G.E., 2004. Nutritional Quality of Fruits, Nuts, and Vegetables and their Importan ce in Human Health. In: USDA ARS Agriculture Handbook Number 66: The Commercial Storage of Fruits, Vegetables, and Florist and Nursery Stocks. http://www.ba.ars.usda.gov/hb66/contents.html . USDA ARS, Washington D.C.. Last accessed December 2006. Kanazawa, K., Sakakibara, H., 2000. High c ontent of dopamine, a strong antioxidant, in cavendish banana. J. Agric. Food Chem., 48, 844-848. Kerbel, E., 2004. Banana and Plantain. In: USDA ARS Agricu lture Handbook Number 66: The Commercial Storage of Fruits, Vege tables, and Florist and Nursery Stocks. http://www.ba.ars.usda .gov/hb66/contents.html . USDA ARS, Washington D.C.. Last accessed December 2006.

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194 BIOGRAPHICAL SKETCH Daniel Stanley was born and raised in St. Croix, U.S. Virgin Islands. He attended Rutgers, The State University of New Jers ey for his undergraduate studies where he earned both a B.S. in environmental and bus iness economics and a B.S. in agricultural sciences. He has also been involved with tr opical agriculture rese arch projects at the University of the Virgin Islands, St. Croix Campus.