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Ripening, volatiles and sensory attributes of West Indian and Guatemalan-West Indian hybrid avocados as affected by 1-me...

Permanent Link: http://ufdc.ufl.edu/UFE0042026/00001

Material Information

Title: Ripening, volatiles and sensory attributes of West Indian and Guatemalan-West Indian hybrid avocados as affected by 1-methylcyclopropene and ethylene.
Physical Description: 1 online resource (213 p.)
Language: english
Creator: Pereira, Marcio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 1mcp, alkanes, americana, aqueous, asynchrony, avocado, betacaryophyllene, booth7, electronic, ethylene, flavor, guatemalan, monroe, nose, persea, ripening, sensory, sesquiterpenes, simmonds, texture, volatiles, westindian
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This study aimed to evaluate the effects of the ethylene inhibitor 1-methylcyclopropene (1-MCP) on ripening, volatile compounds and sensory attributes of avocado. Fruit ripening was strongly affected by postharvest application of aqueous 1-MCP. Concentrations above 225 ug*L^-1 (4.16 mmol m^-3) and 2-min immersion treatments delayed ripening sufficiently to limit shelf life due to significant fruit shriveling and high decay severity. Ripening asynchrony (blossom end softens faster than stem end) occurs naturally and it was consistently exaggerated in all 1-MCP treatments at 200 ug*L^-1 or above; incidence was much lower for 150 ug*L^-1 treatment. However, a 4-day treatment of mid-ripe fruit with ethylene at 100 uL*L^-1 effectively promoted complete recovery from the strong ripening asynchrony caused by aqueous 1-MCP treatment at 900 ug*L^-1. Polygalacturonase activity was not directly related to ripening asynchrony. Total volatile emissions from untreated avocado pulp cubes decreased during ripening. Sesquiterpenes, mainly Beta-caryophyllene, were predominant in mature-green and mid-ripe fruit and minimally detected in ripe fruit. Alkanes were a major group in 'Booth 7' and 'Monroe' at all ripeness stages. The results suggest that ethylene is involved in ester metabolism in 'Simmonds' and of Alpha-copaene metabolism in all cultivars studied. Aqueous 1-MCP at 75 or 150 ug*L^-1 for 1 min significantly increased alkane emission in mid-ripe 'Booth 7' and 'Monroe'. Total volatile emissions were not affected by ethylene or 1-MCP in ripe 'Simmonds' fruit. Electronic nose analysis successfully classified avocado pulp by ripeness stage using fruit either untreated or treated with aqueous 1 MCP. Lower classification power was obtained when all treatments and ripeness stages were involved. Overall, a single postharvest immersion in aqueous 1-MCP (75 or 150 ug*L^-1 for 1 min) effectively extended shelf-life of West Indian and Guatemalan-West Indian hybrid avocados by 20% to 100%. At ripe stage, fruit quality remained marketable, and acceptable sensory attributes of texture, flavor and overall liking were maintained. Best results were observed for Guatemalan-West Indian hybrids and for early-harvested fruit (picking date A).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Marcio Pereira.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Sargent, Steven A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042026:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042026/00001

Material Information

Title: Ripening, volatiles and sensory attributes of West Indian and Guatemalan-West Indian hybrid avocados as affected by 1-methylcyclopropene and ethylene.
Physical Description: 1 online resource (213 p.)
Language: english
Creator: Pereira, Marcio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 1mcp, alkanes, americana, aqueous, asynchrony, avocado, betacaryophyllene, booth7, electronic, ethylene, flavor, guatemalan, monroe, nose, persea, ripening, sensory, sesquiterpenes, simmonds, texture, volatiles, westindian
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This study aimed to evaluate the effects of the ethylene inhibitor 1-methylcyclopropene (1-MCP) on ripening, volatile compounds and sensory attributes of avocado. Fruit ripening was strongly affected by postharvest application of aqueous 1-MCP. Concentrations above 225 ug*L^-1 (4.16 mmol m^-3) and 2-min immersion treatments delayed ripening sufficiently to limit shelf life due to significant fruit shriveling and high decay severity. Ripening asynchrony (blossom end softens faster than stem end) occurs naturally and it was consistently exaggerated in all 1-MCP treatments at 200 ug*L^-1 or above; incidence was much lower for 150 ug*L^-1 treatment. However, a 4-day treatment of mid-ripe fruit with ethylene at 100 uL*L^-1 effectively promoted complete recovery from the strong ripening asynchrony caused by aqueous 1-MCP treatment at 900 ug*L^-1. Polygalacturonase activity was not directly related to ripening asynchrony. Total volatile emissions from untreated avocado pulp cubes decreased during ripening. Sesquiterpenes, mainly Beta-caryophyllene, were predominant in mature-green and mid-ripe fruit and minimally detected in ripe fruit. Alkanes were a major group in 'Booth 7' and 'Monroe' at all ripeness stages. The results suggest that ethylene is involved in ester metabolism in 'Simmonds' and of Alpha-copaene metabolism in all cultivars studied. Aqueous 1-MCP at 75 or 150 ug*L^-1 for 1 min significantly increased alkane emission in mid-ripe 'Booth 7' and 'Monroe'. Total volatile emissions were not affected by ethylene or 1-MCP in ripe 'Simmonds' fruit. Electronic nose analysis successfully classified avocado pulp by ripeness stage using fruit either untreated or treated with aqueous 1 MCP. Lower classification power was obtained when all treatments and ripeness stages were involved. Overall, a single postharvest immersion in aqueous 1-MCP (75 or 150 ug*L^-1 for 1 min) effectively extended shelf-life of West Indian and Guatemalan-West Indian hybrid avocados by 20% to 100%. At ripe stage, fruit quality remained marketable, and acceptable sensory attributes of texture, flavor and overall liking were maintained. Best results were observed for Guatemalan-West Indian hybrids and for early-harvested fruit (picking date A).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Marcio Pereira.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Sargent, Steven A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042026:00001


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1 RIPENING, VOLATILES AND SENSORY ATTRIBUTES OF WEST INDIAN AND GUATEMALAN WEST INDIAN HYBRID AVOCADO S AS AFFECTED BY 1 METHYLCYCLOPROPENE AND ETHYLENE By MARCIO EDUARDO CANTO PEREIRA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Marcio Eduardo Canto Pereira

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3 To my parents, Eldo Pereira and Anamor Soares Canto, as thi s accomplishment is a prize for your love and care and belief in me. To my lovely wife, Belinda, for you are my best friend, my helper and my support, and to my children, Andr and Rebeca, for being such a joy in my life. You are a blessing and I love yo u so much. To Steve and Suzana Sargent, in recognition of all those years that your blessed marriage has touched me and my family and the lives of many other Brazilians with your friendship Christian values and constant support Not to us, oh Lord, not to us But to your name be the glory Because of your love and faithfulness (Psalm 115:1)

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4 ACKNOWLEDGMENTS I thank my God and my Lord and Savior Jesus Christ, for guiding me in this terrific experience, for making me amazed by His wonders, for drawing me closer to Him and making me grow in faith as I discover in Christ the hidden treasures of wisdom and knowledge. I thank deeply my lovely wife, Belinda, and my children, Andr and Rebeca, for walking these steps with me regardless the situation and for t heir overflowing love and care demonstrated for me every day. I extend my gratitude to my parents, Eldo and Anamor, and my sister, Ana Patr cia as well as to my father in law, Barry Thomas, my mother in law, Angela, and my brother in law, Steven, who always supported and prayed for me to accomplish this doctoral degree. I would like to express my gratitude to Dr. Steven Sargent, who, as an advisor, encouraged me constantly, contributing in multiple ways to my personal, academic and professional development I thank him for being a good friend and for making me and my family feel welcomed and shared joyful friendship and fellowship moments with him and his family. I also thank my committee members Dr. Donald J. Huber, Dr. Jeffrey K. Brecht, Dr. Charles A. Sims, Dr. Jonathan Crane and Dr. Celso Moretti, for providing me valuable advice in a friendly atmosphere and making significant contributions to make this a better study. I also thank Dr. Harry Klee, who gently allowed me to conduct all the study on volatil e compounds in his laboratory. I would like to thank Adrian Berry for the numerous times she helped me to accomplish my goals during my doctoral program, from trip arrangements to experiment set up and analyses. Also, to Kim Cordasco and James Lee, who wer e always available

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5 to help whenever I needed it. I extend my sincere gratitude to Denise Tieman and Lorenzo Puentes, who were patient and available to help and to conduct specific steps of volatile analysis and sensory analysis, respectively. I could not have done what I did without them. I thank my employer Embrapa Brazilian Agricultural Research Corporation for the opportunity given to me to develop my doctoral program at the University of Florida, for being my sponsor and supportive in several ways during these years abroad. I would like to thank all the staff and researchers at the Embrapa Cassava & Tropical Fruits Research Center who were very supportive in all those years. I thank the University of Florida and the Horticultural Sciences Department and all the staff for giving me administrative conditions to develop this study and for providing a top excellent academic formation during my doctoral program. Also, I thank my fellow graduate students for their friendship and valuable help in several tasks related to academic courses and experiments. I would like to thank the Redland Citizens Association for the honor of being the recipient of the Charlie Burr Scholarship Award 2008 and all the avocado growers involved in this research who let me select the fruit from their groves or supplied me with quality fruit for this study. I must acknowledge my brothers and sisters in Christ at The Family Church, a place where my family and I enjoyed great fellowship and could keep growing in faith, strengthening our relationship with God. Finally, I thank all the Brazilians who helped me and my family to go through this journey and supported us in many ways with their friendships and time.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 TABLE OF CONTENTS .................................................................................................. 6 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 13 LIST OF ABBREVIATIONS ........................................................................................... 15 ABSTRACT ................................................................................................................... 16 CHAPTER 1 INTRODUCTION .................................................................................................... 18 2 LITERATURE REVIEW .......................................................................................... 20 Avocado Origin and Industry ................................................................................... 20 Fruit Development Ripening and Composition ....................................................... 22 Ethylene Biosynthesis, Action and Inhibition ........................................................... 26 Treatment with Ethylene ................................................................................... 27 Analogs and Inhibitors of Ethylene ................................................................... 28 1 methylcyclopropene ...................................................................................... 29 Efficacy of 1 MCP treatment ...................................................................... 31 Adverse effects of the postharvest treatment with 1MCP ......................... 33 Plant Volatile Compounds ....................................................................................... 34 Volatiles in Food ............................................................................................... 35 Volatiles in Avocado ......................................................................................... 37 Effects of Ethylene and 1MCP on Volatile Production D uring Ripening .......... 38 Methods for Volatile Extraction and Analysis .................................................... 40 Sensory Attributes .................................................................................................. 41 Research Objectives ............................................................................................... 44 3 DETERMINATION OF CONCENTRATION AND EXPOSURE TIME FOR POSTHARVEST ETHYLENE AND/OR AQUEOUS 1 MCP TREATMENT IN AVOCADO .............................................................................................................. 46 Introduction ............................................................................................................. 46 Material and Methods ............................................................................................. 48 Aqueous 1MCP Preparation and Treatment ................................................... 48 Experiment 1 .................................................................................................... 49 Experiment 2 .................................................................................................... 49

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7 Experiment 3 .................................................................................................... 50 Ripening and Quality Parameters ..................................................................... 51 Whole fruit firmness ................................................................................... 51 Peel color ................................................................................................... 51 Respiration and ethylene production rates ................................................. 51 Weight loss ................................................................................................ 52 Decay ......................................................................................................... 52 Statistical Analysis ............................................................................................ 52 Results .................................................................................................................... 53 Experiment 1 .................................................................................................... 53 Experiment 2 .................................................................................................... 55 Experiment 3 .................................................................................................... 56 Discussion .............................................................................................................. 56 Conclusions ............................................................................................................ 61 4 USE OF ETHYLENE TREATMENT TO ALLEVIATE RIPENING ASYNCHRONY FROM 1 MCP EXPOSURE IN AVOCADO ............................................................. 69 Introduction ............................................................................................................. 69 Material and Methods ............................................................................................. 71 Plant Material ................................................................................................... 71 Aqueous 1MCP Preparation and Treatment ................................................... 71 Ethylene Treatment .......................................................................................... 72 Ripening and Quality Parameters ..................................................................... 72 Whole fruit firmness ................................................................................... 72 Peel color ................................................................................................... 73 Respiration and ethylene production rates ................................................. 73 Weight loss ................................................................................................ 74 Pulp firmness ............................................................................................. 74 Polygalacturonase (PG) activity ................................................................. 75 Statistical Analysis ............................................................................................ 75 Results .................................................................................................................... 75 Booth 7 ........................................................................................................... 75 Booth 8 ........................................................................................................... 77 Discussion .............................................................................................................. 79 Conclusions ............................................................................................................ 84 5 CHANGES IN VOLATILES DURING AVOCADO RIPENING AS AFFECTED BY ETHYLENE AND 1 MCP ........................................................................................ 95 Introduction ............................................................................................................. 95 Materi al and Methods ............................................................................................. 97 Phase 1 Effects of Ethylene and 1MCP on Volatile Compounds .................. 97 Phase 2 Aqueous 1MCP Effects on Volatile C ompounds in Three Cultivars ........................................................................................................ 98 Ethylene and Aqueous 1MCP Preparation and Treatment .............................. 98 Ripening and Quality Analysis .......................................................................... 99

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8 Whole fruit firmness ................................................................................. 100 Respiration and ethylene production rates ............................................... 100 V olatile compounds .................................................................................. 101 Statistical Analysis .......................................................................................... 102 Results .................................................................................................................. 102 Phase 1 Effects of Ethylene and 1MCP on Volatile Compounds ................ 102 Whole fruit firmness ................................................................................. 102 Respiration ............................................................................................... 103 Ethylene production ................................................................................. 103 Volatile compounds .................................................................................. 104 Phase 2 Aqueous 1MCP Effects on Volatile Compounds in Three Cultivars ...................................................................................................... 106 Whole fruit firmness ................................................................................. 106 Respiration ............................................................................................... 107 Ethylene ................................................................................................... 107 Volatile compounds .................................................................................. 108 Discussion ............................................................................................................ 111 Co nclusions .......................................................................................................... 116 6 USE OF AN ELECTRONIC NOSE TO CLASSIFY AVOCADO PULP BY RIPENESS STAGE .............................................................................................. 134 Introduction ........................................................................................................... 134 Material and Methods ........................................................................................... 135 Experiment 1 .................................................................................................. 135 Plant material ........................................................................................... 135 Electronic nose set up .............................................................................. 136 Sample preparation and sampling conditions .......................................... 137 Experiment 2 .................................................................................................. 138 Experiment 3 .................................................................................................. 138 Experimental Design and Statistical Analysis ................................................. 139 Results .................................................................................................................. 139 Experiment 1 .................................................................................................. 139 Experiment 2 .................................................................................................. 140 Experiment 3 .................................................................................................. 142 Discussion ............................................................................................................ 143 Conclusions .......................................................................................................... 146 7 SENSORY ATTRIBUTES OF BOOTH 7 AVOCADO FOLLOWING ETHYLENE PRETREATMENT AND/OR EXPOSURE TO GASEOUS OR AQUEOUS 1 MCP ...................................................................................................................... 156 Introduction ........................................................................................................... 156 Material and Methods ........................................................................................... 159 Plant Material ................................................................................................. 159 1 MCP Treatment ........................................................................................... 160 Season 1: Ripening and Quality Parameters .................................................. 161

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9 Whole fruit firmness ................................................................................. 161 Respiration and ethylene production ........................................................ 161 Weight loss .............................................................................................. 162 Texture profile analysis TPA ................................................................. 162 Seasons 1 and 2: Sensory Analysis ............................................................... 162 Statistical Analysis .......................................................................................... 163 Results .................................................................................................................. 164 Season 1: Ripening and Quality Paramet ers .................................................. 164 Whole fruit firmness ................................................................................. 164 Respiration rate and ethylene production ................................................ 164 Weight loss .............................................................................................. 1 65 Texture profile analysis TPA ................................................................. 165 Sensory Analysis ............................................................................................ 166 Season 1 .................................................................................................. 166 Season 2 .................................................................................................. 166 Correlations and effects of gender and avocado eating habit .................. 167 Discussion ............................................................................................................ 167 Conclusions .......................................................................................................... 174 8 FINAL CONCLUSIONS ........................................................................................ 185 APPENDIX A VOLATILE COMPOUNDS DETECTED IN SIMMONDS, BOOTH 7 AND MONROE ............................................................................................................ 187 B SEQUENCE OF QUESTIONS ASKED TO PANELISTS DURING SENSORY ANALYSIS OF FR ESH AVOCADO ...................................................................... 191 C SENSORY ANALYSIS OF SIMMONDS AND BETA ......................................... 193 LIST OF REFERENCES ............................................................................................. 194 BIOGRAPHICAL SKETCH .......................................................................................... 213

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10 LIST OF TABLES Table page 3 1 Days to ripe stage, final weight loss and whole fruit firmness of Monr oe avocado treated with aqueous 1MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) and stored at 20 C (Exp. 1). ............................................................... 62 3 2 Initial and final p eel color of Monroe avocado treated with aqueo us 1 MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) and stored at 20 C (Exp. 1). .... 62 3 3 Peak rate and days to peak respiration and ethylene production of Monroe avocado treated with a queous 1MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) stored at 20 C. (Exp. 1) ...................................................................... 62 3 4 Effect of ethylene pretreatment and concentration of aqueous 1MCP on days to reach ripe stage, f inal weight loss and decay rating of Arue avocado during storage at 20 C (Exp. 2). ........................................................................ 63 3 5 Effect of ethylene pretreatment and concentration of aqueous 1MCP on peak respiration and ethylene production rates of Arue avocado during storage at 20 C (Exp. 2). ................................................................................... 63 3 6 Effect of delay to ethylene application, ethylene pretreatment and concentration of aqueous 1MCP on days to reach ripe stage, final weight loss and decay rating of Simmonds avocado during storage at 20 C (Exp. 3). .............................................................................................................. 64 3 7 Effect of delay to ethylene application, ethylene pretreatment and concentration o f aqueous 1MCP on peak respiration and ethylene production rates of Simmonds avocado during storage at 20 C (Exp. 3). ........ 65 4 1 Peel color of mature green and ripe Booth 7 avocado untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min and stored at 20 C. ................................................................................................................. 86 4 2 Pulp firmness of stem end and blossom end segments of Booth 7 avocado untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min and stored at 20 C. .................................................................................. 87 4 3 PG activity of stem end and blossom end segments of ripe Booth 7 avocado untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min and stored at 20 C. .................................................................................. 88 4 4 Peel color of mature green and ripe Booth 8 avocado untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min and stored at 20 C. ................................................................................................................. 89

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11 4 5 Pulp firmness of stem end and blossom end segments of Booth 8 avocado untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min and stored at 20 C. .................................................................................. 90 4 6 PG activity of stem end and blossom end segments of ripe Booth 8 avocado untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min and stored at 20 C. .................................................................................. 91 5 1 Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds avocado (n=6) (Phase 1, Experiment 1). ................................. 118 5 2 Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds avocado (n=6) (Phase 1, Experiment 2). ................................. 120 5 3 V olatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds avocado (n=6) (Phase 2). ........................................................ 122 5 4 Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR ) and ripe (R) Booth 7 avocado (n=6) (Experiment 4). .................................................... 124 5 5 Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Monroe avocado (n=6) (Experiment 5). .................................................... 126 6 1 Interclass Mahalanobis distances between maturegreen, midripe and ripe Booth 7 avocado pulp. (Training session). ...................................................... 147 6 2 Res ults from the identification session using ripe Booth 7 avocado pulp as test samples. .................................................................................................... 147 6 3 Cross validation for the training session for Simmonds avocado pulp (n=10) from fruit either u ntreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis at three ripeness stages. ........... 148 6 4 Interclass Mahalanobis distances for Simmonds avocado pulp (n=10) from fruit either untreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis at three ripeness stages. ........... 148 6 5 Interclass Mahalanobis dist ances for maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds or Booth 7 avocado pulp. ................................................ 149 6 6 Cross validation for the training session for Booth 7 avocado pulp (n=10) from fruit eit her untreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis at three ripeness stages. ........... 150 6 7 Interclass Mahalanobis distances for Booth 7 avocado pulp (n=10) from fruit either untreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis in three ripeness stages. ........... 150

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12 7 1 Definitionz of Texture Profile Analysis TPA parameters analyzed f or ripe mesocarp tissue withdrawn from mid segment of early (EH) and late harvested (LH) Booth 7 avocado fruit pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). ........................................................ 175 7 2 Texture profile analysis of ripe mesocarp tissue taken from mid segment of early (EH) and lateharvested (LH) Booth 7 avocado fruit pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). ......................... 176 7 3 Average texture, flavor and overall liking scores from 75 untrained panelists for early (EH) and late harvested (LH) Booth 7 avocado fruit pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). ......................... 177 7 4 Average texture, flavor and overall liking scores from 75 untrained panelists for early (EH) and late harvested (LH) Booth 7 avocado fruit treated with 1MCP only (Season 2). ...................................................................................... 177 7 5 Overall Pearson correlation coefficients between parameters of sensory analysis of ripe Booth 7 avocado fruit based on combined scores for both seasons (2008 and 2009) and harvest times (early and lateharvest). ............ 177 A 1 Volatile compounds emitted by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds, Booth 7 and Monroe avocado (n=6) stored at 20 C, and respectiv e descriptors, odor threshold and relative abundance (%) in each ripeness stage for each cultivar (Phase 2 experiments). .................................. 188 C 1 Average texture, flavor and overall liking scores from 75 untrained panelists for Simmonds avocado fruit untreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L1 (A150) for 1 min at 20 C and stored at 20 C. ................................................................................................. 193 C 2 Averag e texture, flavor and overall liking scores from 75 untrained panelists for Beta avocado fruit untreated (control) or immersed in aqueous solution of 1 MCP at 150 g L1 (A150) for 1 min at 20 C and stored at 20 C. ............ 193

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13 LIST OF FIGURES Figure page 3 1 Whole fruit firmness of Monroe avocado treated with aqueous 1MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) and stored at 20 C. .......................... 66 3 2 Limiting quality factors of Monroe avocado treated with aqueous 1MCP during storage at 20 C. ...................................................................................... 67 3 3 Overall coefficient of vari ation (C.V.) for whole fruit firmness of Arue avocado (n=6) pretreated with ethylene (0, 12 or 24 h duration), followed by 24h delay, then treated with aqueous 1MCP (0, 50, 100, 150 or 200 g L1 for 1 min) and stored at 20 C. ........................................................................... 68 3 4 Overall coefficient of variation (C.V.) for whole fruit firmness of Simmonds avocado (n=6) pretreated with ethylene (0, 12 or 24 h duration), followed by 24h interval then treated with aqueous 1MCP (0, 100 or 200 g L1 for 1 min) and stored at 20 C. .................................................................................... 68 4 1 Whole fruit firmness of Booth 7 (A) and Booth 8 (B) fruit untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min at 20 C and stored at 20 C. ................................................................................................... 92 4 2 Respiration ( CO2) and ethylene production (C2H4) rates of Booth 7 (A) and Booth 8 (B) fruit untreated (Control) or treated (1MCP) with aqueous 1MCP at 900 g L1 for 1 min at 20 C and stored at 20 C. ................................. 93 4 3 Weight loss of Booth 7 and Booth 8 fruit untreated (Control ) or treated (1 MCP) with aqueous 1MCP at 900 g L1 for 1 min at 20 C and stored at 20 C. ................................................................................................................. 94 5 1 Steps of fruit processing for volatile collection. ................................................. 128 5 2 Whole fruit firmness of Simmonds avocado in Phase 1, experiments 1 (A) and 2 (B). .......................................................................................................... 129 5 3 Respiration rates of Simmonds avocado in Phase 1, experiments 1 (A ) and 2 (B). ................................................................................................................. 130 5 4 Ethylene production rates of Simmonds avocado in Phase 1, experiments 1 (A) and 2 (B). .................................................................................................... 131 5 5 Whole frui t firmness of Simmonds (A), Booth 7 (B) and Monroe (C) avocado untreated (control) or treated with aqueous 1MCP at 75 (M75) or 150 g L1 (M150) for 1 min at 20 C (Phase 2 experiments). .......................... 132

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14 5 6 Respiration and ethylene production rates of Simmonds (A and B), Booth 7 (C and D) and Monroe (E and F) avocado untreated (control) or treated with aqueous 1MCP at 75 (M75) or 150 g L1 (M150) for 1 min at 20 C (Phase 2 experiments). ................................................................................................. 133 6 1 Ripeness stages of Booth 7 avocado assessed for electronic nose analysis. A) mature green, B) midripe and C) ripe. ........................................................ 151 6 2 Details of the electronic nose analysis. ............................................................. 151 6 3 Canonical Discriminant Analysis Plot of electronic nose readings (n=6) of maturegreen (MG), mid ripe (MR) and ripe (R) Booth 7 avocado pulp. ......... 152 6 4 Canonical Discriminant Analysis Plot of electronic nose readings (n=10) of maturegreen (MatGr), mid ripe (MR) and ripe (R) avocado pulp. .................... 153 6 5 Canonical Discriminant Analysis Plot of electronic nose readings (n=10) of ripe (R) avocado pulp.. ..................................................................................... 154 6 6 Canonical Discriminant Analysis Plot of electronic nose readings (n=10) for maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds (left) or Booth 7 (right) avocado pulp. ......................................................................................... 155 7 1 Details of sensory analysis. .............................................................................. 178 7 2 Whole fruit firmness in early (A) and lateharvested (B) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). 179 7 3 Respiration in early (A) and lateharvested (B) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). .................. 180 7 4 Ethylene production in early (A) and lateharvested (B) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). 181 7 5 Weight loss in early (EH) and lateharvested (LH) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). 182 7 6 Over all frequency (A) and scores (B) given by panelists according to gender on texture, flavor and overall liking of ripe Booth 7 avocado fruit. ................... 183 7 7 Overall frequency of eating avocado (A) and scores (B) given by panelists according to how often they eat avocado on texture, flavor and overall liking of ripe Booth 7 avocado fruit. .......................................................................... 184

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15 LIST OF ABBREVIATION S C Degrees Celsius 1 MCP 1 methylcyclopropene C2H4 Ethylene CO2 Carbon dioxide d Day h Hour min Minute N Newtons R.H. Relative humidity gfw grams of fresh weight a.i. active ingredient WI West Indian G WI GuatemalanWest Indian hybrid

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16 Abstract of Dissertation Presented to the Graduate School of the Un iversity of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RIPENING, VOLATILES AND SENSORY ATTRIBUTES OF WEST INDIAN AND GUATEMALAN WEST INDIAN HYBRID AVOCADOS AS AFFECTED BY 1 METHYLCYCLOPROPENE AND ETHYLENE By Marcio Eduardo Canto Pereira August 2010 Chair: Steven A. Sargent Major: Horticultural Science This study aimed to evaluate the effects of the ethylene inhibitor 1 methylcyclopropene (1MCP) on ripening, volatile compounds and sensory at tributes of avocado. Fruit ripening was strongly affected by postharvest application of aqueous 1MCP. C oncentrations above 225 g L1 (4.16 mmol m3) and 2min immersion treatments delayed ripening sufficiently to limit shelf life due to significant fruit shriveling and high decay severity. Ripening asynchrony (blossom end softens faster than stem end) occurs naturally and it was consistently exaggerated in all 1MCP treatments at 200 g L1 or above; incidence was much lower for 150 g L1 treatment. However, a 4 day treatment of mid ripe fruit with ethylene at 100 L L1 effectively promoted complete recovery from the strong ripening asynchrony caused by aqueous 1MCP treatment at 900 g L1. Polygalacturonase activity was not directly related to ripening asynchrony. Total volatile emissions from untreated avocado pulp cubes decreased during ripening. Sesquiterpenes, mainly cary ophyllene, were predominant in maturegreen

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17 and midripe fruit and minimally detected in ripe fruit. Alkanes were a major group in Booth 7 and Monroe at all ripeness stages. The results suggest that ethylene is involved in ester metabolism in Simmonds and of copaene metabolism in all cultivars studied. Aqueous 1MCP at 75 or 150 g L1 for 1 min significantly increased alkane emission in mid ripe Booth 7 and Monroe. Total volatile emissions were not affected by ethylene or 1MCP in ripe Simmonds fruit. Electronic nose analysis successfully classif ied avocado pulp by ripeness stage using fruit either untreated or treated with aqueous 1MCP. Lower classification power was obtained when all treatments and ripeness stages were involved. Overall, a single postharvest immersion in aqueous 1MCP (75 or 150 g L1 for 1 min ) effective ly extended shelf life of West Indian and Guatemalan West Indian hybrid avocados by 20% to 100%. At ripe stage, fruit quality remained marketable, and acceptable sensory attributes of texture, flavor and overall liking were maint ained. Best results were observed for GuatemalanWest Indian hybrids and for early harvested fruit (picking date A).

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18 CHAPTER 1 INTRODUCTION Avocado is a unique fruit in several aspects. Cell division and growth do not cease throughout development, the fruit accumulates significant amounts of oil in the pulp and the fruit do not ripen while attached to the tree ( Bower and Cutting, 1988) During ripening, this climacteric fruit produces significant amounts of ethylene and it is very sensitive to external applications of this plant hormone. Recently, several studies were conducted with the ethylene inhibitor 1methylcyclopropene (1MCP) to extend shelf life of avocado (Feng et al., 2000; Jeong et al., 2002a; Adkins et al., 2005; Choi et al., 2008) Results show that this compound is very efficient in delaying ripening of avocados. However, depending on the combination of fruit maturity, temperature, 1MCP concentration and treatment duration, fruit may not soften completely and ripening asynchrony has been observed. The compound 1 MCP is registered for commercial postharvest application in avocado (Watkins, 2008) Therefore, other aspects of fruit quality must be evaluated as well. Studies with application of 1MCP in other fruit crops have shown that it can significantly reduce or alter the volatile profile and the aroma of the ripe fruit (Argenta et al., 2003; Zhu et al., 2005; MoyaLeon et al., 2006; Marin et al., 2009) Additionally, sensory attributes such as flavor and texture may be affected and consumer acceptance compromised. These factors were not yet studied for avocados treated with 1 MCP. The lack of this important information for the avocado industry, especially in Florida, motivated this study. D espite the information available on the literature about avocado, most of the available reports are for Mexican types, one of the three known

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19 races of avocado. There is still lack of information on cultivars of the other two races, West Indian and Guatemalan, largely cultivated in Florida. Also, the aqueous formulation of 1MCP has a great potential to be used as a postharvest treatment in avocado and, therefore, the study of the effects of concentration and treatment time is valuable for the avocado industry. The overall hypothesis tested was that a postharvest treatment of aqueous 1 methylcyclopropene significantly affects ripening, the volatile production and profile, and the sensory attributes of West Indian and GuatemalanWest Indian hybrid avocados. The objectives of this research were: 1) to determine concentration and exposure time for postharvest ethylene and/or aqueous 1MCP treatment in West Indian and GuatemalanWest Indian hybrid avocados; 2 ) t o determine whether a delayed and prolong ed ethylene treatment is effective to alleviate ripening async hrony from 1 MCP exposure in avocado; 3 ) to identify the main volatile compounds in West Indian and GuatemalanWest Indian hybrid cultivars at three ripeness stages; 4 ) to investigate the groups of volatile compounds that are enhanced or suppressed by 1MC P and ethylene treatments during ripening of avocado; 5 ) to evaluate the use of an electronic nose to classify avocado pulp by ripeness stage in untreated or treated fruit with aqueous 1 MCP ; 6 ) to evaluate sensory attributes of avocado following ethylene pretreatment and/or exposure to gaseous or aqueous 1MCP

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20 CHAPTER 2 LITERATURE REVIEW Avocado Origin and Industry Avocados are originally from Tropical America and three races of avocados are known: West Indian, Guatemalan and Mexican. Cold tolerance and oil content are higher for Mexican avocados and lower for West Indian avocados while Guatemalan fruit are intermediate. Many existent cultivars are hybrids between two of these races Mexican types are smaller and have higher oil content than West Indian or Guatemalan fruit ( Crane et al., 2007b). The fruit is consumed fresh or to make guacamole and other dishes (Evans and Nalampang, 2006). The interest in avocados is growing mainly due to its nutritional and health benefits (Evans and Nalampang, 2009). The world avocado production was estimated at over 3.5 million tons as of 2008 (FAOSTAT, 2010). Mexico was the largest avocado producer accounting for 31.8% of the world production Chile and Indonesia were the second and third main world producers with 7.1% and 6.4%, respectively The United States was in 9th place, with 114,000 tons, 3.2% of the world production. As of 2007, Mexico was the largest world exporter (34% world exports), followed closely by Chile (32%), but with a much higher value US $62 1 million against US $358 million of the latter country. The USA was the main avocado world importer, reaching near ly 45% of global imports in 2007 (FAOSTAT, 2010) The main cultivar imported was Hass, a GuatemalanMexican hybrid, coming mainly from Mexi co and Chile The U.S. consumption of fresh avocados more than doubled since 1998, particularly due to the growth of the Hispanic population, year round availability of the fruit and advertisement of nutritional and health benefits (Evans and Nalampang, 200 9 ).

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21 The avocado industry in the United States is located mainly in California and Florida with a minimal production in Hawaii (< 0.5%) and the entire production is oriented for the fresh market ( NASS, 20 10) In 200708, California accounted for 85% (165,000 tons) of the USA production and 96% (US $328 million) of the value of production, while Florida had 14.2% (27,500 tons) of the production and 3.5% (US $ 12.1 million) of the total value. In 200809, California production was severely affected by wildfires, generating a 46.7% decrease in production, reflecting in a 39.9% reduction of the countrys production. In California, Mexicantype avocados are grown and the hybrid Hass is grown in more than 90% of the producing acreage ( California Avocado Commiss ion 20 10 ). In Florida, avocado is the main tropical fruit crop, with approximately 3,000 bearing ha as of season 20072008 and $ 12 million value of production (Bronson, 2009) The commercial production is located primarily in Miami Dade and Collier Counti es where G uatemalan (G) or W est I ndian (WI) avocados and their hybrids are produced. Florida has several major and minor cultivars that mature at different times during the season, which extends from late May to March ( Crane et al., 2007a). Most early sea son (late May August) cultivars are WI types and midseason (September October) and late cultivars (November March) are G types or G WI hybrids. Simmonds (WI), Bernecker (WI) and Miguel (G WI) are examples of early season cultivars ; Booth 7 ( G WI), Choquette (G WI) and Lula (G WI), mid season cultivars ; and Monroe (G WI), the major lateseason cultivar ( Tropical Research and Education Center 2008). Harvesting and marketing costs more than double the costs of an avocado production system which is due to the labor intensive operations of picking, handling

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22 and packing, used to avoid bruises and scratches to the fruit (Evans, 2005). The bulk of the avocado produced in Florida is sold outside the state, contributing significantly as a revenue generator for Florida. The nutritional appeal of lower oil and lower calories contents of the Florida avocados than the Hass has been a great marketing tool for the Florida avocado industry, which is investing on fruit quality, shelf life extension and year round availability ( Crane et al., 2007a). An extension of its marketability would represent also an extension of marketing possibilities. Fruit Development, Ripening and Composition The fruit is a berry with a fleshy mesocarp and skin varying from gr een to purple when ripe, depending on the cultivar ( Crane et al., 2007b). Avocado follows a single sigmoidal growth curve, with rapid cell division in the first weeks, followed by growth of the seed and embryo. Later growth is due to mesocarp growth (Piper and Gardner, 1943). Cell division continues while the fruit is attached to the tree, but at a lower rate in mature fruit Avocados do not ripen while on the tree, which is explained in part by continued cell division ( Schroeder, 1958). When the fruit is detached from the tree it becomes sensitive to ethylene and ripens (Gazit and Blumenfeld, 1970). Florida avocados ripen best at temperatures from 16 C to 24 C and the lowest safe storage temperature during ripening is 13 C for W est I ndian cultivars and 4 C for most other Florida cultivars Softening period for Florida cultivars range from 3 to 8 d ( Crane et al., 2007b) The ethylene production for a preclimacteric fruit is very low, but increases rapidly to rates over 100 L C2H4 kg1 h1 when ripening at 20 oC (Kader and Arpaia, 2006). The fruit has a high respiratory rate during ripening, from 40 to 150 mL CO2 kg1 h1 when stored at 20 oC.

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23 Unlike other fleshy fruit, avocados accumulate significant amounts of oil during growth in specialized oil cells called idioblasts (Ortiz et al., 2004). These cells are structurally mature in very small fruit and at this stage they have only a narrow and irregular cytoplasm in the periphery of the cell. They are completely isolated from the rest of the tissues by s uberin, which helps to protect neighboring cells from toxic effects of the oil (Platt Aloia et al., 1983). Oil and moisture content accumulate in an inversely proportional manner, with oil increasing with fruit development (Du Plessis, 1979). Oil content in Florida grown avocados may range from as low as 2% to as high as 17% on average, being lower for West Indian cultivars (Hatton et al., 1964). A gradient in oil content is found in the avocado flesh, with higher contents found in the outer parts (Schroeder, 1987). Fruit dry matter at harvest increases during the same season (Lee et al., 1983) and late fruit has more oil content than early fruit ( Hatton et al., 1964; Lee et al., 1983). Overall changes in dry matter and oil content are much lower after harv est than when fruit is still on the tree (Ozdemir and Topuz, 2004). Because dry matter measurements are faster and accurate and are highly correlated with oil content, percent dry matter is used as a maturity index in California (Woolf et al., 2003; Kader and Arpaia, 2006). In Florida, where the avocado industry is restricted to a smaller area and more uniform climate picking dates are suitable to be used as harvest indexes ( Harding, 1954; Soule and Harding, 1955). Although a minimum oil content is needed for good palatability (Hatton et al., 1964), h arvest maturity for each Florida avocado cultivar is determined by date based on weight or size ( Crane et al., 2007b) as established by the Florida Avocado Administrative

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24 Committee (Government Printing Office, 2009) in a shipping schedule (Florida Avocado Administrative Committee, 2009) Other maturity indices tested had wide variation and were not considered adequate for commercial application (Hatton et al., 1957; Hatton and Campbell, 1959). There are no sig nificant changes in oil content during postharvest handling and the variability among fruits does not promote a good correlation between dry matter and oil content (Jeong et al., 2002 b ). Avocado oil is rich in monounsaturated lipids which are known to im prove the ratio of good (HDL) to bad (LDL) cholesterol, and benefit people with heart diseases, diabetes and high blood pressure (Hall et al., 1980) Lipid content increases during fruit development, mainly due to triglycerides, with oleic acid being largely synthesized (Kikuta and Erickson, 1968; Du Plessis, 1979). Contents of palmitic and linolenic acids decrease with avocado fruit development. Other minor fatty acids contents also decrease or remain the same (Ratovohery et al., 1988). The main fatty acids found in avocado are oleic acid (18:1), palmitic acid (16:0), linoleic acid (18:2), palmitoleic acid (16:1) and linolenic acid (18:3) (Ratovohery et al., 1988; Moreno et al., 2003; Pacetti et al., 2007). The essential fatty acids linoleic (18:2) and linolenic (18:3) are more abundant in the almond, but are found in the pulp in levels of 10 to 17% (Pacetti et al., 2007). Variations in levels of fatty acids may occur due to locations and temperature, and also during season (Kaiser and Wolstenholme, 1993). T he significant content of monounsaturated fat facilitates the absorption of carotenoids and other antioxidant compounds into the bloodstream (Lu et al., 2005; Unlu et al., 2005). Furthermore, a study done on over 100 common foods in the United States, incl uding fruits, vegetables and nuts, reported avocado (Hass) as having the

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25 highest lipophilic antioxidant capacity among fruits and the great majority of the other foods ( Wu et al., 2004 ). The daily consumption of avocado per capita is about 2.7 g per day, which represents 86 mg VCE (vitamin C equivalent) for each 100 g eaten (Chun et al., 2005). It was recently reported that avocado contains over 20 essential nutrients and various potential cancer preventing phytochemicals. In vitro and in vivo studies sug gest that avocado should be added to a list of fruits as a part of a cancer prevention diet (Ding et al., 2007). The concentration of carotenoids and other antioxidants vary from season to season and from sample to sample (Lu et al., 2005). Total carotenoids in the pulp of Hass avocados declined by over 50% during ripening and were found to be as low as 10% of the total present in the skin. Lutein was the most abundant carotenoid in the pulp and the concentration did not change significantly during ripening, although a decreasing gradient was found from the skin towards the seed. Neoxanthin was also present and in highest concentrations in the first 3 d after harvest. Other carotenoids present in the pulp, but consistently in very low concentrations (< 0. 1 g.g1) were betacarotene, alfacarotene, violaxanthin, antheraxanthin and zeaxanthin (Ashton et al., 2006). Sugars accumulate in avocado mainly during the first phases of development and their levels drop towards ripening. The C7 sugars are the most impor tant and persistent until maturation (Liu et al., 1999; Bertling and Bower, 2006). Sugars are most likely used for lipid synthesis in the flesh and starch accumulation in the seed. Five soluble sugars were detected in avocado, constituting 98% of the fruit total soluble sugars: sucrose, fructose, glucose, mannoheptulose and perseitol. After the rapid expansion phase,

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26 levels of fructose and glucose were very low (2 to 4% dry weight). Total sugar levels decrease during postharvest ripening; fructose and glucose are likely used as respiratory substrates. Little starch is accumulated in the skin and pulp. Ripe fruit may have as low as 0.3% starch (Liu et al., 1999). Ethylene Biosynthesis, Action and Inhibition Ethylene has an essential role in ripening of climac teric fruits, not only triggering the process, but also maintaining its normal progress leading to a ripe edible fruit Two systems of ethylene production operate in a climacteric fruit. System 1, during growth and development and the preclimacteric phase when ethylene inhibits ethylene synthesis; and system 2, during the climacteric phase of ripening, when ethylene stimulates its synthesis in an autocatalytic fashion. The transition between the two systems still remains to be completely clarified (Barry and Giovannoni, 2007). Ethylene biosynthesis begins with methionine and involves only three steps: (1) conversion of methionine to s adenosyl L methionine (SAM); (2) formation of 1aminocyclopropane1 carboxylic acid (ACC) by the enzyme 1aminocyclopropan e 1 carboxylase synthase (ACS); (3) conversion of ACC to ethylene by the enzyme ACC Oxydase (ACO). Both ACO and ACC are limiting steps ( Argueso et al. 2007) Once formed, ethylene needs to bind to the receptor for action. Studies indicate that copper is r equired for ethylene binding in receptors and conformational changes of binding domains associated with signaling are not completely known (Binder, 2008). A number of changes during ripening are ultimately the result of induction or suppression of the expr ession of genes by ethylene (Giovannoni, 2004). Nonetheless, ethyleneindependent events also occur during ripening of climacteric fruits (Pech et al., 2008).

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27 Treatment with E thylene E thylene has been used commercially in several commodities to promote uni form ripening and for degreening of citrus peel (Saltveit, 1999). Although very low concentrations are capable of affecting fruit ripening (Wills et al., 2001), ethylene is usually used from 5 to 150 L L1 in a flow through system (Saltveit, 1999). Avocados can be treated commercially with ethylene to increase uniformity of fruit ripening. Treatment with ethylene depends on cultivar, maturity and season the earlier the fruit, the longer the treatment with ethylene. Recommended conditions are 10 to 100 L L1 ethylene at 17 to 20 C, 48 to 72 h for early season, 24 to 48 h for midseason and 12 to 24 h for lateseason fruit (Woolf et al., 2004; Kader and Arpaia, 2006). Sensitivity of avocado fruit to ethylene increases with time after harvest. It was shown that sensitivity to external ethylene treatment was higher 25 h after harvest than 1 h after harvest. Fruit treated with 100 L L1 ethylene for 24 h softened uniformly when treated 2 d after harvest, but was not uniform when treated 1 d after picking and were insensitive when treated 1 h after harvest (Gazit and Blumenfeld, 1970). Similar findings were reported for Hass avocado. External ethylene was not sufficient to trigger the ripening process and promoted reversible increases in respiratory response when application was done 2 h after harvest for 6 h or 12 h. Longer exposures to ethylene (18 and 24 h) led to irreversible s timulation and anticipation of the climacteric peak, accelerating ripening (Eaks, 1966). Application of 10 L L1 ethylene at 20 C for 18 h had no effect on seeded Arad avocado, but hastened significantly the onset of climacteric ethylene peak when applied 24 h after harvest in both seeded and seedless avocado (Hershkovitz et al., 2010). Zauberman et al. (1988) suggested that ethylene is

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28 needed not only to trigger, but also to sustain the ripening process, which would require its continuous presence. The seed is involved in ethylene responsiveness during avocado ripening (Hershkovitz et al., 2010). Treatment of Hass avocado with ethylene at 100 L L1 for 24 h reduced the time (days) to ripen from 13 to 10 d (Adkins et al., 2005). Ethylenetreated Monroe avocado fruit (100 L L1 for 24 h at 13 C) showed significantly more uniform softening than control fruit when stored for 7 or 14 d at 13 C (Jeong et al., 2002a). The dry matter content of fruit stored at 13 C for 7 or 14 d decreased slightly but not significantly for early and lateharvest fruit. There were no significant differences for oil content (about 5.4%) as well. Ethylene exposure prior to cold storage increased chances of chilling injury (Pesis et al., 2002). Treatment with ethylene (100 L L1) increased sensitivity of Fuerte avocados to chilling injury (Lee and Young, 1984). Analogs and I nhibitors of E thylene Analogs can elicit the same physiological effects as ethylene, but in much higher concentration (Burg and Burg, 1967; Saltveit, 1999). Carbon monoxide and acetylene concentrations must be respectively 2,900 and 12,500 times higher. Propylene is the most active ethylene analog known and requires a 130 times higher concentration than ethylene to promote the same effects (Burg and Bur g, 1967). The use of a propylene treatment enables the measurement of endogenous ethylene production during ripening (McMurchie et al., 1972). On the other hand, there are inhibitors of ethylene synthesis or action that can be used to prevent or reduce the effects of ethylene. AVG (Aminoethoxyvinylglycine)

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29 inhibits ethylene synthesis at the step from SAM to ACC (Adams and Yang, 1979). STS (silver thiosulfate) has been used in flowers as an inhibitor of ethylene perception (Veen, 1987). CO2 is a competitive inhibitor of ethylene action and it has been suggested that endogenous levels of CO2 might be responsible for inhibiting the climacteric rise of ethylene in climacteric fruits during the preclimacteric phase (Burg and Burg, 1967). The effectiveness of a compound to serve as an antagonist seems to be dependent on the time that it is bound to the ethylene receptor (Sisler, 2006). V apor pressure differences and hydrophobic interactions could contribute to the effectiveness (Sisler and Serek, 2003) Several studies have shown that other compounds inhibit ethylene action, such as 1alkane substituted cyclopropenes ( Sisler et al., 2003), 2,5NBD, DACP ( Sisler and Serek, 2003), 1 ECP and 1PCP ( Feng et al., 2004) 1 methylcyclopropene The molecule 1methylcyclopropene ( 1 MCP ) is considered the most effective ethylene action inhibitor since it is active at extremely low concentrations, is already commercially available and it is considered to be nontoxic (Sisler, 2006; Environmental Protection Agency, 2008 ). The c ompound is a gas at room temperature (Sisler and Serek, 1997) and is highly efficient at very low concentrations. It tightly binds to the binding site before ethylene is bound suppressing ethylene perception and consequent action (Sisler and Serek, 1999). It has been a powerful tool to better understand the role of ethylene in postharvest metabolism of horticultural products (Huber, 2008). During the last decade the compound 1MCP has been widely used in research to extend postharvest life of a wide range of horticultural products, including vegetables, flowers and climacteric and nonclimacteric fruits (Huber, 2008). Commercial use

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30 started with flowers and more recently became common for apples in the U.S. (Watkins, 200 8 ). In general, the longer the product is treated with 1MCP and the higher the concentration applied, the more pronounced are the effects of the inhibitor. While exposure of Bartlett pears to 0.01 L L1 1 MCP had no effect on ripening, treatment with 1 L L1 1 MCP inhibited softening and exposure to 0.2 to 0.4 L L1 1 MCP promoted better results (Ekman et al., 2004). Increasing the concentration of 1MCP leads to longer delays of ripening in tomatoes (Moretti el al., 2002; Opiyo and Ying, 2005). Softening of guavas treated with 300 nL L1 1 MCP for 6 h was not different from nontreated fruit, but was significantly delayed by the same concentration applied for 12 h or 600 nL L1 1 MCP applied for 6 h (Singh and Pal, 2008). For avocado, i ncreasing 1MCP concentrations (50 or 500 nL L1) was more effective than increasing treatment durations (0 to 36 h). A maximum response for 1 MCP was found to be 500 nL L1 for 18 to 24 h for Hass (Adkins et al., 2005). The concentration and the length of exposure to 1 MCP significantly influenced the effect of the gas in delaying Simmonds avocado fruit ripening, suggesting that treatment with 1 MCP at 0.45 L L1 for 24 h is sufficient to exert maximum delay ( Jeong et al., 2002a). Ripening of Quintal avocados was delayed satisfactorily with 1MCP, and the best concentrations are between 90 and 270 nL L1 (Kluge et al., 2002). Several effects of 1MCP treatment in avocado were reported and include: delay of the onset of climacteric (Feng et al., 2000), delay of color changes (Garca et al., 2005; Hershkovitz et al., 2005) and reduced mesocarp discoloration (Pesis et al., 2002) However, one of the most significant effects is the delay of softening. Firmness of 1-

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31 MCP treated fruit is often similar to nontreated fruit in the first days of ripening but the rate of softening reduces in the later stages of ripening. This is related to the delay of activity of several cell wall enzymes that are involved with softening in avocado, mainly polygalacturonase (PG). This enzyme is strongly suppressed by 1MCP (F eng et al., 2000; Jeong et al., 2002a; Jeong and Huber, 2004; Choi et al., 2008). I t was suggested that PG is important at the later stages of ripening but is not required for the extensive softening of avocado ( Jeong et al., 2002a). A new aqueous formulation of 1MCP was reported as an immersion treatment for plums, extending shelf life most effectively at a concentration of 1,000 g kg1 (Manganaris et al, 2008). Aqueous 1MCP applied to breaker Florida 47 tomatoes was efficient at concentrations as low as 50 g L1 and for 30 s exposure. Considering ripening parameters, best application was 200 g L1 for 1 min (Choi and Huber, 2008). Appli cation of aqueous 1MCP (625 g L1 for 1 min) strongly inhibited softening in avocado (Choi et al., 2008). The aq ueous solution of 1MCP was originally formulated to be used under field or preharvest conditions, but these results suggest that this formulation could also be used as a postharvest treatment (Choi et al., 2008). Efficacy of 1 MCP treatment While ethylene can be bound to the receptor for minutes, a single application of 1MCP can turn the treated product insensitive to ethylene for days (Sisler and Serek, 2003). It is not clear yet whether sensitivity to ethylene returns due to synthesis of new ethylene receptors or due to release of 1MCP from the receptor or if both can happen. Observations made in different studies suggest that 1MCP binding to the ethylene receptor is reversible (Sisler, 2006; Huber, 2008).

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32 The sensitivity and the response of the produc t to ethylene at the time of 1MCP application is a key factor for the efficacy of the treatment or development of the problems. Climacteric fruit at early ripeness stages are more sensitive to 1MCP than those more advanced in ripening, such as the case o f papaya (Manenoi et al., 2007) and tomato (Hurr et al., 2005). Different responses to 1MCP treatments are likely to be related to internal ethylene levels (Ekman et al., 2004; Watkins et al., 2007; Zhang et al., 2009 b ). Additionally, there is strong evidence for enzymic degradation of 1MCP in tissues that can contribute to persistence of or recovery from 1MCP effects (Huber et al., 2010). According to Feng et al. (2000), 1MCP acts as a noncompetitive inhibitor of ethylene in avocado only if applied before ethylene application. Adkins et al. (2005) suggested that there is little effect of ethylene exposure after 1MCP treatment, but some potential effect by apply ing 1 MCP after ethylene exposure. When applying the same gaseous 1 MCP treatment (500 nL L1 for 18 h at 20 C) 0 to 8 d after the same ethylene treatment, 1MCP was effective when applied less than 4 d, but no significant differences were found for Hass avocados treated 4 d after the ethylene treatment and the control. Similarly, it was sugg ested that inhibition of ethylene by 1MCP is non competitive in banana (Jiang et al., 1999); delayed 1MCP treatments were not effective when applied after 3 or 5 d to fruit pretreat ed with ethylene. The use of ethylene has been reported to be inefficient when applied after 1MCP. Papayas immersed in ethephon (100 or 500 L L1 for 5 min or brief dip) up to 1 day after 1 MCP treatment did not show any difference in firmness from those treated with 1 MCP only (Manenoi et al., 2007). Ethylene treatments (100 L L1 for 24 h) applied to

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33 Hass did not influence ripening recov er y when applied up to 14 d after treatment with 1 MCP at 500 nL L1 for 18 h at 20 C (Adkins et al., 2005) E thylene treatment (100 L L1 for 12 h at 20 C) applied to midripe Booth 7 avocado fruit did not promote ripening recovery from an application of 1 MCP (0.9 L L1 for 12 h at 20 C) to pre ripe fruit ( Jeong and Huber, 2004). Jeong and Huber (2004) suggested that ripening recovery in avocado can be only partially amended through short term ethylene exposure and differs significantly for different ripening parameters, but that it is possible that more prolonged or continuous exposure to ethylene may prove more efficacious in reversing the effects of 1 MCP. Adverse effects of the postharvest treatment with 1MCP The efficacy of 1 MCP is so high that is extremely important to define the treatment protocols for concentration, exposure time, temperature and maturity stage to avoid problems during ripening or storage. The literature reports problems such as suppression of peel color development in melon ( Ergun et al., 2005 a ) and banana (Golding et al., 1998), uneven ripening in avocado (Woolf et al., 2005), rubbery texture in papaya (Manenoi et al., 2007; Pereira et al., 2007), failure to soften in pears (Ekman et al., 2004), enhanced flesh disorders in st one fruits (Lurie and Weksler, 2005) and higher decay incidence in strawberry (Jiang et al., 2001), avocado, custard apple, papaya and mango (Hofman et al., 2001). Diene is credited as the main compound involved in the resistance of avocado to postharvest fungal disease (Prusky et al., 1992). The synthesis of the antifungal compound diene can be stimulated by ethylene in idioblasts (LeikinFrenkel and Prusky, 1998). The compound is synthesized mainly in the mesocarp and transported to the

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34 skin. I t has been suggested that the antifungal compound diene needs to be present above a minimum concentration to inhibit growth of Colletotrichum gloeosporioides and consequent development of anthracnose in unripe avocado. Treatment with 1MCP could reduce diene synthes is, favoring anthracnose development (Wang et al., 2006). The higher decay incidence in 1MCP treated fruit can also be a consequence of extended periods of ripening. As reported for Hass avocado, t he greater the period of time (days) necessary to ripen, the greater was the severity of body rots ( Adkins et al. 2005) Hofman et al. (2001) also reported that 1 MCP treatment was associated with slightly increased decay severity caused by Colletotrichum spp. (body rots) and Dothiorella spp. (stem rots); however, i t is believed that the higher incidence of diseases was due to longer ripening time and not because of the product itself (Hofman et al., 2001). A lthough 1MCP can contribute to reduced resistance of fruit to disease, a direct effect of 1 MCP on disease development is not likely (Adkins et al., 2005). Even though limitations exist, the potential of this technology is undeniable and commercial use can be considered for avocado ( Feng et al., 2000; Jeong et al., 2003; Choi et al., 2008). Plant V olatile C ompounds Volatile compounds are largely low molecular weight lipophilic compounds. Plants use these compounds for several reasons, from attraction of pollinators to defense mechanism The main groups of volatiles are the terpenes, phenylpropanoids / benzenoids, fatty acid and amino acid derivatives (Dudareva et al., 2004). The complexity and diversity of plant volatiles is demonstrated in the review by Knudsen et al. (2006), in which an extensive list of 1719 volatile compounds from floral scents is presented.

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35 Volatiles in F ood Flavor of a food is a result of taste (mouth) and smell (nose). Only five basic tastes are known: salt y sweet, sour, bitter and umami (Reineccius, 2006). On the other hand, the human nose is sensitive to thousands of volatile compounds that exist in nature. Some volatiles are liberated by intact food; other volatiles are liberated during mastication and are sensed in the nose by retronasal aroma. Aroma compounds interact with food matrix in several ways, reducing or increasing their volatility (Druaux et al., 1998). Retronasal aroma, the sensation experienced during food consumption when flavor molecules are pulled from the mouth to the nasal cavity, is a great contributor to the overall flavor of a product. The intensity of this sensation depends on the nature of each compound, salivation, time of mastication and food matrix (Mialon and Ebeler, 1997). Although thousands of volatile compounds have been reported, only a small percentage of them contribute to flavor as aroma due to diff erent thresholds of perception in the human nose (Reineccius, 2006). Aroma is one the factors that contributes to acceptability or rejection of a product. But the presence of an aroma compound itself is not enough, since a very high concentration may not be active while others with very low concentration can produce a strong impact (Druaux et al., 1998). A volatile will be an odor active compound when the ratio of its concentration to its odor threshold, expressed in log units, is positive (Baldwin, 2004). In tomato, only a small number of the more than 400 volatiles detected have a positive impact on the flavor profile. The major compounds can be derived from fatty acids, amino acids or carotenoids and are usually associated with ripening. The association w ith ripening would be an indication that the fruit has the highest nutrient bioavailability (Goff and

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36 Klee, 2006). Aldehydes, esters, ketones, terpenoids and sulfur containing compounds are the main classes of compounds for flavor volatiles in fruits (Bald win, 2002). Fatty acids derivatives originate from C18 unsaturated fatty acids including linoleic acid and linolenic acid, which then undergo reactions catalyzed by the enzyme lipoxygenase. The compounds resulting from this first reaction can be further m etabolized by other enzymes, generating other groups of compounds such as aldehydes and their isomers (Dudareva et al., 2006). These volatiles are associated with flavors described as tomato, green or grassy (Goff and Klee, 2006) and are important to the flavor of tomato, cucumber, peppers and other vegetables (Baldwin, 2002). Aldehydes can be further reduced to alcohols on the lipoxygenase pathway, mediated by alcohol dehydrogenases. Later, alcohols can generate esters in reactions mediated by alcohol acyltransferases (Dudareva et al., 2006). In apples, fatty acids are important precursors of esters (Song and Bangerth, 2003). Amino acids can also be precursors for these compounds in fruits (Dudareva et al., 2006), such as in banana (Tressl and Drawert 1973). Esters are frequently associated with fruity notes on fruit aroma (Dixon and Hewett, 2000; Jordn et al., 2001; Jetti et al., 2007; Zellner et al., 2008). Terpenes are the largest group of plant volatiles and include hemiterpenes (C5 compounds), m onoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20). They are derived from the mevalonic acid (MVA) pathway and originated from the isoprenoid (C5 compound) pathway, where precursors are the isopentenyl diphosphate (IPP) and its isomer dimethylall yl diphosphate (DMAPP) (Dudareva et al., 2006). Terpenes are

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37 important aroma compounds in citrus (Flamini et al., 2007; Flamini and Cioni, 2010) and mango (Quijano et al., 2007; Pandit et al., 2009). Volatiles in A vocado Several studies have reported volat iles in avocado leaves and fresh and processed avocado fruit However, there are no studies on changes of volatile composition during ripening. Terpenes are the main group of volatiles in fresh leaves and essential oil and are suggested to be used as means of cultivar race or Persea species identification (Bergh et al., 1973; King and Knight, 1987; King and Knight, 1992; Wu et al., 2007; Joshi et al., 2009). Leaves of Mexicantype avocados are rich in estragol, a compound that gives the anise scent to the l eaves of this race. However, this volatile in West Indian or Guatemalan cultivars is negligible (King and Knight, 1987; King and Knight, 1992; Pino et al., 2006; Wu et al., 2007). Terpenes are also predominant in fruit (Pino, 1997; Sinyinda and Gramshaw, 1998; Pino et al., 2000; Pino et al., 2004). However, estragole is not found in fruit mesocarp and sesquiterpenes are the main type of terpene. For Hass and California, (E)nerolidol was the main compound in the ripe fruit, with lesser amounts of car yophyllene, pinene, trans bergamotene and bisabolene (Pino et al., 2000). For Moro, (Z)nerolidol was the major compound (Pino et al., 2004). The sesquiterpene caryophyllene was found to be the most abundant compound in Israeli avocado mesocarp ripeness stage and cultivar not reported (Sinyinda and Gramshaw, 1998) In this study, terpene hydrocarbons mainly sesquiterpenes formed 80% of total volatiles.

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38 The volatile profile of fruit mesocarp also includes lipidderived compounds that are found in increased amounts with processing. The presence of aldehydes was higher in Israeli avocado mesocarp when cut and left for 2 h at 20 oC (Sinyinda and Gramshaw, 1998). In microwaved avocado puree, aldehydes were found in much higher amounts than in unpr ocessed avocado puree (Lopez et al., 2004). Microwave processing for oil extraction also generates significant amounts lipid degradation aldehydes, such as hexanal, octanal and nonanal (Moreno et al., 2003). Levels of hexanal which is characteristic of o xidation processes were found to be lower in oils which were richer in oleic acid (Haiyan et al., 2007). Effects of E thylene and 1 MCP on V olatile P roduction D uring R ipening During ripening the production of aroma volatile compounds is one of the multipl e physiological changes of climacteric fruit affected by ethylene (Zhu et al., 2005; Barry and Giovannoni, 2007). Not surprisingly, the ethylene inhibitor 1MCP is capable of affecting volatile production as well (Huber, 2008; Watkins, 2008). The literatur e has evidence that ethylene does not regulate all the steps of volatile production. For instance, it was showed that not all steps of the pathways of aroma biosynthesis in apple are regulated by ethylene (Schaffer et al., 2007). In tomatoes, hexanal and hexenal, are among the most important aroma compounds. These are formed by lipid oxidation during fruit maceration via the activity of lipoxygenases on fatty acids. Studies suggest that ethylene mediates regulation of this enzyme and consequently an inhibit ion of ethylene would lead to less volatile production and a change in aroma profile. On the other hand, the enzyme alcohol dehydrogenase (ADH), that converts hexanal and hexenal into hexanol and hexenol, does not seem to be regulated by ethylene (Zhu et al., 2005).

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39 Ester production, mainly butyl and hexyl acetate, increased during ripening of Packhams Triumph pears and were highly correlated with ethylene production (MoyaLeon et al., 2006). The ethylene inhibitor 1 MCP reduced the levels of these compounds and the rate of alcohol production during cold storage, reducing contents of butanol and hexanol. Similarly, 1 MCP treatment delayed the increase in ester and alcohol production in dAnjou pears and reduced the c oncentrations of both groups of comp ounds, as well as aldehydes (Argenta et al., 2003). Esters and alcohols were reduced by 1MCP in Delicious and Golden Delicious apples as well as ketones and aldehydes (Kondo et al., 2005). It was suggested that 1MCP influences the enzyme activity at the lipid catabolism pathway. Lactones, a group of cyclic ester compounds, were significantly suppressed by 1MCP (1000 nL L1 for 12 h at 20 oC) in San Castrese (low aroma) and Ceccona (strong aroma) apricots (Botondi et al., 2003). Terpenes were not affected by ethylene inhibition with 1MCP in apricots (Valds et al., 2009). The production of the sesquiterpene farnesene in pears (Gapper et al., 2006) and apples is greatly reduced by 1MCP (Pechous et al., 2005) and the last step of the pathway for this sesquiterpene biosyntheses is highly induced by ethylene (Schaffer et al., 2007). The ethylene inhibitor 1 MCP stimulated terpene alcohols (linalool and terpenol) in Ceccona apricot (Botondi et al., 2003). In Fortune mandarin, the production of t erpenes was stimulated ethylene, while it was initially reduced by 1MCP treatment but was progressively recovered over time (Herrera et al., 2007). D espite the evidences of changes in volatile compounds in several horticultural products, there are no reports of the influence of 1 MCP or ethylene in the volatile production and profile of avocado during ripening

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40 Methods for V olatile E xtraction and A nalysis The choice of the method used to extract volatiles depends on a number of factors and often two methods are used to improve identification (Reineccius, 2006) Steam distillation is used to carry out the volatiles from the sample followed by solvent extraction (Zhang and Li, 2010). Liquid extraction with solvents highly depends on the polarity of solvents and volatiles in the sample and has a great disadvantage of extracting the sugars and lipids of the sample, which are unwanted for further chromatographic analysis (Reineccius, 2006) In c ontrast to liquid extraction, Solid Phase Micro Extraction SPME is a quick, simple and solventless method to extract volatiles. SPME uses a coated fiber that adsorbs the volatiles and t h is same device is used to collect the volatiles and for injection into a gas chromatograph ( GC ) ( Arthur and Pawliszyn, 1990). SPME is mostly used for static headspace analysis, which may more closely reflect the true flavor profile, but compounds are present at very low levels, if detected at all (Baldwin, 2004). Dynamic headspace analysis (purge & trap) is another technique that uses purified inert gas in a flow through system to collect and concentrate volatiles from the headspace of a sample in a solid trap containing the sorbent such as Tenax (Zhang and Li, 2010). It favors the highest volatile compounds and also permit s concentrat ion of low threshold aroma compounds with higher levels of compounds than static headspace analysis (Reineccius, 2006). Gas chromatography has been employed as a separation and analytical technique for volatile analysis. The advent of Mass Spectrometer ( MS) and its strong capability of compound identification made it possible to couple GC MS to generate a powerful technique for analytical volatile identification and quantification (Zhang and Li, 2010).

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41 More recently, a technique called GC olfactometry mad e possible to join the separation of compounds through gas chromatography and the identification of odor active compounds by the human nose (Zellner et al., 2008) GC olfactometry has made it possible to identify s ome compounds with low thresholds such as potent sulfur and nitrogen compounds that do not form an easily measurable peak in the chromatogram but still have an active aroma (Reineccius and Vicker, 2004) With processing industries becoming automated, the electronic nose is another alternative for nondestructive evaluation of samples according to volatile compounds, simulating an olfactory system. The apparatus contains a carrier gas that takes the sample through the detector which characterizes and quantifies the volatiles from the samples by means of pattern recognition ( Deisingh et al., 2004; Li and Wang, 2006). Although it can detect nonaromatic volatile compounds, the electronic nose does not give information that leads to identification and quantification of individual compounds, but rather discriminates samples based on the recognition of the pattern of total volatiles emitted by the sample (Baldwin, 2004). The equipment has been tested to assess fruit ripening in peaches, nectarines, apples and pears, being able to reasonably predict quality parameters (Brezmes et al., 2005). The electronic nose was also used successfully to separate fruit from different harvest maturities in tomato (G m ez et al., 2006a ) and mango (Lebrun et al., 2008) as well as to distinguish apricot varieties based on their volatile profile (Solis Solis et al., 2007) Sensory A ttributes Appearance, flavor and texture are the main sensory acceptability factors in foods. While the first uses the optical sense, flavor involves chemical senses for taste and odor, and texture is the response of tactile senses to physical stimuli (Bourne, 2004).

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42 These three factors can be evaluated quantitatively by means of several equipments and techniques (Mitcham et al., 1996; Abbott, 1999; Butz et al., 2005). Nonetheless, only a human can evaluate the quality of each of these attributes for consumption through a sensory analysis (Reineccius, 2006). Flavor quality of horticultural products is ultimately defined genetically and its description is very complex. Sensory evaluation of flavor can be done by taste panels generally using a large number of panelists rating their perceptions on a hedonic scale (Baldwin, 2004). Texture has no specific sensory receptors as for taste and aroma. The parameters of texture are perceived when the food is placed and deformed in the mouth. It is an important indicative of produce freshness (Szczesniak, 2002). Freshness of apples was best described by taste, crispiness and juiciness and also appears to be related to aroma (P neau et al., 2006). Sensory studies indicated that the eating quality of blueberries is best related to flavor, but it is also influenced by juiciness, bursting energy, sweetness and appearance (Saftner et al., 2008). A flavored tomato was rated high for sweetness, tomatolike and fruitiness descriptors and low for sour and green tomato, while a poorly flavored tomato was described as sour and green tomato and low in sweetness (Tandon et al., 2003). For avocado flavor, g enerally the higher the fruit oil content, the richer the flavor (Storey et al., 1973; Woolf et al., 2004). In a sensory analysis study of five of the major cultivars grown in California, trained panelists were asked to rate ripe fruits for nuttiness, greenness and wateriness for later correlation with minimum oil content necessary for fruit acceptability O n a 0 to 10 scale, 7 was considered the minimum score for acceptability. For this score, oil content was 8.7% (fresh weight basis) for Bacon, 10%

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43 for Fuerte, 11.2% for Hass, 9% for Pinkerton and 10.3% for Zutano, a nd the equivalent mean percentage dry weight for all cultivars was 20.8% (Lee et al., 1983). A sensory panel evaluated 15 selections of avocado for visible fibers, sweetness, firmness, texture, flavor and overall acceptability. Pulp texture had the highes t correlation coefficient (r = 0.80) with overall acceptability, followed by flavor. Visible fibers was the most negative factor. Even though oil content was associated with flavor (r = 0.63), it was fairly low correlated with acceptability (r = 0.34) (Raz eto et al., 2004). Similar results were found for nine selections and Hass and Bacon, where texture and flavor were positively correlated with acceptability, while fibers and mesocarp discoloration were negatively correlated with acceptability (Villa, 2005). Besides nutty flavor, some avocados can have a slightly higher sugar content that contributes to a sweeter flavor (Shaw et al., 1980) Immature avocados have an unpleasant bitter flavor that leaves a distinctively long aftertaste on the palate. Thi s flavor becomes undetectable when the fruit is ripe and i s associated with the presence of three longchain C17 aliphatic compounds (Brown, 1972). However, bitterness can also be detectable in some cases, even in ripe fruits and it is a negative factor f or sensory acceptability (Shaw et al., 1980) In addition to these flavor characteristics, shriveled fruit are not likely to be ac cepted by consumers of avocado (Storey et al., 1973). Current research on postharvest has shifted to f ocus on maintaining opti mal flavor quality while extending shelf life of a horticultural product not only appearance. It is important to identify optimal handling conditions and to develop technologies that will lead to this goal (Kader, 2008).

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44 Application of 1MCP technology m ay affect sensory acceptability of fruits and vegetables. This technology has been used by apple industries around the world (Watkins, 2008). Anna apple treated with 1MCP was less aromatic, but preferred by panelists over nontreated fruit (Lurie et al. 2002). Similar results were reported later for the same cultivar, and added that the fruit is firmer and juicier than untreated, even though less aromatic (PreAymard et al., 2005). In a sensory test performed for Gala apples, consumers distinguished b etween 1MCP treated and nontreated fruit, but there were no differences for overall liking scores. However, consumers that ate the fruit regularly preferred untreated fruit when compared to those that did not eat it (Marin et al., 2009). Informal sensory analysis of Florida 47 tomato treated with 1MCP revealed that aroma profiles were negatively and irreversibly affected, mainly when treatment was applied in early ripeness stages (Hurr et al., 2005). In Packhams Triumph pears, 1 MCP treatment maintained textural characteristics of the fruit and was preferred by panelists over untreated fruit after 6 months of storage (Moya Leon et al, 2006) Flavor and consumer liking of kiwi was not affected by 1MCP treatment (Harker et al., 2008a). Additionally, p anelists did not notice differences in aroma, color and firmness of mango treated with 1MCP over the untreated fruit (Cocozza et al., 2004). Research O bjectives Despite the information available on the literature about avocado, most of the available repor ts are for Mexican types, one of the three known races of avocado. There is still lack of information on cultivars of the other two races, West Indian and Guatemalan, largely cultivated in Florida. Additionally, although 1MCP effectively delay s avocado ri pening, there is no information on its effects on volatile compounds

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45 and sensory parameters. The lack of this important information for the avocado industry, especially in Florida, motivated this study. Also, the aqueous formulation of 1MCP has a great potential to be used as a postharvest treatment in avocado and, therefore, the study of the effects of concentration and treatment time is valuable information for the avocado industry. The objectives of this research were: 1) to determine concentration and exposure time for postharvest ethylene and/or aqueous 1MCP treatment in West Indian and GuatemalanWest Indian hybrid avocados; 2) t o determine whether a delayed and prolong ed ethylene treatment is effective to alleviate ripening asynchrony from 1 MCP ex posure in avocado; 3) to identify the main volatile compounds in West Indian and GuatemalanWest Indian hybrid cultivars at three ripeness stages; 4 ) to investigate the groups of volatile compounds that are enhanced or suppressed by 1MCP and ethylene trea tments during ripening of avocado; 5) to evaluate the use of an electronic nose to classify avocado pulp by ripeness stage in fruit untreated or treated with aqueous 1 MCP; 6) to evaluate sensory attributes of avocado following ethylene pretreatment and/or exposure to gaseous or aqueous 1MCP

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46 CHAPTER 3 DETERMINATION OF CONCENTRATION AND EXPOS URE TIME FOR POSTHARVEST ETHYLENE AND/OR AQUEOUS 1 MCP TREATMENT IN AVO CADO Introduction The avocado industry in the United States is mainly sustained by the produc tion of the States of California and Florida, responding respectively for 85 % and 14 2 % of the country production (NASS, 2010 ). The California industry is strongly dependent (90% acreage) on Hass a small (5 to 12 oz) GuatemalanMexican hybrid which is p roduced nearly year round. In Florida, about 60 early mid and lateseason West Indian and Guatemalan cultivars and hybrids, larger in size (from a few ounces to five lbs), are grown in Florida and the season extends from May to early March (Evans and Na lampang, 2006). The avocado industry is an important revenue generator for Florida since about 80% of the fruit are sold outside the state, with a farm gate value of about $ 12 mi llion (Crane et al., 2007a ). Avocados can be treated commercially with ethylene to increase uniformity of fruit ripening (Saltveit, 1999) Sensitivity of avocado fruit to ethylene increases with time after harvest (Eaks, 1966; Gazit and Blumenfeld, 1970; Hershkovitz et al., 2010). Treatment with ethylene depends on cultivar, maturi ty and season the earlier the fruit, the longer the treatment with ethylene. Recommended conditions are 10 to 100 L L1 ethylene at 17 to 20 C, 48 to 72 h for early season, 24 to 48 h for midseason or 12 to 24 h for lateseason fruit (Woolf et al., 2004; Kader and Arpaia, 2006). Treatment of Hass avocado with ethylene at 100 L L1 for 24 h reduced the time (days) to ripen from 13 to 10 d (Adkins et al., 2005). Ethylenetreated Monroe avocado fruit (100 L L1 for 24 h at 13 C) showed significantly more uniform softening than control fruit when stored for 7 or

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47 14 d at 13 C (Jeong et al., 2002a). Zauberman et al. (1988) suggested that ethylene is needed not only to trigger, but also to sustain the ripening process, which would require its continuous presence. E thylene also hastens ripening, which is undesirable when shelf life extension is the purpose. The attempts to extend avocado shelf life include the use of 1m ethylcyclopropene (1 MCP), a cyclic olefin likely to be the main ethylene antagonist compound in the near future (Sisler, 2006). It has been extensively tested and used as a postharvest treatment of horticultural products (Huber, 2008; Watkins, 2006 2008) to extend shelf life of both climacteric (Manenoi et al., 2007; Mattheis, 2008; Singh and Pal, 2008) and nonclimacteric fruits (Cai et al., 2006 ; Mao et al., 2004). Avocado is one of the several horticultural crops registered for commercial use of 1 MCP in the United States and around the world (Watkins, 2008). Studies have shown that ga seous 1MCP is very effective for delaying ripening of avocado. For the cultivar Hass, significant delays of ripening occur from concentrations as low as 15 nL L1 gaseous 1MCP applied for 24 h (Feng et al., 2000). I ncreasing 1MCP concentration (50 or 500 nL L1) was more effective for Hass avocado than increasing treatment duration (0 to 36 h) (Adkins et al., 2005). However, reported treatments for maximum delay of ripening differ, varying from 50 to 500 nL L1 from 12 to 24 h (Adkins et al., 2005; F eng e t al., 2000; Woolf et al., 2005). For Florida grown avocados, a relatively low concentration of 90 nL L1 was not effective for Simmonds but a maximum delay of ripening was obtained with 450 nL L1 for 24 h at 20 C ( Jeong et al., 2002a) Ripening w as significantly delayed in Tower II (Jeong et al., 2003) and Booth 7 (Jeong et al.,

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48 2003; Jeong and Huber, 2004) treated with 900 nL L1 for 12 h, and Donnie avocado treated with 1,000 nL L1 for 24 h at 7 C (Arias et al., 2005). Studies done with a new aqueous formulation of 1MCP demonstrated a great potential to be used as an alternative postharvest treatment to gaseous 1MCP For instance, a single application of 625 g L1 aqueous 1MCP for 1 min in tomato was comparable in efficacy to a 9h exposure to 500 nL L1 gaseous 1MCP (Choi et al., 2008). Significant shelf life extension of plum was observed with an immersion treatment in aqueous 1 MCP at 1,000 g kg1 (Manganaris et al, 2008). Aqueous 1MCP applied to breaker stage Florida 47 tomat oes was eff ective at concentrations as low as 50 g L1 and for 30 s exposure; best application was 200 g L1 for 1 min (Choi and Huber, 2008). For Hass avocado, an application of aqueous 1MCP at 625 g L1 for 1 min strongly delayed ripening (Choi et al., 2008). Th e objective of this study was to determine concentration and exposure time for postharvest ethylene and/or aqueous 1MCP treatment in West Indian and Guatemalan West Indian hybrid avocados. Material and Methods Aqueous 1 MCP P reparation and T reatment A queous 1MCP was prepared from formulation AFxRD 300 (2% a.i., AgroFresh, Inc. Philadelphia, PA ) according to Choi and Huber (2008) For each desired concentration, adequate amounts of the powder were weighed and suspended in 30 L of distilled w ater in 50L plastic containers. The solution was swirled gently for 1 min and used within 10 and 45 minutes after preparation.

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49 Groups of eight to 12 fruit were placed in mesh bags and completely immersed in the respective solution for the desired time. B ags were gently agitated to ensure full contact of solution with fruit surface. After removal from the solution, the fruit were allowed to drain excess solution and immediately dried with a paper towel. Experiment 1 'Monroe' avocado ( Persea americana Mill. ) a late season cultivar ( Tropical Research and Education Center 2008) was harvested in November 2007 on date A according to the Avocado Shipping Schedule for Florida (Florida Avocado Administrative Committee, 2009) at maturegreen stage in Homestead, FL, returned the same day to the Postharvest Horticulture Laboratory in Gainesville, FL, and held overnight at 20 C The following day fruit were s orted for absence of major defects and diseases and randomly separated into groups for treatment applications. F ruit were treated with aqueous 1MCP [ 225, 450 or 900 g L1 a.i. (4.16, 8.32 or 16.64 mmol m3 a.i.)] for 1 or 2 min at 20 C. Immediately after treatment, fruit ripening was monitored at 20 C (84% R H ) for whole fruit firmness weight loss, peel color respiration and ethylene production. Control fruit (not exposed to 1MCP) were stored under identical conditions. Experiment 2 This experiment was designed to evaluate lower concentrations of 1MCP than those used in the first experiment and also to determine if a treatment with ethylene would help to synchronize ripening and reduce variability within a lot of fruit. West Indian Arue avocado, an early season cultivar ( Tropical Research and Education Center 2008) was harvested at maturegreen stage on date A in May 2008, from a commercial grower located in Homestead, FL. Fruit were transported the same

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50 day to Gainesville, FL, and held overnight at 20 C Treatments were applied 24 h after harvest The treatments included exposure time to ethylene ( 100 L L1 for 0, 12 or 24 h at 20 C ) prior to application of several concentrations of aqueous 1MCP [ 0, 50, 100, 150 and 200 g L1 a.i. (0, 0.92, 1.85, 2.54 and 3.70 mmol m3 a.i.)] for 1 min at 20 C An interval of 24 h was observed between the ethylene (12 or 24 h) and 1MCP treatments. F ruit ripening was monitored at 20 C and based on of whole fruit firmness weight loss, peel color respiration, ethylene production and decay Control fruit (not exposed to 1MCP or ethylene) were maintained under ident ical storage conditions. Experiment 3 Another experiment was conducted to evaluate pretreat ment with ethylene and lower concentrations of 1MCP with the West Indian Simmonds avocado ( Tropical Research and Education Center 2008) Fruit were harvested on date A in June 2008 at maturegreen stage from a commercial grower located in Homestead, FL, transported in the same day to Gainesville, FL. The treatments were combinations of application delay after harvest (12 or 24 h), time of exposure to ethylene (100 L L1 for 0, 12 or 24 h at 20 C ) and concentrations of aqueous 1MCP [ 0, 100 and 200 g L1 a.i. (0, 1.85 and 3.70 mmol m3 a.i.)] for 1 min at 20 C An interval of 24 h between the ethylene and 1 MCP treatments was observed. F ruit ripening was monitored at 20 C and based on whole fruit firmness weight loss, peel color respiration ethylene production and decay Control fruit (not exposed to 1MCP or ethylene) were maintained under identical storage conditions.

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51 Ripening and Q uality Parameters Whole f ruit f irmness Firmness was determined by a nondestructive compression test on whole, unpeeled fruit using an Instron Universal Testing Instrument (Model 4411, Canton, MA, USA) fitted with a flat plate probe (5 cm diameter) and 50kg load cell. After establishing zero force contact between the probe and the equatorial region of the fruit, the probe was driven with a crosshead speed of 20 mm min1. The force was recorded at 2.5 mm deformation and was determined at two points on the equatorial region of each fruit, with a 90 angle between points. The same four fruit of each treatment were measured repeatedly every other day until they reached the full ripe stage. Fruit were considered commercially ripe upon reaching 10 to 20 N firmness. Peel color The same fr uits used for firmness determination were used to measure peel color at the equatorial region (two readings per fruit), recorded every other day with a Minolta Chroma Meter CR 400 (Konica Minolta Sensing, Inc., Japan) operating with a C illuminant and d/0 diffuse illuminating / 0 viewing angle and equipped with a 11mm diameter light projection tube aperture. The Chroma Meter was calibrated with a white standard tile. The CIELAB values L* (lightness), a* and b* were measured. The results are presented as l ightness ( L ), chroma value ( C ) and hue angle (h) The chroma value and hue angle were c alculated from the measured a and b values using the formulas C *=( a *2+ b *2)1/2 and h =arc tangent ( b* / a* ) (McGuire, 1992). Respiration and ethylene production rates R espiration (as CO2 evolution) and ethylene production rates were measured daily, using the same four fruit for each treatment. Fruit were individually sealed for 20

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52 min in 2 L plastic containers prior to sampling. A 1m L gas sample was withdrawn by a plast ic syringe through a rubber septum for analysis. Carbon dioxide (CO2) was determined using a Gow Mac gas chromatograph (Series 580, Bridge Water, NJ, USA) equipped with a thermal conductivity detector (TCD). Ethylene was measured by injecting a 1.0 m L gas sample into a Tracor gas chromatograph (Tremetrics, Austin, TX, USA) equipped with a flame ionization detector. Weight loss Weight loss was calculated (fresh weight basis) using the Equation 3 1: (3 1) where: WL is the weight loss given in percentage, Wo is the initial weight and Wf is the final weight. Decay The severity of decay was evaluated for each fruit according to a subjective, visual scale, where: 0 = no decay; 1 = trace, up to 1 spot observed; 2 = slight, up to three little spots (< 10 mm) or on e medium spot (10 to 20 mm) observed; 3 = medium, spots increased in size (> 20 mm); 4 = severe, spots increased in size and occurrence of mycelium or sporulation. Fruit of a treatment were considered unmarketable when the average score was higher than 2.5. The results are shown as the average of the scores given for each treatment. Pathogens were identified by the UF Plant Disease Clinic at Gainesville, FL. Statistical A nalysis The experiments were conducted in a completely randomized design. Experiments 2 and 3 were conducted in factorial schemes of, respectively, 3 x 5 and 3 x 3 (delay to

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53 ethylene application x concentration of 1MCP). For whole fruit firmness, weight loss and decay, four replicates were used in Experiment 1 and eight in Experiments 2 and 3, while six replicates were used for respiration and ethylene production in all experiments. Statistical procedures were performed using the Statistical Analysis Software SAS version 9.1.3 (SAS Institute Inc., Cary, NC). Differences between means wer e determined using Duncans M ultiple R ange T est. Results Experiment 1 Un treated fruit softened from an initial firmness of 200 N to 10 to 20 N at full ripe stage within 14 d after treatment (Figure 3 1; Table 31) Softening was strongly affected by all aq ueous 1 MCP treatments that delayed ripening 12 to 16 d beyond the control. Fruit immersed in 225 g L1 aqueous 1MCP for 1 min had delayed ripening with fairly uniform mesocarp softening. Fruit immersed in aqueous 1 MCP at 900 g L1 for 1 min or in 225, 450 or 900 g L1 for 2 min maintained firmness values higher than 20 N for 28 d, exhibited severe decay and did not reach the firmness of a ripe fruit. Ripening asynchrony (stem end remains firmer than blossom end) of fruit treated with aqueous 1MCP was enhanced with increased immersion time and 1MCP concentration. When sliced ripening asynchrony was apparent in that affected fruit had much firmer pulp at the stem end (Figure 3 2A) than at the blossom end (Figure 3 2B) In fact, puncture tests made on crosssections of ripe fruits from all treatments revealed that pulp firmness at the blossom end was comparable to that of a ripe nontreated fruit while the stem end was not completely soft (data not shown).

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54 Non treated control fruit had nearly 9% weight loss when ripe (Table 31) Treated fruit had slightly lower rate of weight loss than control but significantly higher weight loss was noticed in ripe fruit due to extended ripening time. As a consequence fruit were significantly shriveled at the end of the experiment (Figure 3 2C) I n control fruit peel c olor change d from a darker to a lighter green h decreased (Table 3 2) at slower rates during the first 10 d and then Hue angle decreased sharply in the last 4 d when fruit were ripe (data not shown) There were no significant statistical differences between 1MCP treatments and the control for all color parameters. However, fruit treated with 450 or 900 g L1 aqueous 1MCP for 2 min were significantly greener than treatments at 225 g L1 for 1 or 2 m in. Control fruit reached p eak respiration and ethylene production rates 11 d after treatment (Table 33) F or the various 1MCP treatments peak rates were reduced by 16% to 40% (respiration) and 26% to 44% (ethylene), even though statistical di fferences ( P <0.05) were found only for peak respiration rates. The respiration peak rate was negatively correlated with 1MCP concentration and application time. Time to respiration and ethylene production peak rates for treated fruit was significantly delayed as co mpared with control fruit Respiration peak rate s for the 1MCP treated fruit were observed 12 to 15 d later than control, while ethylene production peak s were delayed by 13 to 15 d Decay severity was another limiting quality factor for 1MCP treated fruit, with anthracnose ( Colletotrichum sp.) and stem end rot ( Diplodia sp.) as the main diseases. While ripe control fruit had low incidence of decay when ripe, d ecay in treated fruit was

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55 first observed in this study after 18 d of storage (data not shown). T reated fruit that did not ripe n were limited by decay at 28 to 30 d of storage (Figure 3 2D) Experiment 2 Control Arue f ruit ripened within 9 d and exposure to ethylene did not hasten ripening (Table 34). 1 MCP delayed ripening from 2 to 4 d regardless of length of ethylene exposure. The highest concentration of 1MCP (200 g L1) delayed ripening but ripening asynchrony was observed on the majority of fruit. This disorder was observed in much lower incidence for fruit treated with 150 g L1 but did not occur in 50 and 100 g L1 treatments (data not shown). Pretreat ment of Arue with ethylene did not reduce ripening variability within fruit not treated with 1MCP. The lowest variation was observed for fruit pretreat ed with ethylene for 12 h and 1 MCP at 100 or 150 g L1 (Figure 3 3). For the West Indian Arue, there were no significant differences among treatments for peak respiration and ethylene production rates, except for ethylene in fruit treated 24 h after harvest for 24 h (Table 35). Peak rates for nontreated control higher than the other cultivars Avocados tr eated only with 1MCP had suppressed peak respiration rates (28.4%) and ethylene production rates (24.6%). Longer ethylene exposure did not influence significantly the peak rates, but tended to reduce them. Weight loss was lower for nontreated control fruit, reaching 5.7% when fruit were ripe (Table 34). Treatments with higher concentrations of 1MCP had higher weight loss. Pretreat ment with ethylene on weight loss did not show a clear trend. As for decay, there were no overall difference due to treatment, and mean scores for all treatments were marketable (Table 34).

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56 Experiment 3 Control Simmonds fruit ripened in 9 d, and treatment with 1MCP delayed ripening by 4 d (Table 36). A 24 h delay prior to 24h ethylene treatment hastened ripening by 2 d. A 24 h delay followed by 24h pretreat ment with ethylene resulted in the least variability between Simmonds fruit not treated with 1MCP, therefore, promoting better uniformity of ripening. However, the 24h ethylene treatment increased variability for 1 MCP treated fruit (Figure 34). Treatment with 1 MCP at 200 g L1 generally increased the variability within Simmonds fruit pretreat ed with ethylene. Respiration of untreated Simmonds avocado was more affected by the 24h delay to ethylene treatment (Table 3 7). Ethylene production peak rates were generally not affected by treatments, although a significant effect of 1MCP concentration was observed for fruit treated for 24 h with ethylene, 24 h after harvest. Weight loss was more affected by the length of ethylene treatment than by 1MCP concentration, being lower for longer fruit exposures (Table 36). Some differences in decay severity were observed, although a clear trend was not defined. All fruit were below the threshold of decay for unmarketable fruit (Table 36). Discussion The results of this study cl early demonstrated that aqueous applications of 1MCP on avocado were as effective as the gaseous applications previously reported in the literature (Adkins et al., 2005; Arias et al., 2005; Feng et al., 2000; Hofmann et al., 2001; Jeong and Huber, 2004; J eong et al., 2002a). This new formulation could be explored as an alternative postharvest treatment for West Indian Guatemalan and GuatemalanWest Indian hybrid avocados.

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57 Softening was strongly affected by aqueous 1 MCP and this effect was more noticeabl e for higher concentrations and longer treatment durations. Softening suppression bec a me more evident in the later stages of ripening as observed in other studies (Jeong et al., 2002; Jeong and Huber, 2004). This is credited in part to suppression of the activity of the enzyme polygalacturonase (Choi et al., 2008; Jeong and Huber, 2004; Jeong et al., 2002, 2003). In this study, 1MCP t reated Monroe fruit soften ed faster during the first 12 d of storage, then slowed until the end of the experiment (Figure 3 1) A s imilar softening pattern was reported for Floridagrown Simmonds ( Jeong et al., 2002a) and Booth 7 (Jeong and Huber, 2004) avocado treated respectively with 450 or 900 nL L1 gaseous 1MCP for 12 h at 20 C. While 1 MCP can be effective at advanced stages of ripening (Huber, 2008), sensitivity of climacteric fruit to 1 MCP is reported to be higher at early stages of ripening ; however, softening problems such as uneven ripening, rubbery texture and ripening asynchrony have been reported (Jeong and Huber, 2004; Hurr et al., 2005; Manenoi et al., 2007, Pereira et al., 2007). In the present study, pre climacteric avocados were treated 24 h after harvest. Ripening asynchrony of fruit treated with aqueous 1MCP was exacerbated with increased immers ion time and 1MCP concentration for Monroe. This was also observed on some occasions for Arue and Simmonds avocado treated with 200 g L1 aqueous 1 MCP and in much lower incidence for fruit treated with 150 g L1. Increas ed concentrations of gaseous 1MCP also led to higher incidence of uneven ripening in Hass avocado (Woolf et al., 2005). S oftening asynchrony was also reported for avocado and, more notably for tomato when the fruit was partially (50% external surface) immersed in a 625 g L1 aqueous 1-

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58 MCP solution for 1 min (Choi et al., 2008). In that study, the authors also observed that uptake or efficacy of 1MCP did not appe ar to be influenced by the stem end scar in tomato, and suggest ed uniform application of the solution to the entire fruit for optimum effect. Simmonds fruit responded better to ethylene pretreatment than Arue avocado, even though both cultivars are We st Indian types. There was generally less variability in whole fruit firmness for Simmonds fruit with increased delay after harvest and longer exposure time (Figure 34). Also, Simmonds fruit ripened faster under these treatment conditions (Table 36). These results are in agreement with other studies using Mexicantype avocados, in which s ensitivity of avocado fruit to ethylene increase d with increased delay after harvest ( Eaks, 1966; Gazit and Blumenfeld, 1970) and that longer exposure times were more effective than shorter ones (Eaks, 1966; Zauberman et al., 1988) Longer ethylene treatment more effectively promoted uniform ripening in Monroe avocado (100 L L1 ethylene for 24 h at 13 C) ( Jeong et al., 2002a). 1 MCP treated fruit had significantly ( P <0.05) higher weight loss when ripe due to extended ripening time (Table 36) This was noticed more evidently in Experiment 1 for Monroe fruit treated wit h higher concentrations of 1MCP that, as a consequence, were significantly shriveled by the end of the experiment (Figure 3 2C) Treatment of Magaa mamey sapote (Ergun et al., 2005b) and Tower II avocado (Jeong et al., 2003) with wax after gaseous 1MCP treatment significantly reduced weight loss during storage, indicating that the addition of this coating can help reduce shriveling and weight loss Additionally, as shown in the experiments with Arue and Simmonds, lower concentrations of 1MCP eff ectively extended shelf life without evident shriveling.

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59 The results from this study suggest that peel color change in avocado was more sensitive to application of 1MCP during early stages of ripening, while softening was more affected at later stages of ripening. Peel color change in Monroe fruit treated with 1 MCP was delayed in the earlier stages of ripening (data not shown) when changes in whole fruit firmness are less noticeable (Figure 31). Additionally, final peel color of 1MCP treated fruit did not differ from that of untreated Monroe fruit, even for those fruit that did not ripen to desired ripefruit firmness. These observations may be related to differential tissue (skin and pulp) responses to ethylene, as suggested for Galia and cantaloupe melons (Ergun et al., 2005a; Pech et al., 2008). Also, studies using genetic manipulation and 1MCP indicate that ethylene does not completely regulate all ripening processes and that levels of ethylene dependency are not the same during ripening (Barry and Giovannoni, 2007; Pech et al., 2008). Since color changes and softening involve different genetic regulation, ethylene sensitivity and downstream responses are also important to consider. Similar to the findings of this study, softening was a lso stro ng ly suppress ed in pear (Ekman et al., 2004), tomato (Hurr et al., 2005) and papaya (Manenoi et al., 2007; Pereira et al., 2007) fruit that attained full peel color change. In pear, the difference in the time between color change and softening was generall y greater with increased storage period (Ekman et al., 2004). The significant reduction of peak respiration rate noticed for Monroe fruit treated with high concentrations of 1MCP (Table 33) was not observed for low concentration treatments applied for Arue (Table 35) and Simmonds (Table 37). Reduction of the respiration rate up to 60% was reported for Joanna Red plum treated with up to 10,000

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60 g L1 aqueous 1MCP, as well as a significant delay and reduction of ethylene production in a dosedependent manner (Manganaris et al., 2008). Significant suppression of respiration and ethylene production rates has been reported for tomato after treatmen t with 200, 400 or 600 g L1 aqueous 1MCP (Choi and Huber, 2008) and after treatment with 500 nL L1 gaseous 1MCP for 24 h, in which rates were reduced by approximately 80% (Guilln et al., 2007). Decay was another limiting quality factor for 1 MCP tre ated fruit for Monroe. For Arue (Table 34) and Simmonds (Table 36), ripe fruit for all treatments were considered marketable, with decay scores below the marketability threshold. The main diseases observed were stem end rot ( Diplodia sp.) and anthr acnose ( Colletotrichum sp.). The resistance of avocados to anthracnose is dependent on adequate levels of the antifungal compound AFD ( (Z,Z) 1 acetoxy 2 hydroxy 4 oxo heneicosa12,15diene) that is produced by the fruit; reduction in AFD during ripening is hastened by 1MCP treatment (Wang et al., 2006). However, the limitations from decay in Monroe fruit appear to be primarily due to excessive delay of ripening caused by 1MCP rather than solely due to a reduction in AFD levels. Similar findings were reported for Hass avocado for which levels of disease in fruit treated with 1MCP we re more likely due to prolong ed storage ( Adkins et al., 2005; Hofman et al., 2001). Additionally, decay may not occur if initial AFD levels are high enough so that they woul d not be reduced sufficiently below the threshold needed for Colletotrichum infection to occur, as observed for Fuerte (Wang et al., 2006)

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61 Conclusions The results of this study clearly demonstrated that the aqueous formulation of 1 MCP was effective as a postharvest treatment to delay ripening of West Indian and GuatemalanWest Indian hybrid avocados. Strongest effects were associated with higher concentrations and longer immersion times in the aqueous 1MCP solution. Treatments strongly affected softening. Ripening asynchrony (stem end firmer than blossom end) was consistently observed in all 1MCP treatments above 200 g L1, and greater for higher concentrations and longer treatment duration. Excessive delay of ripening caused by all 2min immersion t reatments and by 900 g L1 for 1 min led to significant shriveled appearance of the fruit and high decay severity, limiting shelf life. The longer the delay to ethylene treatment (24 h) and the longer the exposure time of the fruit (24 h), the better the fruit responded to ethylene treatment. However, Simmonds was more affected than Arue, the former showing less variability in whole fruit firmness as compared with untreated fruit. 1MCP treatment of fruit pretreat ed with ethylene may increase variabil ity of ripening within a fruit. It can be concluded from these studies that a single postharvest immersion in aqueous 1MCP ( 50 to 150 g L1 for 1 min ) effective ly extended shelf life of West Indian and GuatemalanWest Indian hybrid avocados from 2 d (22%) to 4 d (44%) Nevertheless, since responses of fruit varied with cultivar, specific conditions must be developed for each one

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62 Table 3 1. Days to ripe stage final weight loss and whole fruit firmness of Monroe avocado treated with aqueous 1MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) and stored at 20 C (Exp. 1) Treatment Days to ripe stage or to limit of marketability z We ight loss (%) Final Firmness (N) Control 14d 8.8b 15.5d 225 1 min 26c 14.8a 16.4d 450 1 min 30 a 16.2a 17.9d 900 1 min 30ab 16.2a 32.5bc 225 2 min 30ab 16.4a 20.9cd 450 2 min 28b 15.7a 39.1ab 900 2 min 28b 14.2a 48.5a zMeans followed by the same small letter in the column do not differ significantly according to Duncans Multiple Range Test (P< 0.05) (n=4). Table 32 Initial and final p eel color of Monroe avocado treated with aqueous 1MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) and stored at 20 C (Exp. 1) Treatment L* z a* b* Chroma Hue Angle Initial 38.3 0.7 14.8 0.9 22.9 1.4 27.3 1.7 122.9 0.3 Control 43.3 ab 15.4 28.0ab 31.9ab 118.9abc 225 1 min 44.4a 15.3 30.1a 33.8a 117.0c 450 1 min 43.8a 15.6 30.3a 34.0a 117.4bc 900 1 min 43.0ab 14.9 27.4ab 32.4ab 117.5bc 225 2 min 44.4a 15.0 30.3a 33.8a 116.4c 450 2 min 39.3b 14.0 23.4b 27.2b 121.2a 900 2 min 40.6ab 14.6 25.5ab 29.4ab 119.9ab zMeans followed by the same small letter in the column do not differ significantly according to Duncans Multiple Range Test ( P <0.05). (n=4) Table 33. Peak rate and days to peak respiration and ethylene production of Monroe avocado treated with aqueous 1MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) stored at 20 C. (Exp. 1) Respiration Ethylene Treatment Peak rate z (mg CO2 kg1 h1) Days to peak Peak rate ( L C 2 H 4 kg 1 h 1 ) Days to peak Control 124.2a 11d 299.4a 11c 225 1 min 103.0b 23c 176.8a 24b 450 1 min 10 3.7ab 25b 187.7a 24b 900 1 min 86.8b 23c 166.7a 24b 225 2 min 89.3bc 23c 204.1a 24b 450 2 min 73.7c 23c 197.8a 26a 900 2 min 78.6c 26a 210.5a 26a zMeans followed by the same small letter in the column do not differ significantly according to D uncans Multiple Range Test ( P <0.05) (n=4).

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63 Table 34. Effect of ethylene pretreatment and concentration of aqueous 1MCP on days to reach ripe stage, final weight loss and decay rating of Arue avocado during storage at 20 C (Exp. 2). Parameterz Ethyl ene treatmenty (h) 1 MCP ( g L 1 ) 0 50 100 150 200 Days to ripe stage 0 9 11 11 13 13 12 9 11 11 13 13 24 9 11 11 13 13 Final weight loss (%) 0 5.7cAB 6.3bcA 6.1bcA 7.0abAB 8.0aA 12 5.2cB 6.6bA 6.1bA 7.9aA 7.4aA 24 6.0bA 6.4abA 6.5abA 6.7abB 7.3aA Decay rating (0 4) x 0 1.25aA 0.63aB 0.75aA 1.00aA 1.13aA 12 0.50aA 0.88aAB 0.71aA 1.00aA 1.50aA 24 0.63aA 1.75aA 0.88aA 1.25aA 1.88aA zFor each parameter, m eans followed by the same small letter within the same row or by the same capital letter with in the same column do not differ significantly according to Duncans Multiple Range Test ( P < 0.05) (n=8). yFruit were pretreated with ethylene, followed by 24 h interval then treated with aqueous 1MCP and stored at 20 C. xSubjective, visual scale, where: 0 = no decay; 1 = trace, up to 1 spot observed; 2 = slight, up to three little spots (< 10 mm) or one medium spot (10 to 20 mm) observed; 3 = medium, spots increased in size (> 20 mm); 4 = severe, spots increased in size and occurrence of mycelium or sporulation. Fruit were considered unmarketable when the average score for a treatment was higher than 2.5. Table 35. Effect of ethylene pretreatment and concentration of aqueous 1MCP on peak respiration and ethylene production rates of Arue avocado during storage at 20 C (Exp. 2). Parameterz Ethylene treatmenty (h) 1 MCP ( g L 1 ) 0 50 100 150 200 Respiration (mg CO2 kg1 h1) 0 272.6aA 229.8aA 211.1aA 202.9aA 195.2aA 12 221.1aA 228.7aA 188.3aA 186.3aA 236.1aA 24 201.9aA 20 8.1aA 199.6aA 163.2aA 158.2aA Ethylene ( L C2H4 kg1 h1) 0 363.9aA 341.0aA 307.5aA 301.2aA 274.4aA 12 357.6aA 278.9aA 282.6aA 267.6aA 267.4aA 24 299.9abA 321.3aA 285.2abA 255.8abA 194.5bA zFor each parameter, m eans followed by the same small letter within the same row or by the same capital letter with in the same column do not differ significantly according to Duncans Multiple Range Test ( P < 0.05) (n=6) yFruit were pretreated with ethylene, followed by 24 h interval then treated with aqueous 1 MCP and stored at 20 C.

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64 Table 36. Effect of delay to ethylene application, ethylene pretreatment and concentration of aqueous 1MCP on days to reach ripe stage, final weight loss and decay rating of Simmonds avocado during storage at 20 C (Exp. 3 ). Parameterz Ethylene Treatment y 1 MCP ( g L 1 ) Delay after harvest (h) Duration (h) 0 100 200 Days to ripe stage 0 9 13 13 12 12 11 11 11 24 9 9 9 Final weight loss (%) 0 5.7bB 7.9aA 7.9aA 12 12 7.1aA 6.5aB 6.9aB 24 5.5aB 5.6aC 6. 0aB Decay Rating (0 4) x 0 1.17abA 1.67aA 0.33bA 12 12 0.50aA 0.33aB 0.00aA 24 0.50aA 0.67aAB 0.83aA Days to ripe stage 0 9 13 13 24 12 11 13 9 24 7 9 9 Final weight loss (%) 0 6.7aA 7.6aA 7.4aA 24 12 6.9aA 7.2aA 5.5bB 24 4.3abB 3.9bB 4.8aB Decay Rating (0 4) 0 0.33aA 0.83aAB 0.33aA 24 12 0.67aA 1.67aA 0.83aA 24 0.00bA 0.00bB 0.67aA zFor each parameter, in each level of D elay after harvest m eans followed by the same small letter within the same row or by t he same capital letter in the same column do not differ significantly according to Duncans Multiple Range Test ( P <0.05) (n=6). yFruit were pretreated with ethylene delayed by 12 or 24 h after harvest, followed by 24h interval then treated with aqueous 1MCP and stored at 20 C. xSubjective, visual scale, where: 0 = no decay; 1 = trace, up to 1 spot observed; 2 = slight, up to three little spots (< 10 mm) or one medium spot (10 to 20 mm) observed; 3 = medium, spots increased in size (> 20 mm); 4 = severe, spots increased in size and occurrence of mycelium or sporulation. Fruit were considered unmarketable when the average score for a treatment was higher than 2.5.

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65 Table 37. Effect of delay to ethylene application, ethylene pretreatment and concentration of aqueous 1MCP on peak respiration and ethylene production rates of Simmonds avocado during storage at 20 C (Exp. 3). Parameterz Ethylene Treatment y 1 MCP ( g L 1 ) Delay after harvest (h) Duration (h) 0 100 200 Respiration (mg CO2 kg1 h1) 0 216.6aA 170.7aA 148.8aA 12 12 205.5aB 194.5aA 176.2aA 24 200.1aB 215.3aA 195.5aA Ethylene ( L C2H4 kg1 h1) 0 271.7aA 237.0aA 185.6aA 12 12 219.5abA 195.7bA 267.7aA 24 202.0aA 154.2aA 217.5aA Respiration (mg CO2 kg1 h1) 0 217.9aC 197.4aB 164.8aA 24 12 237.6aB 180.8aB 204.9aA 24 264.9aA 273.0aA 205.5bA Ethylene ( L C2H4 kg1 h1) 0 213.3aB 262.5aA 198.9aA 24 12 188.5aB 192.8aA 18 6.6aA 24 309.9aA 226.7bA 120.2cA zFor each parameter, in each level of D elay after harvest m eans followed by the same small letter within the same row or by the same capital letter in the same column do not differ significantly according to Duncans Multiple Range Test ( P <0.05) (n=6) yFruit were pretreated with ethylene delayed by 12 or 24 h after harvest, followed by 24h interval then treated with aqueous 1MCP and stored at 20 C.

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66 Figure 31. Whole fruit firmness of Monroe avocado treated with aqueous 1MCP (225, 450 or 900 g L1 for 1 or 2 min at 20 C) and stored at 20 C. Vertical bars represent standard error (n=4) Dotted line represents firmness threshold (20 N) for ripe fruit.

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67 A B C D Figure 32 Limiting quality factors of Monroe avocado treated with aqueous 1MCP during storage at 20 C. Asynchrony: A) Cross section of the stem end region pulp was firm and peel adhered to the pulp; B) cross section of the blossom end region pulp was soft and peel detached from pul p; C) shriveling and D) decay at the end of the experimental period.

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68 Figure 33 Overall coefficient of variation (C.V.) for w hole fruit firmness of Arue avocado (n=6) pretreat ed with ethylene (0, 12 or 24 h duration ) followed by 24h delay, then t reated with aqueous 1 MCP (0, 50, 100, 150 or 200 g L1 for 1 min) and stored at 20 C. A B Figure 34 Overall coefficient of variation (C.V.) for w hole fruit firmness of Simmonds avocado (n=6) pretreat ed with ethylene (0, 12 or 24 h duration ) fo llowed by 24h interval then treated with aqueous 1 MCP (0, 100 or 200 g L1 for 1 min) and stored at 20 C. Ethylene applied following delay of 12 h (A) or 24 h (B) after harvest.

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69 CHAPTER 4 USE OF ETHYLENE TREATMENT TO ALLEVIATE RIPENIN G ASYNCHRONY FROM 1 MCP EXPOSURE IN AVOC ADO Introduction During the last decade the compound 1methylcyclopropene (1MCP) has been widely used in research to extend postharvest life of a wide range of horticultural products, including vegetables, flowers, climacteric and non climacteric fruits (Huber, 2008). The molecule 1 MCP is currently considered the most effective ethylene action inhibitor since it is active at extremely low concentrations, is considered to be nontoxic and is already commercially available (Sisler, 2006; Environmental Protection Agency, 2008). Commercial use started with application to floral crops and in recent years became a common treatment for apples in production areas around the world (Watkins, 2008). While ethylene can be bound to the receptor for minutes, a single application of 1MCP can turn the treated product insensitive to ethylene for days (Sisler and Serek, 2003). It is not clear yet whether sensitivity to ethylene returns due to synthesis of new ethylene receptors or due to release of 1MCP from the receptor or if both occur Observations made in different studies suggest that 1MCP binding to the ethylene receptor is reversible (Sisler, 2006; Huber, 2008). The literature reports problems in produce quality originat ing from postharvest 1 MCP treatments such as suppression of surface color development in melon (Ergun et al., 2005 a ) and banana (Golding et al., 1998), uneven ripening in avocado (Woolf et al., 2005), rubbery texture in papaya (Manenoi et al., 2007; Pereira et al., 2007), failure to soften in pears (Ekman et al., 2004), enhanced flesh disorders in stone fruits (Lurie and

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70 Weksler, 2005) and higher decay incidence in strawberry (Jiang et al., 2001), avocado, custard apple, papaya and mango (Hofman et al., 2001). Softening is s trongly affected by 1MCP treatments in avocado, especially in the later stages of ripening. This is related to the delay of activity of several cell wall enzymes that are involved with softening in avocado, mainly polygalacturonase (PG). This enzyme is st rongly suppressed by 1MCP (Feng et al., 2000; Jeong et al., 2002a; Jeong and Huber, 2004; Choi et al., 2008), but since the fruit reaches ripefruit firmness, it is suggested that PG is important at the later stages of ripening but is not required for the extensive softening of avocado (Jeong et al., 2002a). The use of ethylene treatment has been reported to be inefficient when applied after 1 MCP. Papayas treated with ethephon (100 or 500 L L1 for 5 min or brief dip) up to 1 day after 1MCP treatment did not show any difference in firmness from those not treated with ethephon (Manenoi et al., 2007). Ethylene treatments (100 L L1 for 24 h) applied to Hass did not effective ly promote ri pening recover y when applied up to 14 days after treatment with 1MCP at 500 nL L1 for 18 h at 20C (Adkins et al., 2005). E thylene treatment (100 L L1 for 12 h at 20C) applied to midripe Booth 7 avocado fruit did not effectively promote ripening recovery from an application of 1MCP (0.9 L L1 for 12 h at 20C) to preripe fruit (Jeong and Huber, 2004). It was suggested by Jeong and Huber (2004) that ripening recovery in avocado can be only partially amended through short term ethylene application and that the extent of recovery differs significantly for different ripening parameters However the authors also suggested that more prolonged or continuous exposure to ethylene may prove more efficacious in reversing the effects of 1MCP.

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71 Even though li mitations exist, the potential of 1 MCP technology is undeniable and commercial use was suggested for avocado (Feng et al., 2000; Jeong et al., 2003; Choi et al., 2008). This study was designed to determine whether a delayed and prolong ed ethylene treatmen t is effective to alleviate ripening asynchrony from 1 MCP exposure in avocado. Material and Methods Plant M aterial Two mid season avocado cultivars were selected for this study. Both are GuatemalanWest Indian hybrids and are grown on a major commercial s cale in Florida ( Tropical Research and Education Center 2008) Booth 7 fruit were harvested o n October 2009 from an experimental planting at the Tropical Research Education Center, in Homestead, FL. Fruit were harvested on date D (Florida Avocado Admini strative Committee, 2009). Fruit were harvested at maturegreen stage early in the morning and immediately transported to the Postharvest Horticulture Laboratory in Gainesville, FL. Booth 8 fruit were obtained on date D (Florida Avocado Administrative Committee, 2009) from a commercial grower in the same city These fruit were harvested and stored at 13 C before being transported to Gainesville, FL, 24 h after harvest. Upon arrival fruit were held overnight at 20 C and then were selected for absence of major defects and diseases before treatment Aqueous 1 MCP P reparation and T reatment Aqueous 1MCP was prepared from formulation AFxRD 300 (2% a.i., AgroFresh, Inc., Philadelphia, PA) according to Choi and Huber (2008). For each desired concentration, adequate amounts of the powder were weighed and suspended in 30 L

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72 of distilled water in 50L plastic containers. The solution was swirled gently for 1 min and used within 10 and 45 minutes after preparation. Groups of eight to 12 fruit were placed in mesh bags and completely immersed in 900 g L1 (16.64 mmol m3 a.i.) aqueous 1MCP solution for 1 min. Bags were gently agitated to ensure full contact of solution with fruit surface. After removal from the solution, fruit were allowed to drain excess solution and immediately dried with a paper towel F ruit ripening was monitored at 20 C and based on whole fruit firmness, peel color, respiration, ethylene production pulp firmness and weight loss Control fruit (not exposed to 1MCP) were maintained under identi cal storage conditions. E thylene T reatment When 1 MCP treated fruit reached midripe stage, with 120 N (MR120) or 80 N (MR80) of whole fruit firmness, they were exposed to ethylene ( 100 L L1) for 2 or 4 d at 20 C in a 174L metal chamber in a flow through system. After treatment fruit were stored at 20 C until ripe. A group of 1 MCP treated fruit unexposed to ethylene was also kept at 20 C. Ripe fruit were assessed for peel color, p ulp firmness and polygalacturonase activity as described below. Ripening and Quality P arameters F ruit ripening was monitored at 20 C and based on whole fruit firmness, peel color, respiration, ethylene production pulp firmness and weight loss Control fr uit (not exposed to 1MCP) were maintained under identical storage conditions. Whole f ruit f irmness F irmness was determined by a nondestructive compression test on whole, unpeeled fruit using an Instron Universal Testing Instrument (Model 4411, Canton, MA USA) fitted with a flat plate probe (5 cm diameter) and 50kg load cell. After establishing

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73 zero force contact between the probe and the equatorial region of the fruit, the probe was driven with a crosshead speed of 2 0 mm min1. The force was recorded at 2.5 mm deformation and was determined at two points on the equatorial region of each fruit with a 90 angle between points The same four fruit of each treatment were measured repeatedly every other day until they reached the full ripe stage. Fruit were considered com mercially ripe upon reaching 10 to 15 N firmness at the equator. Peel color P eel color was determined at the equatorial region (two readings per fruit), with a Minolta Chroma Meter CR 400 (Konica Minolta Sensing, Inc., Japan) operating with a C illuminant and d/0 diffuse illuminating / 0 viewing angle and equipped with a 11mm diameter light projection tube aperture. The Chroma Meter was calibrated with a white standard tile. The CIELAB values L* (lightness), a* and b* were measured. The resu lts are presented as lightness (L*), chroma value (C*) and hue angle (h). The chroma value and hue angle were calculated from the measured a* and b* values using the formulas C*=(a*2+ b*2)1/2 and h=arc tangent (b*/a*) (McGuire, 1992). Respiration and ethy lene production rates Respiration (as CO2 evolution) and ethylene production rates were measured daily, using the same four fruit for each treatment. Fruit were individually sealed for 20 min in 2 L plastic containers prior to sampling. A 1m L gas sample w as withdrawn by a plastic syringe through a rubber septum for analysis. The concentration of gases in the sample was determined using a Varian gas chromatograph ( CP 3800, Middelburg, The Netherlands) equipped with Valco valves system (Houston, Texas, USA). CO2 was separated in a Hayesep Q column (Ultimetal, 1.0 m*1/8; 80100 mesh) and detected by a thermal conductivity detector (TCD) Ethylene was separated in a Molsieve column

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74 (Ultimetal, 1.5m*1/8; 13x80100 mesh) and a P ulse D ischarge H elium I onization D etector (PDHID) was used for detection. The conditions of the run were as follows: injector at 220 C; column oven at 50 C; TCD at 130 C and PDHID at 120 C; filament at 180 C. Ultra Pure Helium was used as carrier gas at 20 mL min1. The equipment was calibrated daily with a gaseous standard mix composed of 1.1 L L1 C2H4, 1.03% CO2, 19.98% O2 and N2 balance. Weight loss Weight loss was calculated (fresh weight basis) using the Equation 4 1: (4 1) where: WL is the weight loss given in percentage, Wo is the initial weight and Wf is the final weight. Pulp f irmne ss Cross section slices of 1.5 cm from both stem end and blossom end were used for this analysis. The extremities of the fruit were cut off and the slices were taken avoiding the seed cavity. Pulp f irmness was determined by a puncture test using an Instron Universal Testing Instrument (Model 4411, Canton, MA, USA) fitted with a 8 mmdiameter convex probe and 50kg load cell. The slice was placed on a flat surface for measurement in the center of the exposed tissue. After establishing zero force contact betw een the probe and the mesocarp tissue, the probe was driven with a crosshead speed of 5 0 mm min1 for 5 mm depth, puncturing the mesocarp tissue in an insideout (seed cavity to extremity) direction. The maximum force was recorded.

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75 Polygalacturonase (PG) a ctivity This analysis was performed separately for both stem end half and blossom end half of ripe fruit. Each fruit was peeled and divided in halves transversally. Each half was cut in pieces and frozen ( 30 C) separately for further analysis. Polygalact uronase (PG, E.C. 3.2.1.15) activity was assayed as described by Jeong et al. (2002a). The activity was determined after incubation of 100 L of the cell free protein extract with 500 L of polygalacturonic acid dissolved in 40 mM NaAc, pH 4.5, for 2 h at 34 C. Uronic acid reducing groups were measured in a microplate reader for absorbance readings at 600 nm using the method of Milner and Avigad (1967). Total protein content was determined using the bicchinoninic acid BCA method (Smith et al., 1985) wi th bovine serum as standard. PG activity was expressed as molecular D galacturonic acid equivalents produced per kilogram of protein per minute. Statistical A nalysis The experiments were conducted in a completely randomized design with six treatments and f ive replicates Statistical procedures were performed using the Statistical Analysis Software SAS version 9.1 (SAS Institute Inc., Cary, NC). Differences between means were determined using Duncans M ultiple R ange T est. Results Booth 7 Untreated control Booth 7 fruit had initial whole fruit firmness of 200 N and softened to 10 N in 12 d (Figure 4 1A). Application of 900 L L1 aqueous 1MCP for 1 min delayed ripening by another 10 d. Firmness of Booth 7 fruit treated with 1MCP was similar to untreated control until 4 d after treatment. Softening of treated fruit continued at a lower rate after 10 d when fruit was at 81 N.

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76 Peel color changes in Booth 7 control fruit were from a darker (lower L* and higher h) to a lighter green (higher L* and lower h) (Table 41). Treated fruit not exposed to ethylene had darker peel (lower L*) than control; ethyleneexposed fruit at mid ripe stage at 120 N for 2 (MR120 2d) or 4 d (MR120 4d) and at 80 N for 4 d (MR80 4d) were more yellow than fruit treated with 1MCP only. Respiration rate of control Booth 7 started to increase 4 d after storage at 20 C (Figure 4 2A). Sharpest inc rease of the rate was observed from 7 to 9 d. Peak respiration rate of 238 mg CO2 kg1 h1 was observed at 11 d while peak ethylene production rate of 122 L C2H4 kg1 h1 was observed at 10 d For 1 MCP treated fruit, rates started increasing after 12 d Rates increased to 204 mg CO2 kg1 h1 by the last day of the experiment, however, a clear respiration peak was not noticed. Ethylene production of treated fruit was significantly lower than control, reaching a peak rate of 70 L C2H4 kg1 h1 at 19 d aft er treatment. The sharpest increase of ethylene production was delayed by 7 d as compared with nontreated fruit, but the sharp decline noticed for control fruit was not observed in treated fruit. For Booth 7 control fruit, the initial pulp firmness at t he stem end was 24% higher than that of blossom end (Table 42 ). A difference of more than 20% between those two segments was observed in the other three ripeness stages when fruit was assessed for the analysis. Treated fruit also had firmer stem end pulp at intermediate ripeness stages (120 N and 80 N whole fruit firmness) and higher than control. When treated fruit reached 80 N whole fruit firmness, pulp firmness for both ends was similar to pulp firmness of control at 120 N whole fruit firmness. At ripe stage, treated fruit showed

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77 pronounced ripening asynchrony, with both stem and blossom ends pulp firmness higher than control. Ethylene treatment effectively overcame the exacerbated ripening asynchrony caused by the application of 1MCP in Booth 7 (Table 4 2 ). Fruit from MR120 2d, MR1204d and MR804d treatments softened with pulp firmness similar to untreated control in both stem and blossom ends. Treatment MR802d was not effective and stem end pulp firmness was similar to unexposed treated fruit. Pol ygalacturonase (PG) activity of ripe untreated control fruit was comparable for either stem or blossom end (Table 43 ). Although PG activity in treated fruit was not significantly different from control, it was significantly lower in the stem end than in t he blossom end. A significant difference in PG activity between stem and blossom ends was also observed for MR802d fruit. PG activity of the stem end of fruit from MR120 treatments was significantly higher than untreated control and unexposed fruit. Ethyl ene treatment generally increased PG activity at the blossom end when compared with untreated control. Fruit treated with aqueous 1MCP had twice the weight loss of control fruit (Figure 4 3). Shriveling was noticed occasionally in ripe treated fruit. Boo th 8 Untreated control Booth 8 fruit had initial whole fruit firmness of 189 N and ripened in only 8 d, faster than Booth 7 (Figure 41B). Application of 900 L L1 aqueous 1MCP for 1 min delayed ripening by 8 d. Softening of 1MCP treated Booth 8 had a pattern similar to Booth 7. While firmness of control fruit declined sharply in the

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78 first 4 d softening of treated fruit occurred faster until 6 d after treatment, when fruit was at 79 N and at lower rates until ripened. Peel color in Booth 8 was not affected as much as in Booth 7. The only significant differences were found for L* and C* of MR804d (Table 44 ). No differences in h were observed. Increased respiration rates were observed for untreated fruit since the beginning of storage and p eak rate of 187 mg CO2 kg1 h1 was observed at 5 d (Figure 4 2B). Ethylene production increased rapidly, reaching a peak of 83 L C2H4 kg1 h1 in only 4 d of storage. Treated fruit had slightly lower rates for both gases and the increase in the respiratory rate paralleled the increase in ethylene production rate, this one delayed by 8 d by 1 MCP application. Peak rates were observed at 13 d after 1 MCP treatment, with 155 mg CO2 kg1 h1 and 75 L C2H4 kg1 h1. Ripening asynchrony also occurred in Booth 8 (Table 45 ). A 64% difference in pulp firmness was observed for fruit at 120 N whole fruit firmness. However, control fruit stem end softened significantly at 80 N; pulp firmness of ripe control was significantly higher than that of the blossom end. 1MCP treatment significantly affected pulp firmness, and both ends remained much firmer than those for the untreated control. Most noticeable differences occurred for the blossom end when fruit were at 120 N whole fruit firmness. For treated fruit at 80 N w hole fruit firmness, pulp firmness in both ends was three times higher than untreated control. As observed for Booth 7, treated fruit at ripe stage showed strong ripening asynchrony. Exposure of treated fruit to ethylene effectively promoted stem end sof tening only for fruit from treatments MR1204d and MR804d, exposed to ethylene for 4 d (Table 4-

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79 5 ). For those exposed for 2 d pulp firmness at the stem end was similar to unexposed fruit. PG activity in both stem and blossom ends of untreated control fruit was slightly higher than observed for Booth 7 (Table 46). However, the activity was not significantly affected by 1MCP or ethylene exposure. As observed for Booth 7, fruit treated with aqueous 1MCP had twice the weight loss of control fruit. Dis cussion The cultivars in this study have the same origin, being GuatemalanWest Indian hybrids (Crane et al., 2007 a ), but there were several differences in postharvest behavior. The softening pattern observed for Booth 7 was similar to that observed by Jeong and Huber (2004). The faster ripening period observed for untreated Booth 8 is in agreement with Hatton et al. (1964) who reported 5 to 9 d at 21.1 C for this cultivar to ripen. L pez (1998) also reports ripening of Booth 8 averaging from 6 to 9 d but does not specify the storage temperature. The present study showed that postharvest application of aqueous 1MCP significantly delays ripening of Booth 7 and Booth 8 avocado. The efficacy of this formulation as a postharvest treatment has been reported for Hass avocado (Choi et al., 2008), Florida 47 (Choi and Huber, 2008) and Sanibel tomato (Choi et al., 2008) and for Joanna Red plums (Manganaris et al., 2008). In this study the most evident effect of the postharvest immersion in aqueo us 1 MCP (900 g L1 for 1 min) for both Booth 7 and Booth 8 was the delay of fruit softening. This was confirmed by the persistently high values of pulp firmness and the incidence of strong ripening

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80 asynchrony. Peel color, respiration, ethylene production and PG ac tivity were more affected by 1MCP treatment in Booth 7 than Booth 8. In this study, Booth 8 was treated 48 h after harvest and showed much earlier onset of climacteric than Booth 7 which was treated 24 h after harvest. Additionally, although both cultivars were late harvested, Booth 8 was harvested two months after the last date (date D) of its harvesting schedule (Florida Avocado Administrative Committee, 2009), while Booth 7 only two weeks after. The more advanced ripeness stage of Booth 8 might have contributed to a lower effect of 1MCP, as observed in other studies with avocado (Adkins et al., 2005; Choi et al., 2008). Ripening asynchrony occurred in both cultivars (Tables 43 and 4 4), confirming that the regular ripening in avocado occurs from the blossom end to the stem end of the fruit. The same pattern of ripening also occurs in tomato and it was suggested that the asynchronous ripening must be caused by differential regulation of ethylene sensitivity (Ciardi and Klee, 2001). In avocado, the reason for ripening asynchrony might reside in the presence of the seed, since it influences both fruit development (Blumenfeld and Gazit, 1974) and ripening (Hershkovitz et al., 2009, 2010). It was reported that Ettinger avocado stored for 10 d at 20 C had double the ethylene production in the inner pulp close to the seed base as that measured for whole fruit (Hershkovitz et al., 2009). Application of 1MCP was equally effective to delay ripening and downregulate the expression of ethylene rela ted genes in both seeded and seedless Arad avocado (Hershkovitz et al., 2010). However, embryo growth, which is stimulated by ethylene and delayed by 1MCP treatment, increased the response of the mesocarp tissue to ethylene, being higher close to the base of the seed (Hershkovitz et al., 2009).

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81 Although ripening asynchrony was observed for untreated control fruit, 1MCP application exaggerated the difference in pulp firmness between the stem end and the blossom end of treated fruit. Hass avocado parti ally immersed in aqueous 1MCP had significant suppression of ripening in the treated side, leading to ripening asynchrony (Choi et al., 2008). The authors report ed that the uptake or efficacy of the aqueous 1MCP did not appear to be influenced by the stem end scar and that 1MCP effects on fully immersed fruit were exerted well within if not throughout the tissues. Choi et al. (2008) also suggest that 1MCP has limited diffusive capacity. In the present study, 1MCP influenced both stem and blossom end, delaying softening of the pulp. Therefore, since in the present study fruit were fully immersed in the aqueous 1 MCP solution, it is unlikely that the observed ripening asynchrony was due to a higher uptake of 1MCP in the stem end. Schroeder (1985, 1987) reported that oil content was higher near the distal end of the seed and that ripening occurs first in the blossom end of the fruit, suggesting the relationship of higher oil contents to earlier ripening. Two gradients of dry matter and oil content were reported for Hass avocado mesocarp tissue, one with decreasing contents from the stem end to the seed cavity and the other from the distal end (base) of the seed to the blossom end of the fruit (Schroeder, 1985, 1987). In this present study, the blossom en d of 1 MCP treated fruit softened normally when compared with untreated fruit, indicating that oil content would not be the main reason for a stronger effect of 1 MCP exaggerating ripening asynchrony. Choi and Huber (2009) reported that gaseous 1MCP is so rbed rapidly to nonspecific sites in avocado exocarp and the oil content positively influences rates of sorption in the mesocarp tissue. However, these

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82 authors suggest that 1MCP sorbed to oil is likely of minimal biological significance due to rapid out gassing upon transfer to 1MCP free environments. Studies have shown that the activity of the enzyme polygalacturonase (PG) is more affected by 1MCP applications than other cell wall enzymes (Jeong et al., 2002a; Jeong and Huber, 2004). Choi et al. (2008) fully or partially immersed Hass avocado fruit in aqueous 1MCP, and observed that PG activity was lower than in untreated fruit. In the present study PG activity in Booth 7 was lower in the stem end than in the blossom end of treated fruit (Table 43 ), indicating a slower ripening at the stem end. However, although PG activity was affected by 1MCP, it was not likely to be the main reason for ripening asynchrony occurrence since PG activity in treated fruit did not differ from control. In fact, stron g ripening asynchrony was also observed for Booth 8 and there were no significant differences in PG activity between treated fruit and control. Additionally, softening of Simmonds ( Jeong et al., 2002a) and Booth 7 (Jeong and Huber, 2004) treated with 1 MCP ultimately softened to values comparable to those of control fruit, even with a strong suppression of PG activity in these fruit. Earlier studies have shown that ethylene treatment applied to fruit treated with 1 MCP did not completely revert the e ffects of the ethylene inhibitor. Hass avocado treated with 500 nL L1 gaseous 1MCP for 18 h was largely unaffected by ethylene treatment at 100 L L1 for 24 h at 20 C when applied up to 14 d after 1 MCP (Adkins et al., 2005). Treatment of midripe (75 to 90 N whole fruit firmness) Booth 7 fruit with ethylene treatment at 100 L L1 for 12 h at 20 C was also largely ineffective to overcome the effects of gaseous 1MCP at 900 nL L1 for 12 h. One of the factors that may have contributed to this lack of response may have been the use of high

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83 concentrations of 1 MCP since many studies have shown that the effects of 1MCP are strongly correlated with higher concentrations (Jiang et al., 1999; Feng et al., 2000; Moretti et al., 2002; Adkins et al., 2005; Manenoi et al., 2007; Choi and Huber, 2008; Singh and Pal, 2008). Another reason for the unsuccessful results would be a short time ethylene exposure (up to 24 h). Commercial applications of ethylene to promote uniform ripening of several climacteric fruit not treated with 1 MCP often require days of application (Saltveit, 1999). For avocado not treated with 1MCP, it is suggested that continuous presence of ethylene is required for fruit ripening (Zauberman et al., 1998). In the present study, fruit at t wo ripeness stages (120 or 80 N whole fruit firmness) treated with 900 g L1 aqueous 1MCP then exposed to ethylene for 4 d effectively overcame the pronounced ripening asynchrony caused by 1MCP treatment. This result was observed for both cultivars. Ethylene exposure for 2 d was not effective, except for Booth 7 treated at 120 N whole fruit firmness. Ethylene exposure stimulated PG activity in the stem end and blossom end of treated Booth 7 fruit. This result is in agreement with the study of Jeong and Huber (2004) that reported that ethylene treatment of 1MCP treated fruit enhanced levels of PG activity in Booth 7. However, the success of recovery from strong ripening asynchrony may not be attributable to PG activity, since Booth 8 softened as control under exposure to ethylene for 4 d, but PG activity was not sign ificantly affected (Table 46). The recovery from the effects of 1MCP on ripening asynchrony may be related to ethylene sensitivity. It has been reported that 1MCP prevents ethyleneinduced receptor degradation in tomato, which controls the timing of rip ening in this fruit (Kevany

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84 et al., 2007). These authors also demonstrated that ethylene treatment depletes levels of receptors, which would lead to higher sensitivity to ethylene since the receptors are negative regulators of ethylene signaling (Binder, 2 008; Cara and Giovannoni, 2008). Hershkovitz et al. (2009) reported a higher expression of the gene PaETR ( Persea americana ethylene receptor) close to the base of the seed that may be related to higher ethylenemediated receptor degradation, as suggested by Kevany et al. (2007) for tomato. The PaETR transcript is up regulated at the onset of ripening and hyper regulated by ethylene treatment (Hershkovitz et al., 2009, 2010). Additionally, Huber et al. (2010) raised the hypothesis that 1MCP metabolism, mos t likely through enzymic oxidation, would affect tissue sensitivity particularly if receptor associated 1MCP can be targeted for degradation. Conclusions This study demonstrated that aqueous 1MCP effectively delayed ripening of Booth 7 and Booth 8 avocado, both GuatemalanWest Indian hybrid cultivars, delaying changes in peel color and retarding the onset of respiration and ethylene climacterics. However, there were marked effects on fruit softening and Booth 7 was more affected than Booth 8. The results also showed that ripening occurred faster in the blossom end and slower in the stem end, characterizing a ripening asynchrony. Application of 900 g L1 aqueous 1MCP caused strong differences between pulp firmness from stem and blossom end, exaggerating ripening asynchrony. E thylene treatment (100 L L1) for 4 d when 1MCP treated fruit was at 120 N or 80 N whole fruit firmness was capable of overcoming the ripening asynchrony and promoting adequate

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85 softening in both ends. Ethylene treatment for 2 d was not effective, except for Booth 7 at 120 N whole fruit firmness. The results of the present study showed that a 4day treatment with ethylene at 100 g L1 effectively promoted complete recovery from the strong ripening asynchrony caused by 1MCP treatment. It is plausible that this treatment duration was sufficient to stimulate sensitivity recovery, which might begin in the blossom end and reach levels that would also affect the stem end segment. PG activity was stimulated by ethylene treatmen t in Booth 7, but was not directly related to ripening asynchrony.

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86 Table 4 1 Peel color of mature green and ripe Booth 7 avocado untreated (Control) or treated (1MCP) with aqueous 1 MCP at 900 g L1 for 1 min and stored at 20 C. Treatmentz Days of ethylene treatment Whole fruit firmness at ethylene treatment (N) L*y C* h Mature green 39.41.9 28.73.0 123.01.1 Ripe Control 0 44.2a 33.2ab 119.6ab 1 MCP 0 41.8b 30.5b 120.7a MR120 2d 2 120 45.5a 35.2a 118.1bc MR80 2d 2 80 43.6ab 33.1ab 119.3ab MR120 4d 4 120 44.8a 33.7ab 118.3bc MR80 4d 4 80 45.7a 36.3a 117.1c zWhen 1MCP treated fruit reached midripe stage, with 120 N (MR120) or 80 N (MR80) of whole fruit firmness, they were exposed to ethylene (100 L L-1) for two (2d) or four days (4d) at 20 C followed by storage at 20 C until ripe. Part of 1 MCP treated fruit (1MCP) unexposed to ethylene was kept at 20 C. yMeans followed by the same small letter within the same column do not differ significantl y according to Du ncans Multiple Range Test ( P < 0.05) (n=6).

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87 Table 4 2 Pulp firmness of stem end and blossom end segments of Booth 7 avocado untreated (Control) or treated (1MCP) with aqueous 1 MCP at 900 g L1 for 1 min and stored at 20 C. Ripenes s stagez Days to reach ripeness stage Days of ethylene treatment Whole fruit firmness at ethylene treatment (N) Whole fruit firmness at pulp firmness analysis (N) Pulp firmness at the stem end segment (N) y x Pulp firmness at the blossom end segment (N) Mature green Control 0 0 200 20 6.7 A 157.8 B Mid ripe Control 5 0 120 172.3abA 135.9abA Control 7 0 80 154.5bA 120.5bB 1 MCP 7 0 120 180.3aA 150.2aB 1 MCP 10 0 80 172.0abA 135.4abB Ripe w Control 12 0 10 to 15 2.0bA 1.4bB 1 MCP 22 0 10 to 15 31.0aA 3.3aB MR120 2d 22 2 120 10 to 15 3.0bA 1.5bB MR80 2d 22 2 80 10 to 15 28.1aA 3.1aB MR120 4d 20 4 120 10 to 15 3.4bA 1.5bB MR80 4d 22 4 80 10 to 15 4.7bA 1.5bB zWhen 1MCP treated fruit reached midripe stage, with 120 N (MR120) or 80 N (MR80) of whole fruit firmness, they were exposed to ethylene (100 L L-1) for two (2d) or four days (4d) at 20 C followed by storage at 20 C until ripe. Part of 1 MCP treated fruit (1MCP) unexposed to ethylene was kept at 20 C. yFor each parameter, m eans followed by the same small letter within the same level of Rip eness stage (maturegreen, midripe or ripe) or by the same capital letter in the same row do not differ significantly according to Duncans Multiple Range Test ( P < 0.05) (n=5). xSegments were analyzed at maturegreen, at 120 N and 80 N whole fruit firmnes s (mid ripe) and 10 to 15 N (ripe) stages. Segments for MR120 and MR80 treatments were analyzed only at ripe stage. wFruit were considered ripe upon reaching 10 to 15 N whole fruit firmness.

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88 Table 4 3 PG activity of stem end and blossom end segments of ripe Booth 7 avocado untreated (Control) or treated (1MCP) with aqueous 1 MCP at 900 g L1 for 1 min and stored at 20 C. Treatmentz Days of ethylene treatment Whole fruit firmness at ethylene treatment (N) PG activity y (mole kg 1 min 1 ) x 10 5 St em end Blossom end Control 0 35.18cdA 36.79bA 1 MCP 0 29.63dB 40.47abA MR120 2d 2 120 N 47.38aA 44.53abB MR80 2d 2 80 N 31.44dB 48.07aA MR120 4d 4 120 N 44.69abA 45.49aA MR80 4d 4 80 N 40.61bcA 44.93abA zWhen 1MCP treated fruit reached midripe stage, with 120 N (MR120) or 80 N (MR80) of whole fruit firmness, they were exposed to ethylene (100 L L-1) for two (2d) or four days (4d) at 20 C followed by storage at 20 C until ripe. Part of 1 MCP treated fruit (1MCP) unexposed to ethylene was kept at 20 C. yMeans followed by the same small letter in the same column or by the same capital letter in the same row do not differ significantly according to Duncans Multiple Range Test ( P < 0.05) (n=3)

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89 Table 4 4 Peel color of mature green and ripe Booth 8 avocado untreated (Control) or treated (1MCP) with aqueous 1 MCP at 900 g L1 for 1 min and s tored at 20 C. Treatmentz Days of ethylene treatment Whole fruit firmness at ethylene treatment (N) L*y C* h Mature green 38.21.2 25.72.7 120.71.1 Ripe Control 0 42.8a 31.3a 116.8a 1 MCP 0 39.8ab 26.6ab 119.4a MR120 2d 2 120 41.1ab 29.5ab 117.1a MR80 2d 2 80 39.5ab 27.4ab 116.9a MR120 4d 4 120 42.1ab 30.1ab 116.4a MR80 2d 4 80 38.5b 23.8b 120.1a zWhen 1MCP treated fruit reached midripe stage, with 120 N (MR120) or 80 N (MR80) of whole fruit firmness, they were exposed to ethylene (100 L L-1) for two (2d) or four days (4d) at 20 C followed by storage at 20 C until ripe. Part of 1 MCP treated fruit (1MCP) unexposed to ethylene was kept at 20 C. yMeans followed by the same small letter within the same column do not differ significantly according to Du ncans Multiple Range Test ( P < 0.05) (n=6).

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90 Table 4 5 Pulp firmness of stem end and blossom end segments of Booth 8 avocado untreated (Control) or treated (1MCP) with aqueous 1 MCP at 900 g L1 for 1 min and stored at 20 C Ripeness stagez Days to reach ripeness stage Days of ethylene treatment Whole fruit firmness at ethylene treatment (N) Whole fruit firmness at pulp firmness analysis (N) Pulp firmness at the stem end segment (N) y, x Pulp firmness at the blossom end seg ment (N) Mature green Control 0 0 200 193.0 A 185.8 A Mid ripe Control 2 0 120 152.6aA 54.6cB Control 3 0 80 42.9bA 32.6cA 1 MCP 4 0 120 169.1aA 155.1aA 1 MCP 6 0 80 128.1aA 102.3bA Ripe w Control 8 0 1 0 to 15 3.2bA 2.2aB 1 MCP 16 0 10 to 15 27.0aA 2.6aB MR120 2d 16 2 120 10 to 15 19.9aA 2.7aB MR80 2d 16 2 80 10 to 15 16.8aA 3.2aB MR120 4d 16 4 120 10 to 15 4.1bA 2.2aA MR80 4d 16 4 80 10 to 15 5.3bA 2.7aA zWhen 1MCP treated fruit reached midrip e stage, with 120 N (MR120) or 80 N (MR80) of whole fruit firmness, they were exposed to ethylene (100 L L-1) for two (2d) or four days (4d) at 20 C followed by storage at 20 C until ripe. Part of 1 MCP treated fruit (1MCP) unexposed to ethylene was kept at 20 C. yFor each parameter, m eans followed by the same small letter within the same level of Rip eness stage (maturegreen, midripe or ripe) or by the same capital letter in the same row do not differ significantly according to Duncans Multiple Range Test ( P < 0.05) (n=5). xSegments were analyzed at maturegreen, at 120 N and 80 N whole fruit firmnes s (mid ripe) and 10 to 15 N (ripe) stages. Segments for MR120 and MR80 treatments were analyzed only at ripe stage. wFruit were considered ripe upon reaching 10 to 15 N whole fruit firmness.

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91 Table 4 6 PG activity of stem end and blossom end segments of r ipe Booth 8 avocado untreated (Control) or treated (1MCP) with aqueous 1 MCP at 900 g L1 for 1 min and stored at 20 C Treatmentz Days of ethylene treatment Whole fruit firmness at ethylene treatment (N) PG activityy (mole kg1 min1) x 105 Stem end Blossom end Control 0 41.94aA 43.46aA 1 MCP 0 43.53aA 46.14aA MR120 2d 2 120 N 37.37aA 40.32aA MR80 2d 2 80 N 46.16aA 47.50aA MR120 4d 4 120 N 40.20aA 39.92aA MR80 4d 4 80 N 38.05aA 41.43aA zWhen 1MCP treated fruit reached midripe stage, w ith 120 N (MR120) or 80 N (MR80) of whole fruit firmness, they were exposed to ethylene (100 L L-1) for two (2d) or four days (4d) at 20 C followed by storage at 20 C until ripe. Part of 1 MCP treated fruit (1MCP) unexposed to ethylene was kept at 20 C. yMeans followed by the same small letter in the same column or by the same capital letter in the same row do not differ significantly according to Duncans Multiple Range Test ( P < 0.05) (n=3)

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92 A B Figure 4 1 Whole fruit firm ness of Booth 7 (A) and Booth 8 (B) fruit untreated (Control) or treated (1 MCP) with aqueous 1MCP at 900 g L1 for 1 min at 20 C and stored at 20 C. Vertical bars represent standard error (n=10). Dotted line represents firmness threshold (15 N) for ripe fruit.

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93 A B Figure 4 2 Respiration ( CO2) and ethylene production ( C2H4) rates of Booth 7 (A) and Booth 8 (B) fruit untreated (Control) or treated (1 MCP) with aqueous 1MCP at 900 g L1 for 1 min at 20 C and stored at 20 C. Vertica l bars represent standard error (n=6).

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94 Figure 4 3 Weight loss of Booth 7 and Booth 8 fruit untreated (Control) or treated (1 MCP) with aqueous 1MCP at 900 g L1 for 1 min at 20 C and stored at 20 C. Vertica l bars represent standard error (n=10).

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95 CHAPTER 5 CHANGES IN VOLATILES DURING AVOCADO RIPENING AS AFFECTED BY ETHYLENE AND 1 MCP Introduction Volatile compounds are largely low molecular weight lipophilic compounds (Pichersky et al., 2006) Plants use these compounds for several reasons, from attraction of pollinators to defense mechanism The main groups of volatiles are the terpenes, phenylpropanoids / benzenoids, fatty acid and amino acid derivatives (Dudareva et al., 2004). Terpenes are the largest group of plant volatiles, including hemiterpenes (C5 compounds), monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) (Dudareva et al., 2006), important aroma compounds in citrus (Flamini et al., 2007; Flamini and Cioni, 2010) an d mango (Quijano et al., 2007; Pandit et al., 2009). Fatty acids and amino acids can be precursors for esters in fruits (Song and Bangerth, 2003; Dudareva et al., 2006), compounds frequently associated with fruity notes on fruit aroma (Tressl and Drawert, 1973; Dixon and Hewett, 2000; Jordn et al., 2001; Jetti et al., 2007). Fatty acids can also undergo reactions catalyzed by the enzyme lipoxygenase and other enzymes, generating compounds such as aldehydes and their isomers (Dudareva et al., 2006), which are associated with flavors described as tomato, green or grassy (Goff and Klee, 2006) and are important to the flavor of tomato, cucumber, peppers and other vegetables (Baldwin, 2002). Aldehydes can be further reduced to alcohols and these can later generate esters (Dudareva et al., 2006). Several studies have reported volatiles in avocado leaves, and in fresh and processed avocado fruit Terpenes are the main group of volatiles in fresh leaves and essential oil and are suggested to be used as means of cultivar race or Persea species identification (Bergh et al., 1973; King and Knight, 1987; King and Knight, 1992; Wu et

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96 al., 2007; Joshi et al., 2009). Terpenes are also predominant in fruit (Pino, 1997; Sinyinda and Gramshaw, 1998; Pino et al., 2000; P ino et al., 2004). For Hass and California, trans nerolidol was the main compound in the ripe fruit, with lesser amounts of caryophyllene, pinene, trans bergamotene and bisabolene (Pino et al., 2000). For Moro, cis nerolidol was the major com pound (Pino et al., 2004). The sesquiterpene caryophyllene was found to be the most abundant compound in Israeli avocado mesocarp neither ripeness stage nor cultivar reported (Sinyinda and Gramshaw, 1998) In that study, terpene hydrocarbons mainly s esquiterpenes formed 80% of total volatiles. The volatile profile of fruit mesocarp also includes lipid derived compounds that are found in increased amounts with processing, especially aldehydes (Sinyinda and Gramshaw, 1998; Moreno et al., 2003; Lopez et al., 2004; Haiyan et al., 2007). Despite the information on volatiles in avocado, they mostly focus on Mexicantype avocados and a study of changes in volatiles during ripening has not been reported. During ripening, the production of aroma volatile compounds is one of the multiple physiological changes of climacteric fruit affected by ethylene (Zhu et al., 2005; Barry and Giovannoni, 2007). The literature also has evidence that ethylene does not regulate all the steps of volatile production (Flores et al ., 2002; Defilippi et al., 2005a ; Zhu et al., 2005; Schaffer et al., 2007). Not surprisingly, the ethylene inhibitor 1 MCP, which strongly affects avocado ripening delaying softening, peel color changes, enzyme activity and the onset of climacteric (Feng et al., 2000; Jeong et al., 2002; Adkins et al., 2005; Choi et al., 2008), is capable of affecting volatile production in treated products as well (Huber, 2008; Watkins, 2008). Application of 1MCP reduced or delayed ester and

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97 alcohol production in pears ( A rgenta et al., 2003; Moya Leon et al., 2006) and apples (Kondo et al., 2005) reduced aldehydes in pears (Argenta et al., 2003) and apples (Kondo et al., 2005) Compounds of a same chemical group can be affected differently (Argenta et al., 2003). The reported effects of 1 MCP on terpenes were none in apricots ( Valds et al., 2009) light in mandarin (Herrera et al., 2007) and strong in reduction of the sesquiterpene farnesene in pears (Gapper et al., 2006) and apples (Pechous et al., 2005). The knowledge of volatile suppression by ethylene inhibitors is important since changes in volatile profile can significantly affect sensory acceptability of a product. However, the literature does not contain information on the effects of 1MCP on avocado volatile pro duction during ripening. Therefore, this study aimed to identify the main volatile compounds in West Indian and GuatemalanWest Indian hybrid cultivars at three ripeness stages and to investigate the groups of volatile compounds that are enhanced or suppressed by 1MCP and ethylene treatments during ripening Material and Methods Phase 1 Effects of E thylene and 1 MCP on V olatile C ompounds The first phase of this study involved t wo experiments (July, 2008 and July, 2009) carried out using 'Simmonds' avocad o ( Persea americana Mill.), a major West Indian early season commercial cultivar in Florida ( Tropical Research and Education Center 2008). Fruit were harvested on date B (Florida Avocado Administrative Committee, 2009) at mature green stage early in the morning from a commercial grower in Miami Dade County, FL, and immediately transported 6 h to the Postharvest Horticulture Laboratory in Gainesville, FL. Upon arrival fruit were left overnight at 20C and then were selected for absence of major defects and diseases and randomly separated into groups for treatment application. For Experiment 1 (July 2008) treatments were: C1

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98 untreated control; E24 ethylene treatment at 100 L L1 for 24 h at 20 C; MCP no ethylene pretreatment; immersion in aqueous formulation of 1MCP at 150 g L1 (2.77 mmol m3 a.i.) for 1 min at 20 C. In Experiment 2 (July 2009) treatments were: C2 untreated control; E12 ethylene treatment at 100 L L1 for 12 h, E12+MCP ethylene treatment for 12 h + 12 h air + aqueous 1MCP at 150 g L1 (2.77 mmol m3 a.i.) for 1 min. Phase 2 Aqueous 1 MCP E ffects on V olatile C ompounds in T hree C ultivars In the second phase of this study three independent tes ts were conducted using the following cultivars: Simmonds (August 2009); Booth 7 (October 2009), a GuatemalanWest Indian hybrid and major midseason cultivar in Florida; and Monroe (November 2009), a GuatemalanWest Indian hybrid and a major latese ason cultivar in Florida ( Tropical Research and Education Center 2008 ). Fruit were harvested at maturegreen stage early in the morning from a commercial grower in Miami Dade County FL, and immediately transported to the Postharvest Horticulture Laboratory in Gainesville, FL. Upon arrival fruit were left overnight at 20C and then were selected for absence of major defects and diseases and separated in groups for treatment application. For all three experiments, treatments were untreated control and immersion in aqueous formulation of 1MCP at 75 (M75) or 150 g L1 (M150) (1.39 or 2.77 mmol m3 a.i.) for 1 min at 20 C. E thylene and A queous 1 MCP P reparation and T reatment For Phase 1 treatments E12, E12+MCP and E24, ethylene (100 L L1 at 20 C ) was applied for the desired time 24 h after harvest in a flow t hrough system, in a 174 L

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99 metal chamber, where fruit were placed for treatment. Phase 2 experiments were treated only with 1MCP 24 h after harvest. A queous 1MCP was prepared from formulation AFxRD 300 (2% a.i., AgroFresh, Inc. Philadelphia, PA ) according to Choi and Huber (2008) For each desired concentration, adequate amounts of the powder were weighed and suspended in 30 L of distilled water in 50L plastic containers. The solution was swirled gently for 1 min and used within 10 and 45 minutes after preparation. Groups of eight to 12 fruit were placed in mesh bags and completely immersed in the respective solution for the desired time. Bags were gently agitated to ensure full contact of solution with fruit surface. After removal from the solution, the fruit were allowed to drain excess solution and immediately dried with a paper towel. F ruit ripening was monitored at 20 C / 92% 3% R.H. and based on whole fruit firmness, respiration and ethylene production. Control fruit (not exposed to 1MCP or ethyl ene) were maintained under identical storage conditions. Initial quality analysis was done in fruit 24 h after harvest. Fruit were assessed for volatile compounds in three ripeness stages, as described below. Ripening and Quality Analysis F ruit ripening w as monitored at 20 C / 92% 3% R.H. and based on whole fruit firmness, respiration and ethylene production. Control fruit (not exposed to 1MCP or ethylene) were maintained under identical storage conditions. Initial quality analysis was done in fruit 24 h after harvest. Fruit were assessed for volatile compounds in three ripeness stages, as described below.

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100 Whole fruit firmness Firmness was determined by a nondestructive compression test on whole, unpeeled fruit using an Instron Universal Testing Instru ment (Model 4411, Canton, MA, USA) fitted with a flat plate probe (5 0 mm diameter) and 50kg load cell. After establishing zero force contact between the probe and the equatorial region of the fruit, the probe was driven with a crosshead speed of 20 mm min1. The force was recorded at 2.5 mm deformation and was determined at two points on the equatorial region of each fruit, with a 90 angle between points. The same four fruit of each treatment were measured repeatedly every other day until they reached the fullripe stage. Fruit were considered commercially ripe upon reaching 10 to 15 N firmness. Respiration and ethylene production rates R espiration (as CO2 evolution) and ethylene production rates were measured daily, using the same six fruit for each treatment held at 20 C Fruit were individually sealed for 20 min in 2L plastic containers prior to sampling. Using a plastic syringe, a 1 mL gas sample was withdrawn from the container through a rubber septum and used for analysis For the first experiment (Simmonds, July 2008), CO2 was determined using a Gow Mac gas chromatograph (Series 580, Bridge Water, NJ, USA) equipped with a thermal conductivity detector (TCD) Ethylene was measured by injecting a 1.0 mL gas sample into a Tracor gas chromatograph (T remetrics, Austin, TX, USA) equipped with a flame ionization detector. For the other experiments, the concentration of gases was determined using a Varian gas chromatograph ( CP 3800 Middelburg, The Netherlands) equipped with Valco valves system (Houston, Texas, USA). CO2 was separated in a Hayesep Q

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101 column (Ultimetal, 1.0 m*1/8; 80100 mesh) and detected by a thermal conductivity detector (TCD) Ethylene was separated in a Molsieve column (Ultimetal, 1.5m*1/8; 13x80100 mesh) and a P ulse D ischarge H eliu m I onization D etector (PDHID) was used for detection. The conditions of the run were as follows: injector at 220 C; column oven at 50 C; TCD at 130 C and PDHID at 120 C; filament at 180 C. Ultra Pure Helium was used as carrier gas at 20 mL min1. The equipment was calibrated daily with a gaseous standard mix composed of 1.1 L L1 C2H4, 1.03% CO2, 19.98% O2 and N2 balance. Volatile compounds Fruit were assessed for volatile analysis at maturegreen, midripe (half of initial fruit firmness) and ripe ripeness stages. Mature green fruit were only assessed for untreated control fruit, as an initial reference. For each ripeness stage, six fruit were individually peeled, halved, deseeded and the pulp chopped into pieces of approximately 1 cm3 (Figure 51A to 5 1D) and kept in hermetically sealed plastic bags (ZipLoc type) until pr eparation for volatile collection. An interval of 60 to 90 min was maintained between chopping and the beginning of volatile collection for all samples in this study, except for the midripe stage of treatment MCP in Experiment 1 (Simmonds, July 2008), f or which collection began 3 h after chopping due to an unexpected equipment unavailability. For each fruit, a sample of 100 g of chopped pulp was enclosed in glass tubes (2.5 cm i.d. x 60 cm) for volatile s collection (Figure 5 1E) adapted from Schmelz et al. (2003) and Tieman et al. (2006) Pulp temperature at the beginning of volatile collection was 20 C to 23 C Air filtered from a hydrocarbon trap (Agilent, Palo Alto, CA) flowed through the tubes at 618 mL min1 with the aid of a

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102 vacuum pump for 1 h and volatiles collected on a 30 mg Super Q column (80/100 mesh, Alltech, Deerfield, IL). After volatile collection, nonyl acetate (400 ng in 5 L methylene chloride) was added to the column as an internal standard and volatiles were eluted with 150 L methyl ene chloride. Volatiles (2 L of total eluted sample) were separated and quantified on an Agilent 6890N gas chromatograph (DB 5 column) and final levels were calculated as ng gfw1 h1. Identification of peaks was done with retention times compared to know n standards (SigmaAldrich, St. Louis, MO) and/or GC MS. Statistical A nalysis The experiments were conducted using a completely randomized design with seven treatments and six replicates Statistical procedures were performed using the Statistical Analysis Software SAS version 9.1.3 (SAS Institute Inc., Cary, NC). Volatile data were log transformed (Log X+1) for mean separation. Differences between means were determined using Duncans M ultiple R ange T est. Results A full list of all compounds detected in the cultivars in this study, with their respective descriptors, is provided (Appendix A 1 ). Phase 1 Effects of E thylene and 1 MCP on V olatile C ompounds Whole fruit firmness In 2008, untreated control (C1) fruit had initial whole fruit firmness of 119 N, softened to mid ripe stage (half of initial whole fruit firmness) in 3 d and reached ripe fruit firmness (10 to 15 N) in 7 d (Figure 5 2A). Fruit exposed to ethylene for 24 h (E24) reached midripe stage in less than 3 d and were ripe 2 d earlier than control. Delay of ripening was observed for fruit treated with 1MCP only (1 MCP), with mid ripe stage reached in 4 d and ripe stage in 9 d after treatment.

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103 In 2009, untreated control (C2) fruit softened from initial whole fruit firmness of 124 N to the mid ripe stage (64 N) within only 2 d of storage (Figure 52B). Fruit continued softening quickly from 2 to 4 d and reached ripe fruit firmness within 6 d of storage. Softening of fruit exposed to ethylene for 12 h (E12) was very similar to control, reachi ng mid ripe and ripe stages at the same time as control fruit. Fruit treated with ethylene for 12 h followed by 12 air then treated with 1MCP (E12+MCP) initially softened faster during the first 2 d Afterward, fruit softened at slower rates, reaching mid ripe and ripe stages 3 and 8 d respectively, after treatment. Respiration Respiration of untreated control (C1) fruit in 2008 increased from 49 mg CO2 kg1 h1 initial rate to a peak rate of 186 mg CO2 kg1 h1 after 5 d of storage (Figure 5 3A). E24 had higher respiration rates than control since the first day of storage and reached a peak rate of 241 mg CO2 kg1 h1 on day 4. In contrast, respiration rates for 1 MCP fruit were lower than control since the first day of storage, started to increase on 3 d and reached a peak of 185 mg CO2 kg1 h1 7 d after treatment. For 2009, control fruit (C2) initial respiration rates were slightly higher than those of the first experiment, with 64 mg CO2 kg1 h1 (Figure 5 3B). Rates increased daily until reaching a peak of 214 mg CO2 kg1 h1 within 4 d of storage. E12 fruit had similar peak rate (218 mg CO2 kg1 h1), reached 1 d before control fruit. First increments in respiration rate of E12+MCP fruit were observed with 5 d of storage, reaching a peak of 166 mg CO2 kg1 h1 2 d after. Ethylene production Climacteric ethylene production was first noticed within 3 d of storage in C1 fruit (2008) and increased rapidly from 40 L kg1 h1 to a peak rate of 233 L C2H4 kg1 h1

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104 on the next day (Figure 54A). The onset of ethylene climacteric in E24 fruit was anticipated. Increased rates were observed since the first day of storage until reaching a peak of 186 L C2H4 kg1 h1 in 3 d. 1 MCP delayed the ethylene climacteric peak by 2 d when compared with control fruit, with a rate of 180 L C2H4 kg1 h1 6 d after treatment. In 2009, ethylene production in control fruit had a sharp increase from 30 L C2H4 kg1 h1 on day 2 to a peak of 241 L C2H4 kg1 h1 on day 4 of storage (Figure 54B). E12 fruit had accentuated increase from 17 to 185 L C2H4 kg1 h1 between 2 and 3 d of storage and reached a peak of 208 L C2H4 kg1 h1 in day 4. Treatment E12+MCP delayed the increase of ethylene rates by 2 d as compared with control, with largest increase between days 5 (70 L C2H4 kg1 h1) and 6 (214 L C2H4 kg1 h1), when the peak was observed. Volatile compounds A total of 27 volatile compounds were identified in Simmonds avocado and the profile of compounds changed significantly during ripening in both 2008 (Table 51) and 2009 (Table 52) experiments Volatiles of mature green fruit were mostly sesquiterpenes (SQT) (72.8% 74% of total volatiles TOV), a group of 15 compounds. Total aldehydes (ALD), mostly represented by hexanal, was the second most abundant group of compounds (15% 16.9%). The largest emissions were observed for copaene (16% 23.5%), caryophyllene (20.1% 21.7%), hexanal (13.7% 15.2%) and cubebene (9.9% 11.8%). In the midripe stage, the volatile profile remained similar to maturegreen stage, with abundance of SQT and ALD. In 2008, lower emissions of alcohols (ALC) (mainly due to 1hexanol), esters (EST) (due to hexyl acetate) and monoterpenes (MOT) ( myrcene and Cis ocimene) for midripe fruit. In 2009, only 1-

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105 hexanol and hexyl acetate had significant lower emissions when compared to maturegreen fruit. Th e most significant change in volatile emissions was observed from midripe to ripe fruit, when a significant reduction of 95% and 83.5% in TOV was observed respectively in 2008 and 2009. Only 9 compounds were detected, with Cis 3 hexenal and Cis 3 hexen1 ol being the most abundant, contributing to ALD and ALC as the main groups of volatiles emitted by the ripe fruit. EST and MOT were not detected and alkanes (dodecane) and sesquiterpenes ( caryophyllene and cubebene) were minimally present (1.5% or less). The effects of ethylene and 1MCP were apparent for the midripe stage. In 2008, E24 midripe fruit had similar volatile profile as compared with midripe control fruit (Table 51). ES T was the only group of volatiles with reduced emissions in E24 fruit and TOV was not significantly different from each other and from maturegreen fruit. Fruit from 1 MCP treatment had reduced TOV in midripe fruit when compared to maturegreen fruit, but was statistically similar to control and E24 midripe fruit. The emissions of ALD, EST, MOT and SQT in 1 MCP mid ripe fruit were lower than those of untreated control, most likely due to the delay in the analysis. The rate of emission of some volatile com pounds was significantly reduced in midripe E24 fruit (Table 51): 1 hexanol, myrcene, hexyl acetate, hexyl butyrate, copaene, bergamotene and muurolene. In 2009, TOV emission by midripe fruit was not affected by ethylene or 1MCP treatments (Table 5 2). However, E12 fruit had greater emissions of ALC and EST than C2 fruit in the midripe stage, but not significantly different than maturegreen stage.

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106 E12+MCP did not affect groups of volatiles in midripe fruit when compared with control, but reduced emissions were observed for ALC and MOT when compared to maturegreen fruit. E12 fruit had significantly increased emissions of Cis 3 hexen1 ol, 1 hexanol and hexyl acetate when compared to midripe C2 and E12+MCP fruit. For E12+MCP were observed reduced emission of Cis ocimene and increased rate of cubebene. No other sesquiterpenes were affected by E12 or E12+MCP in midripe stage. In 2008, KET, represented by 3pentanone, was the only group with rate increased by E24 in the ripe stage (Table 51 ). Higher emission of Cis 3 hexenal was observed in ripe 1MCP fruit. No other significant differences were observed. In 2009 there were no significant differences among treatments in ripe fruit for emission rates of total volatiles, group totals and indiv idual compounds (Table 52). Phase 2 Aqueous 1 MCP E ffects on V olatile C ompounds in T hree C ultivars Whole fruit firmness Untreated Simmonds cultivar softened fairly quickly, within 6 d of storage (Figure 5 5A), as was observed in Phase 1 experiments. Application of 1MCP at 75 (M75) or 150 g L1 (M150) for 1 min delayed ripening by 2 or 4 d respectively. The initial firmness of Booth 7 (190 N) was higher than in Simmonds, and softening to ripefruit firmness (10 to 15 N) occurred within 10 d of s torage (Figure 5 5B). Softening of M75 and M150 fruit was similar to control until 4 d of storage, diverging thereafter. However, firmness of treated fruit was similar until 8 d of storage, when the effect of concentration started to appear. Ripening of M75 and M150 fruit was delayed respectively by 4 and 8 d Monroe had the highest initial firmness (268 N) and the longest ripening time (12 d)

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107 of the three cultivars (Figure 55C). M75 delayed ripening in 6 d, while M150 in 4 d. Differences in firmness bet ween fruit treated with M75 and M150 started to appear within 8 d of storage, when M150 fruit stayed firmer than M75 until they reached the ripe stage. Respiration Respiration rates of Simmonds increased from the first day until 5 d of storage, when a pe ak rate of 203 mg CO2 kg1 h1 was observed (Figure 56A). Treatments M75 and M150 delayed the onset of climacteric respiration and peak rates were observed respectively 2 and 4 d after control. For untreated Booth 7 control fruit, respiration rates incr eased continuously, peaking at 202 mg CO2 kg1 h1 within 9 d of storage (Figure 56C). Respiration of M75 and M150 remained at low levels for 7 and 10 d respectively, and peak rates were observed in day 13 (176 mg CO2 kg1 h1) for M75 and day 17 (173 mg CO2 kg1 h1) for M150 fruit. Control fruit Monroe had increased respiration rate until 7 d of storage, peaking at 150 mg CO2 kg1 h1 (Figure 5 6E). Not only was the peak lower than the other two cultivars, but it was also earlier, 5 d before reaching the ripe stage, contrasting with only 1 d observed for Simmonds and Booth 7. Climacteric respiration of Monroe was delayed by 1MCP treatments. M75 reached peak rate (167 mg CO2 kg1 h1) at 12 d after treatment, oscillating thereafter. M150 peak rat e was observed at 19 d after treatment, and did not decrease until reaching the ripe stage. Ethylene Ethylene rates in Simmonds increased from 2 L C2H4 kg1 h1 at day 2 to a peak rate of 181 L C2H4 kg1 h1 at day 4 of storage (Figure 56B). Treatments M75 and M150 delayed the onset of climacteric respiration and ethylene production, with stronger

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108 reduction of ethylene rates than respiration. B ooth 7 control fruit had sharply increased ethylene rates from 21 L C2H4 kg1 h1 in 7 d of storage to a peak rate of 128 L C2H4 kg1 h1 in the next day (Figure 56D). M75 fruit had slightly increased ethylene rates from 6 to 10 d after treatment, but rates increased rapidly in day 11, peaking at 119 L C2H4 kg1 h1 in the next day. The most accentuated increase in ethylene production of M150 fruit occurred from 12 to 14 d after treatment, when a peak of 104 L C2H4 kg1 h1 was observed. For Monroe, ethylene increased sharply in 5 d of storage in control fruit, peaking at 93 L C2H4 kg1 h1, the lowest of all cultivars studied, in day 7 (Figure 56F). M75 and M150 delayed the onset of climacteric and peak rates were observed at 11 d (110 L C2H4 kg1 h1) and 17 d (100 L C2H4 kg1 h1) after treatment. Despite the delay, peak rates were not reduced when compared with control, as observed for the other two cultivars. Volatile compounds Simmonds As observed in previous experiments (Phase 1), SQT was the most abundant group of volatiles (53%) in maturegreen Simmonds fruit, followed by ALD (23%) (Table 53). Hexanal, copaene and caryophyllene had the highest rates in both maturegreen and midripe untreated fruit. However, TOV of untreated frui t decreased significantly in midripe fruit, affected by the significant reduction in these two main groups of volatiles. Ripe fruit had ALD (49.6%) and ALC (35.3%) as the main groups of volatiles, mainly due to Cis 3 hexenal and Cis 3 hexen1 ol, respecti vely. Application of 1MCP significantly affected volatile profile in mid ripe fruit. No significant differences were found for TOV and for all groups of compounds between control and M75. Except for total alkanes (ALK) and KET, emissions of all other groups

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109 of volatiles in control and M75 midripe fruit were significantly lower than maturegreen fruit. However, M150 maintained higher rates of emission for these groups and TOV in mid ripe fruit, although there was a reduction in MOT and SQT when compared to maturegreen fruit. Individually, the emission of several compounds in the midripe fruit was higher with increased concentration of 1MCP. In ripe fruit, TOV were not significantly affected by 1MCP treatments. M75 fruit had higher emissions of ALK (dodecane). M150 had reduced ketones (3pentanone) when compared to M75. Both 1MCP treatments had higher emissions of 3methyl 3 buten1 ol. Booth 7 The volatile profile of Booth 7 (Table 54) fruit was different than that observed for Simmonds (Table 53), with 34 compounds detected during ripening. SQT constituted the most abundant group of volatiles (78.5%) in maturegreen fruit, followed by ALK (11.6%), with ALD accounting for only 3.1%. Five sesquiterpenes detected in Booth 7 were not present in Simmonds and four sesquiterpenes of Simmonds were not in Booth 7. The sesquiterpene caryophyllene was by far the most abundant compound (50.8%) in this ripeness stage, followed by Cis farnesene (4.9%) and myrcene (4.8%). The alkanes undecane and tridecane, not detected in Simmonds, as well as dodecane and the sesquiterpenes cubebene and bisabolene were also emitted in higher rates than the other compounds. Hexanal, a great contributor (38.4 ng gfw1 h1) to total volatile emissions by matur e green Simmonds, was found in much lower rate (5.3 ng gfw1 h1) in mature green Booth 7. In mid ripe fruit, the emissions of ALD, MOT and SQT were significantly reduced when compared with maturegreen fruit, mainly due to reductions in hexanal, my rcene and caryophyllene. ALK emissions were still high. The profile of control ripe fruit was

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110 predominantly of ALK (75%), followed by ALC (10.9%). Two alcohols and one aldehyde not found in Simmonds were present only in ripe Booth 7 fruit: 3methyl 1 butanol, 2methyl 1 butanol and 2 methyl 2 butena l. Additionally, more esters compounds were found in ripe Booth 7 than in Simmonds. None of the monoterpenes and most sesquiterpenes were not detected in ripe fruit. M75 and M150 significantly affected volatile profile in mid ripe fruit, with higher emissions of TOV than control fruit. However, this difference was due to the emissions of ALK that were significantly higher in both M75 and M150 fruit. These treatments had higher emissions of all alkanes ( decane, undecane, dodecane and tridecane) and hexanal. Rates of some sesquiterpenes ( cadinene, Cis bisabolene) were reduced by 1 MCP treatments while other had increased emissions with M150 ( copaene, caryophyllene). In the ripe fruit, there was dif ference in TOV of treated fruit as compared with control, mainly due to higher ALK. Monroe The volatile profile of Monroe avocado had 25 compounds detected during ripening (Table 55). Only 13 compounds were in common with the other two cultivars, 7 of the m in common only with Booth 7 (Table 54). Mature green fruit had mostly SQT (66%) and ALK (32.3%). caryophyllene was the most abundant compound (29.6%), followed by copaene (19.5%) and the alkanes undecane, dodecane and tridecane. Hexanal was the only aldehyde detected and in lower rate than Booth 7. Although hexanal and some sesquiterpenes had si gnificantly lower emissions in mid ripe fruit when compared to maturegreen stage, TOV was not affected. In the ripe fruit, TOV was only 21.1% of the rate observed for maturegreen

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111 fruit. ALK was the predominant group (80.8%), followed by EST and ALC. No K ET or MOT were detected. M75 and M150 midripe fruit had significantly higher rates of hexanal and ALK. In this ripeness stage, TOV and four sesquiterpenes in M150 fruit were higher than control rates. In the ripe fruit, although ALK were similar to control, they contributed to lower TOV, as well as EST. Discussion TOV of avocado decreased during ripening in all experiments, mainly during the mid ripe to the ripe stage, presenting an opposite pattern when compared to other fruits such as mango (Singh et al., 2004), apple (Defilippi et al., 2004) and tomato (Tieman et al., 2006; Mathieu et al., 2009). According to volatile aroma descriptors (Appendix A 1), the aroma of avocado is mostly woody and green in maturegreen and midripe stages, while in ripe frui t green, sweet and fruity notes are present. The main reason for the drop in total volatile emissions by avocado during ripening was SQT, the most abundant group of volatiles in maturegreen and midripe fruit. These compounds are mostly described as woody. While they accounted for more than 50% of the TOV of these ripeness stages, they dropped to less than 2% in ripe fruit, for which several of these compounds were not detected. In Kensington Pride mango, Lalel et al. (2003) observed sesquiterpenes as t he major group of compounds in early ripeness stage, but they significantly decreased in ripe fruit, while esters increased. In the present study, when the increase of ALC, EST and KET in ripe fruit was observed, it was of much lower intensity than the dec rease in SQT. Tests made by Platt and Thompson (1992) indicated the presence of sesquiterpene hydroperoxides and possibly other terpenes in idioblasts of mature,

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112 unripe avocado. The idioblasts are specialized cells for oil accumulation in avocado (Platt A loia et al., 1983) and may participate in plant defense (Rodriguez Saona and Trumble, 2000) as do the terpenes (Dudareva et al., 2006). Since the production and emission of volatiles is developmentally regulated in plants (Duduareva et al., 2004), the lack of sesquiterpenes in ripe avocado fruit suggests that these compounds are indeed related to plant defense. This regulation in avocado may involve terpene synthases, the main enzymes involved in terpene synthesis (Tholl, 2006). In mango, the expression of mono and sesquiterpene synthases lowered during ripening of Alphonso (Pandit et al., 2010), while for Kensignton Pride a significant reduction in the emission of sesquiterpenes during ripening was reported (Lalel et al., 2003). In Chardonnay grape ( Lucker et al., 2004) and Valencia orange (SharonAsa et al., 2003), specific terpene synthases were developmentally regulated, and in orange it was responsive to ethylene. Significant negative correlations ( 0.60, weakest) between ethylene and SQT were observed for all experiments (data not shown), indicating an influence of ethylene in SQT emission. Treatment E24 in Simmonds reduced emissions of the sesquiterpenes copaene, bergamotene and muurolene in midripe fruit (Table 51) but they were not affected by E12 in 2009 (Table 52) Treatment E12+MCP did not affect sesquiterpene emissions in 2009 (Table 52) except for copaene, when rate was higher than control. In Phase 2, increased concentration of 1MCP maintained higher emissions of several sesquiterpenes in midripe Simmonds (Table 53) and a few in Booth 7 (Table 54) and Monroe (Table 55) indicating participation of ethylene regulating their biosynthesis. However, the only compound commonly affected was -

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113 copaene. Altogether, thes e results indicate that the reduction in copaene emission is regulated by ethylene. In 2008, ethylene production in E24 fruit at midripe stage was higher (147 L C2H4 kg1 h1) than control (68 L C2H4 kg1 h1), while EST emissions were significantly lower than control in E24. In 2009, EST in midripe fruit were significantly higher in E12, while ethylene production was lower than control. Therefore, the increase in ethylene production may have hastened the reduction in EST in midripe fruit. It was ex pected that 1 MCP treated fruit would have higher EST emissions due to lower ethylene production, but that did not occur in Phase 1 experiments most likely due to the delay in the analysis. However, higher 1MCP treatment in Simmonds in Phase 2 maintained higher emissions of EST in the midripe fruit. The most affected ester in this cultivar was hexyl acetate. This compound is derived from hexanol and it is regulated by ethylene in melons (Flores et al., 2002) and apples (Schaffer et al., 2007). Altogether, the results of the present study suggest the involvement ethylene in the regulation of esters in Simmonds avocado. The volatile profile of avocado differed among Simmonds (West Indian), Booth 7 (Guatemalan West Indian hybrid) and Monroe (Guatemalan West Indian hybrid). Two major factors distinguished the cultivars. First, Simmonds had high emissions of volatiles derived from the action of the enzyme lipoxygenase (Chen et al., 2004) on the fatty acids linolenic acid (hexanal) and linoleic ac id ( Cis 3 hexenal and Cis 3 hexen1 ol). Booth 7 and Monroe had very reduced emissions when compared to Simmonds. Since these compounds have low odor threshold (Appendix A), it is likely that they contribute with green notes to the avocado aroma, especially for Simmonds.

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114 It is worth mentioning that hexanal emissions in Simmonds were higher in maturegreen and midripe stages and significantly reduced in ripe fruit, while the other two compounds followed opposite trends. Although differences in fat ty acid composition may exist (Ozdemir and Topuz, 2004), this observation is consistent with the differential activity of lipoxygenases in avocado observed by Nel et al. (1984), that vacuolebound lipoxygenase decreased in activity during ripening, while m embranebound showed a steady increase. It is possible that the vacuolebound lipoxygenase has linolenic acid as substrate, while the membranebound would target linoleic acid. Kiwi (Zhang et al., 2009a ) and tomato (Chen et al., 2004) have different types of lipoxygenases acting in a different manner during ripening. A second distinguishing difference among the cultivars was the lower presence of alkanes in Simmonds than in Booth 7 or Monroe. While the West Indian cultivar had only one alkane detected (dodecane), the GuatemalanWest Indian hybrids had four compounds (decane, undecane, dodecane and tridecane). Alkanes were also reported as volatile compounds of guava (Idstein and Schreier, 1985a), mango (Idstein and Schreier, 1985b; Pandit et al., 2009) and peach (Sumitani et al., 1994). Long chain alkanes are often associated with elongationdecarboxylation of fatty acids (Kates and Wassef, 1970) and differences in alkane composition in leaves were reported among Persea species and cultivars (Scora et al., 1975). The differences among cultivars suggest that different mechanisms of volatile synthesis occur. Midripe M75 and M150 fruit had the highest emissions of alkanes during ripening of Booth 7 and Monroe. The ethylene inhibition promoted by 1MCP may have favored this pathway in these two cultivars.

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115 Another aspect was regarding composition of esters. While hexyl esters, detected in Simmonds and Booth 7, and methyl isovalerate decreased during ripening, methyl butyrate, detected in Monroe, and ethyl esters, detected in Booth 7 increased during ripening, being present mainly in ripe fruit. This differential emission observed for ester compounds influenced EST in ripeness stages. In Simmonds and Booth 7, EST decreased during ripening, while in Monroe, EST increased. Similarly, esters increased or decreased during ripening (Argenta et al., 2003) or storage (MoyaLeon et al., 2006) of pears. Depending on the precursor, different esters will be formed in ripening fruit (Zhu et al., 2005). Reg ulation of ester production may involve ethylenedependent (Golding et al ., 1999; Defilippi et al., 2005b) or ethyleneindependent steps (Flores et al., 2002). Several compounds were detected in only one cultivar. In Simmonds: 3methyl 3 buten1 ol, Cis 3 hexen1 ol, Cis ocimene, hexyl butyrate, elemene and Trans nerolidol. In Booth 7: 2 methyl 1 butanol, 2methyl 2 butena l, ethyl crotonate, ethyl isovalerate, ethyl tiglate, selinene and Cis bisabolene. In Monroe: methyl butyrate, Ylangene and Valencene. Additi onally, the alkanes decane, undecane and tridecane, and the sesquiterpenes alloaromadendrene, elemene and germacrene B were detected only in the GuatemalanWest Indian hybrids. The identification of peculiarities in fruit volatile composition may be us eful for classification purposes, as reported for avocado leaves. Terpenes are the main group of volatiles in fresh leaves and essential oil and are suggested to be used as means of cultivar race or Persea species identification (Bergh et al., 1973; King and Knight, 1987; King and Knight, 1992; Wu et al., 2007; Joshi et al., 2009). Leaves of Mexicantype

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116 avocados are rich in estragol, a compound that gives the anise scent to the leaves of this race. This volatile in West Indian or Guatemalan cultivars and h ybrids is negligible (King and Knight, 1987; King and Knight, 1992; Pino et al., 2006; Wu et al., 2007). Therefore, the results of the present study contribute to a potential identification of the origin of the cultivar based on fruit volatile composition. Conclusions Total volatile emission s in West Indian and GuatemalanWest Indian hybrid avocados decreased during ripening, and volatile profile differed among cultivars and ripeness stages. Sesquiterpenes were the main group of volatile compounds emitted by these avocado cultivars, followed by aldehydes in Simmonds or alkanes in Booth 7 and Monroe. caryophyllene was the most abundant compound in all cultivars. Total mono and sesquiterpenes, total aldehydes and total alkanes decreased during ripening, while total ketones increased. The composition of alcohols and esters of the fruit determined an increase or decrease of total alcohols and total esters. The West Indian Simmonds was characterized by a much higher rate of hexanal in maturegreen and m id ripe fruit than the GuatemalanWest India n hybrids Booth 7 and Monroe, while these cultivars had more alkanes and in higher emission than Simmonds. Some compounds, including sesquiterpenes, were exclusive to one cultivar and t he results of this st udy may also contribute to identify the origin of the cultivar s based on their volatile composition. The results suggest that ethylene is involved in the regulation of esters in Simmonds avocado, which are reduced during ripening. 1MCP increased signifi cantly the emission of alkanes in Booth 7 and Monroe, indicating that it may have favored

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117 this pathway in these two cultivars. Reduction of the sesquiterpene copaene was stimulated by ethylene or inhibited by 1MCP, suggesting ethylenedependent regulation of this compound. Total volatile emissions was not affected by ethylene or 1MCP in ripe fruit in Simmonds, but was higher in 1MCP treated ripe Booth 7 and Monroe higher due to higher emission of alkanes.

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118 Table 51 Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds avocado (n=6) (Phase 1, Experiment 1): untreated control (C1), ethylene treatment for 24 h (E2 4) and aqueous 1MCP (1 MCP) at 150 g L1 for 1 min at 20 C and stored at 20 C. Volatile compoundz MG MR R C1 E24 1 MCP C1 E24 1 MCP ALCOHOLS 1 hexanol 4.92A 2.02B 0.84C 0.32CD 0.13D 0.05D 0.00D 3 Methyl 3 buten 1 ol 0.00B 0.00B 0.00B 0.00B 0.58A 0.60A 0.74A Cis 3 hexen 1 o l 0.85AB 0.87AB 0.57BC 0.03C 2.00A 1.55A 1.10AB ALDEHYDES Cis 3 hexenal 2.42B 1.62B 2.09B 0.30C 2.61B 2.14B 4.64A Hexanal 22.25A 12.58AB 11.92BC 4.91C 1.07D 0.79D 0.91D ALKANES Dodecane 0.40A 0.38A 0.31A 0.32A 0.02B 0.0 1B 0.01B ESTERS Farnesyl acetate 0.43A 0.48A 0.48A 0.22B 0.00C 0.00C 0.00C Hexyl acetate 2.79A 1.04B 0.33C 0.06C 0.00C 0.00C 0.00C Hexyl butyrate 0.85B 1.41A 1.02B 0.46C 0.00D 0.00D 0.00D KETONES 3 pentanone 0.22C 0.09 C 0.14C 0.10C 0.75B 1.15A 0.74B MONOTERPENES myrcene 1.42A 0.71B 0.41C 0.64B 0.00D 0.00D 0.00D L imonene 0.38B 0.96A 0.79A 0.31B 0.00C 0.00C 0.00C Cis ocimene 1.02A 0.56B 0.50B 0.57B 0.00C 0.00C 0.00C SESQ UITERPENES bergamotene 6.94A 6.03A 4.73B 4.42B 0.00C 0.00C 0.00C bisabolene 0.98A 1.01A 1.04A 0.67B 0.00C 0.00C 0.00C cadinene 2.02A 2.17A 2.26A 1.42B 0.00C 0.00C 0.00C caryophyllene 2.14A 2.50A 2.12A 1.47B 0.00C 0.00C 0.00C

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119 Table 51. Continued. Volati le compound MG MR R C1 E24 1 MCP C1 E24 1 MCP caryophyllene 31.76AB 32.06A 28.30AB 22.49B 0.06C 0.09C 0.06C copaene 34.33A 28.51A 20.14B 20.53B 0.00C 0.01C 0.00C cubebene 0.66B 0.95A 0.99A 0.70B 0.00C 0.00C 0.00C cubebene 14.48B 20.77A 21.24 A 11.66B 0.04C 0.08C 0.04C elemene 0.20B 0.28A 0.29A 0.18B 0.00C 0.00C 0.00C farnesene 4.59A 3.92A 4.08A 2.57B 0.00C 0.00C 0.00C Cis farnesene 1.24AB 1.07AB 1.69A 0.90B 0.00C 0.00C 0.00C muurolene 0.41A 0.35A 0.28B 0.23B 0.00C 0.00C 0.00C tran s nerolidol 3.37A 3.24A 3.84A 1.77B 0.00C 0.00C 0.00C sesquiphellandrene 5.10A 4.95A 4.48A 3.44B 0.00C 0.00C 0.00C TOTALS Total a lcohols (ALC) 5.77A 2.89B 1.41B 0.35B 2.71B 2.20BC 1.84BC Total a ldehydes (ALD) 24.67A 14.20AB 14.01BC 5. 21CD 3.68D 2.93D 5.56CD Total a lkanes (ALK) 0.40A 0.38A 0.31A 0.32A 0.02B 0.01B 0.01B Total esters (EST) 4.08A 2.92B 1.83C 0.74D 0.00E 0.00E 0.00E Total ketones (KET) 0.22C 0.09C 0.14C 0.10C 0.75B 1.15A 0.74B Total m onoterpenes (MOT) 2.81A 2.23B 1.70BC 1.52C 0.00D 0.00D 0.00D Total s esquiterpenes (SQT) 108.21A 107.83A 95.47A 72.46B 0.11C 0.18C 0.09C TOTAL volatiles (TOV) 146.16A 130.54AB 114.87AB 80.70B 7.26C 6.47C 8.24C zMeans followed by the same letter in the same row do not differ significantly according to Duncans Multiple Range Test (P< 0.05) (n=6)

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120 Table 52. Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds avocado (n=6) (Phase 1, Experiment 2): untreated control (C2), ethylene treatment for 12 h (E1 2) and ethylene treatment for 12 h + 12 h air + aqueous 1MCP (E12+MCP) at 150 g L1 for 1 min at 20 C and stored at 20 C. Volatile compoundz MG MR R C 2 E 12 E12+ MCP C 2 E 12 E12+ MCP ALCOHOLS 1 hexanol 6.58A 4.21B 10.92A 2.21B 0.80B 1.05B 0.55B 3 Methyl 3 buten 1 ol 0.03B 0.04B 0.11B 0.03B 1.15A 1.18A 1.12A Cis 3 hexen 1 ol 0.95BC 0.73C 2.93B 0.39C 11.59A 17.35A 12.84A ALDEHYDES Cis 3 hexenal 2.05CD 1.38D 3.94BC 2.07CD 5.50AB 8.38A 8.60A Hexanal 21.17A 16.02A 22.06A 18.51A 2.00B 2.53B 2.00B ALKANES Dodecane 1.39A 1.35A 1.30A 1.41A 0. 24B 0.22B 0.24B ESTERS Hexyl acetate 2.64A 1.59B 4.17A 0.85B 0.00B 0.00B 0.00B Hexyl butyrate 1.69A 1.48A 3.08A 1.51A 0.00B 0.00B 0.00B Farnesyl acetate 0.35A 0.33A 0.38A 0.34A 0.00B 0.00B 0.00B KETONES 3 pentanone 0.0 2B 0.09AB 0.15A 0.08AB 0.05B 0.07AB 0.06B MONOTERPEN E S myrcene 2.06A 1.82AB 1.85A 1.15B 0.00C 0.00C 0.00C L imonene 1.54A 0.59AB 0.51AB 0.39BC 0.00C 0.00C 0.00C Cis ocimene 1.60AB 1.76A 1.41AB 1.25B 0.00C 0.00C 0.00C SESQUI TERPENES bergamotene 8.46A 8.37A 8.54A 7.15A 0.00B 0.00B 0.00B bisabolene 2.35A 2.09A 1.84A 1.71A 0.00B 0.00B 0.00B cadinene 3.35A 2.95A 3.13A 2.91A 0.00B 0.00B 0.00B caryophyllene 2.83A 2.30A 2.19A 2.14A 0.00B 0.00B 0.00B

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121 Table 52 C ontinued. Volatile compoundz MG MR R C 2 E 12 E12+ MCP C 2 E 12 E12+ MCP caryophyllene 31.08A 25.13A 24.70A 22.70A 0.01B 0.02B 0.01B copaene 24.67A 21.32A 25.26A 19.83A 0.00B 0.00B 0.00B cubebene 0.93AB 0.83B 1.06AB 1.20A 0.00C 0.00C 0.00C cubeben e 18.17A 15.92A 15.68A 16.46A 0.00B 0.01B 0.00B elemene 0.51A 0.52A 0.49A 0.54A 0.00B 0.00B 0.00B farnesene 7.76A 6.96A 7.46A 6.62A 0.00B 0.00B 0.00B Cis farnesene 2.14A 1.61A 1.54A 1.34A 0.00B 0.00B 0.00B muurolene 0.50A 0.35A 0.33A 0.27A 0.00 B 0.00B 0.00B trans nerolidol 3.77A 3.62A 3.77A 3.55A 0.00B 0.00B 0.00B sesquiphellandrene 5.92A 5.41A 6.10A 5.35A 0.00B 0.00B 0.00B TOTALS Total a lcohols (ALC) 7.55B 4.98C 13.96AB 2.63C 13.53AB 19.58A 14.52AB Total a ldehydes (ALD) 2 3.22A 17.39ABC 26.00A 20.58AB 7.50C 10.91BC 10.69BC Total a lkanes (ALK) 1.39A 1.35A 1.30A 1.41A 0.24B 0.22B 0.24B Total esters (EST) 4.68AB 3.40B 7.63A 2.70B 0.00C 0.00C 0.00C Total ketones (KET) 0.02B 0.09AB 0.15A 0.08AB 0.05B 0.07AB 0.06B Total m onot erpenes (MOT) 5.19A 4.17AB 3.78AB 2.79B 0.00C 0.00C 0.01C Total s esquiterpenes (SQT) 112.42A 97.41A 102.09A 91.77A 0.01B 0.02B 0.01B TOTAL volatiles (TOV) 154.48A 128.79A 154.90A 121.96A 21.34B 30.81B 25.43B zMeans followed by the same capital letter in the same row do not differ significantly according to Duncans Multiple Range Test (P< 0.05) (n=6)

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122 Table 53. Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds avocado (n=6) (Phase 2): untreated control (M0) or treated with aqueous 1MCP at 75 (M75) or 150 g L1 (M150) for 1 min at 20 C and stored at 20 C. Volatile compoundz MG MR R M0 M75 M150 M0 M75 M150 ALCOHOLS 1 hexanol 16.91A 1.56B 3.45B 13.90A 0.61B 0.75B 0.59B 3 Methyl 3 buten 1 ol 0.04C 0.01C 0.01C 0.00C 0.40B 0.79A 0.67A Cis 3 hex en 1 ol 2.37CD 0.39E 0.96DE 5.24BC 8.85AB 11.55A 11.38A ALDEHYDES Cis 3 hexenal 3.79BC 1.57D 2.27CD 4.64BC 9.83A 10.50A 7.18AB Hexanal 38.38A 10.46B 16.95B 36.99A 4.05BC 3.89BC 2.26C ALKANES Dodecane 8.26B 11.40AB 12.94 A 8.55AB 1.08D 2.33B 0.92D ESTERS Farnesyl acetate 0.33A 0.16B 0.20B 0.24B 0.00C 0.00C 0.00C Hexyl acetate 8.38A 0.65BC 1.47B 7.20A 0.00C 0.00C 0.00C Hexyl butyrate 2.45A 0.64CD 1.26BC 2.02AB 0.00D 0.00D 0.00D Methyl isovalerate 0.14A 0.12A 0.16A 0.12A 0.17A 0.14A 0.11A KETONES 3 pentanone 0.44C 0.17C 0.19C 0.44C 2.87AB 3.29A 2.11B MONOTERPENES myrcene 2.03A 0.50C 0.76BC 0.92B 0.00D 0.00D 0.00D L imonene 0.66A 0.17B 0.27B 0.27B 0.00C 0.00C 0.00C Ci s ocimene 1.99A 0.62C 1.06BC 1.03B 0.00D 0.00D 0.00D SESQUITERPENES bergamotene 6.59A 2.74C 3.86BC 4.74B 0.00D 0.00D 0.00D bisabolene 1.56A 0.62C 0.86BC 0.90B 0.00D 0.00D 0.00D

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123 Table 53 Continued. Volatile compoundz MG MR R M0 M75 M150 M0 M75 M150 cadinene 2.11A 1.02C 1.34BC 1.41B 0.00D 0.00D 0.00D caryophyllene 2.26A 1.28B 1.55B 1.58B 0.00C 0.00C 0.00C caryophyllene 24.38A 12.73C 15.32BC 16.99B 0.08D 0.18D 0.29D copaene 31.12A 9.86C 12.74C 18.24B 0.00D 0.00D 0.00 D cubebene 0.86A 0.57B 0.84A 0.75AB 0.00C 0.00C 0.00C cubebene 13.34A 7.91B 9.52B 10.08B 0.02C 0.06C 0.02C elemene 0.33A 0.21B 0.23B 0.27AB 0.00C 0.00C 0.00C farnesene 5.50A 2.42C 3.27BC 3.70B 0.00D 0.00D 0.00D Cis farnesene 1.16A 0.48C 0.60 BC 0.75B 0.00D 0.00D 0.00D muurolene 0.34A 0.19B 0.25AB 0.35AB 0.00C 0.00C 0.00C trans nerolidol 3.18A 1.42C 1.74BC 2.07B 0.00D 0.00D 0.00D sesquiphellandrene 4.42A 2.02C 2.76BC 3.11B 0.00D 0.0D0 0.00D TOTALS Total a lcohols (ALC) 1 9.32A 1.95B 4.41B 19.15A 9.86A 13.09A 12.65A Total a ldehydes (ALD) 42.17A 12.04B 19.21B 41.63A 13.87B 14.39B 9.45B Total a lkanes (ALK) 8.26B 11.40AB 12.94A 8.55AB 1.08D 2.33C 0.92D Total esters (EST) 11.30A 1.57BC 3.09B 9.57A 0.17C 0.14C 0.11C Total ke tones (KET) 0.44C 0.17C 0.19C 0.44C 2.87AB 3.29A 2.11B Total m onoterpenes (MOT) 4.68A 1.29C 2.10BC 2.22B 0.00D 0.00D 0.00D Total s esquiterpenes (SQT) 97.13A 43.47C 54.89BC 64.94B 0.11D 0.25D 0.04D TOTAL volatiles (TOV) 183.31A 71.89C 96.82BC 146.50AB 27 .96D 33.49D 25.28D zMeans followed by the same capital letter in the same row do not differ significantly according to Duncans Multiple Range Test (P< 0.05) (n=6)

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124 Table 54. Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Booth 7 avocado (n=6) (Experiment 4): untreated control (M0) or treated with aqueous 1MCP at 75 (M75) or 150 g L1 (M150) for 1 min at 20 C and stored at 20 C. Volatile compoundz MG MR R M0 M75 M150 M0 M75 M150 ALCOHOLS 1 hexanol 0.33A 0.00B 0.11B 0.00B 0.02B 0.03B 0.06B 2 Methyl 1 but anol 0.00C 0.00C 0.00C 0.00C 0.15A 0.09AB 0.05BC 3 Methyl 1 b ut anol 0.00B 0.00B 0.00B 0.00B 0.47A 0.41A 0.34A ALDEHYDES 2 Methyl 2 buten a l 0.00B 0.00B 0.00B 0.00B 0.11A 0.03B 0.04B Cis 3 hexenal 0.47A 0.12C 0.16C 0.19C 0.08C 0.21BC 0.39AB Hexanal 5.28A 1.20BC 1.89B 1.96B 0.30D 0.39D 0.46CD ALKANES Decane 0.89B 0.73B 5.07A 2.92A 0.23B 0.44B 0.57B Dodecane 8.34B 9.71B 32.68A 29.70A 2.11D 3.71C 4.27C Tridecane 6.44B 8.24B 25.20A 23.83A 1.54D 2.72C 2.77C Undecane 6.08B 4.50BC 20.97A 16.43A 1.24E 2.16DE 2.72CD ESTERS Ethyl crotonate 0.00B 0.00B 0.00B 0.00B 0.14A 0.22A 0.18A Ethyl isovalerate 0.00C 0.14A 0.13A 0.13A 0.04BC 0.09AB 0.02BC Ethyl tiglate 0.00B 0.00B 0.00B 0.00B 0.24A 0.35A 0.20A Farnesyl acetate 0.22A 0.14B 0.10B 0.10B 0.00C 0.00C 0.00C Hexyl acetat e 0.30A 0.00B 0.00B 0.00B 0.00B 0.00B 0.00B Methyl isovalerate 0.33A 0.32A 0.38A 0.52A 0.00B 0.00B 0.00B KETONES 3 pentanone 0.00B 0.00B 0.00B 0.00B 0.02B 0.09A 0.10A MONOTERPENES myrcene 9.03A 5.74B 5.77B 6.40AB 0.00C 0.00C 0.00C L imonene 2.62A 1.53B 1.38B 1.72B 0.00C 0.00C 0.00C

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125 Table 54 Continued. Volatile compoundz MG MR R M0 M75 M150 M0 M75 M150 SESQUITERPENES All o aromadendrene 0.87A 1.08A 0.86A 0.88A 0.00C 0.00C 0.00C bergamotene 3.05A 2.23B 2.14B 2.22B 0.00C 0.00C 0.00C Cis bisabolene 1.28A 1.12A 0.90B 0.91B 0.00C 0.00C 0.00C bisabolene 7.40A 6.27AB 5.13B 5.34B 0.00C 0.00C 0.00C cadinene 1.95A 1.71A 1.40B 1.43B 0.00C 0.00C 0.00C caryophyllene 1.65AB 1.09B 1.51AB 1.85A 0.00C 0.00C 0.00C caryophyllene 95.15A 70.00B 67.74B 75.70B 0.09C 0.12C 0.13C copaene 4.62A 3.24B 4.15AB 4.21A 0.00C 0.00C 0.00C cubebene 8.09A 6.99AB 5.51B 6.22AB 0.00C 0.00C 0.00C elemene 0.56A 0.39B 0.32B 0.31B 0.0 0C 0.00C 0.00C farnesene 6.48A 5.87AB 5.38AB 4.59B 0.00C 0.00C 0.00C Cis farnesene 9.18A 8.99A 6.95A 7.39A 0.00B 0.00B 0.00B Germacrene B 0.41A 0.30B 0.24C 0.26BC 0.00D 0.00D 0.00D muurolene 3.06A 2.15A 2.28A 2.33A 0.00B 0.00B 0.00B selinene 0 .60A 0.40B 0.30B 0.37B 0.03C 0.03C 0.03C sesquiphellandrene 2.68A 2.15B 1.99B 2.05B 0.00C 0.00C 0.00C TOTALS Total alcohols (ALC) 0.33AB 0.00B 0.11B 0.00B 0. 63 A 0.5 2 A 0.4 5 A Total aldehydes (ALD) 5.75A 1.32BC 2.05BC 2.15B 0. 49 D 0. 62 CD 0.8 9 CD Total a lkanes (ALK) 21.76B 23.17B 83.92A 72.88A 5.11D 9.04C 10.33C Total esters (EST) 0.86A 0.60AB 0.62AB 0.75AB 0.43B 0.65AB 0.39B Total ketones (KET) 0.00B 0.00B 0.00B 0.00B 0.02B 0.09A 0.10A Total m onoterpenes (MOT) 1.64A 7.27B 7.15B 8.12AB 0 .00C 0.00C 0.00C Total s esquiterpenes (SQT) 147.01A 113.97B 106.78B 116.07B 0.12C 0.15C 0.17C TOTAL volatiles (TOV) 187.34AB 146.33B 200.63A 199.96A 6.81D 11.08C 12.33C zMeans followed by the same capital letter in the same row do not differ significant ly according to Duncans Multiple Range Test (P< 0.05) (n=6)

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126 Table 55. Volatiles emitted ( ng gfw1 h1) by maturegreen (MG), mid ripe (MR) and ripe (R) Monroe avocado (n=6) (Experiment 5): untreated control (M0) or treated with aqueous 1MCP at 75 (M 75) or 150 g L1 (M150) for 1 min at 20 C and stored at 20 C. Volatile compoundz MG MR R M0 M75 M150 M0 M75 M150 ALCOHOLS 3 methyl 1 butanol 0.00 B 0.00B 0.00B 0.00B 1.40A 1.28A 0.30AB ALDEHYDES Hexanal 1.24 A 0.37B 0.99A 1.06A 0.27B 0 .35B 0.20B ALKANES Decane 2.03 B 1.64BC 3.79A 4.79A 1.32BCD 1.24CD 0.95D Dodecane 20.45 B 20.74B 36.38A 33.91A 11.41C 10.10C 8.90C Tridecane 17.44 B 18.22B 25.82A 23.15A 7.55C 6.32C 6.16C Undecane 12.42 B 10.09B 20.86A 21.51A 7.32C 6.59C 5 .32C ESTERS Methyl butyrate 0.00 C 0.00C 0.00C 0.00C 4.33A 1.13B 1.31B Methyl isovalerate 0.57 A 0.12B 0.16B 0.14B 0.00C 0.00C 0.00C MONOTERPENES myrcene 0.84 A 0.53A 0.78A 0.88A 0.00B 0.00B 0.00B SESQUITERPENES Allo aromadendrene 2.31 AB 2.12B 2.13B 2.85A 0.00C 0.00C 0.00C bergamotene 0.95 A 0.80A 0.81A 0.94A 0.00B 0.00B 0.00B c adinene 2.45 A 2.07B 1.65C 1.94B 0.00D 0.00D 0.00D caryophyllene 2.81 A 2.74A 2.60A 3.12A 0.00B 0.00B 0.00B caryophyllene 47.89 A 46.64A 45.60A 53.59A 0.18B 0.13B 0.20B c opaene 31.55 A 26.35B 25.43B 31.54A 0.04C 0.03C 0.05C cubebene 0.98 B 1.01B 0.96B 1.28A 0.09C 0.07C 0.09C cubebene 1.22 A 1.4 5A 1.24A 1.61A 0.00B 0.00B 0.00B elemene 0.18 B 0.28A 0.29A 0.37A 0.00C 0.00C 0.00C Cis farnesene 1.53 A 1.12B 1.02B 0.95B 0.00C 0.00C 0.00C

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127 Table 55 Continued. Volatile compoundz MG MR R M0 M75 M150 M0 M75 M150 Germacrene B 0.44 A 0.36A 0.31A 0 .36A 0.00B 0.00B 0.00B farnesene 1.87 A 1.56A 1.36A 1.56A 0.00B 0.00B 0.00B muurolene 0.19 A 0.16AB 0.11B 0.12B 0.00C 0.00C 0.00C sesquiphellandrene 1.22 A 1.17A 1.14A 1.33A 0.00B 0.00B 0.00B Valencene 0.75 AB 0.70AB 0.63B 0.86A 0.27C 0.29C 0.28C Yl angene 10.64 A 9.10B 9.87AB 10.84A 0.00C 0.00C 0.00C TOTALS Total a lcohols (ALC) 0.00 B 0.00B 0.00B 0.00B 1.40A 1.28A 0.30AB Total a ldehydes (ALD) 1.24 A 0.37B 0.99A 1.06A 0.27B 0.35B 0.20B Total a lkanes (ALK) 52.33 B 50.68B 86.85A 83.36A 2 7.61C 24.24C 21.33C Total esters (EST) 0.57 BC 0.12C 0.16C 0.14C 4.33A 1.13B 1.31B Total ketones (KET) 0.00A 0.00A 0.00A 0.00A 0.00A 0.00A 0.00A Total m onoterpenes (MOT) 0.84 A 0.53A 0.78A 0.88A 0.00B 0.00B 0.00B Total s esquiterpenes (SQT) 106.98 A 97.62A 95.15A 113.25A 0.58B 0.53B 0.62B TOTAL volatiles (TOV) 161.96 AB 149.33B 183.94AB 198.68A 34.18C 27.54D 23.76D zMeans followed by the same capital letter in the same row do not differ significantly according to Duncans Multiple Range Test (P< 0.05) (n=6)

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128 A B C D E Figure 51. Steps of fruit processing for volatile collection: peeled fruit (A); fruit cut in halves (B); deseeded fruit (C); chopped pulp (D); chopped pulp enclosed in glass tubes for volatile collection (E).

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129 A B Figure 5 2 Whole fruit firm ness of Simmonds avocado in Phase 1, experiments 1 (A) and 2 (B). Treatments were: untreated control (C1 or C2), ethylene for 12 h (E12) or 24 h (E24), aqueous 1MCP at 150 g L1 for 1 min at 20 C (1 MCP) and ethylene for 12 h + 12 h air + aqueous 1 MCP at 150 g L1 for 1 min at 20 C (E12+MCP). All fruit were stored at 20 C. Vertical bars represent standard errors (n=6). Dotted lines represent midripe stage (upper) and firmness threshold (15 N) for ripe fruit (lower).

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130 A B Figure 5 3 Respiration rates of Simmonds avocado in Phase 1, experiments 1 (A) and 2 (B). Treatments were: untreated control (C1 or C2), ethylene for 12 h (E12) or 24 h (E24), aqueous 1MCP at 150 g L1 for 1 min at 20 C (1 MCP) and ethylene for 12 h + 12 h air + aqueous 1MCP at 150 g L1 for 1 min at 20 C (E12+MCP). All fruit were stored at 20 C. Vertical bars represent standard errors (n=6).

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131 A B Figure 5 4 Ethylene production rates of Simmonds avocado in Phase 1, experiments 1 (A) and 2 (B). Treatments were: untreated control (C1 or C2), ethylene for 12 h (E12) or 24 h (E24), aqueous 1MCP at 150 g L1 for 1 min at 20 C (1 MCP) and ethylene for 12 h + 12 h air + aqueous 1MCP at 150 g L1 for 1 min at 20 C (E12+MCP). All fruit were stored at 20 C. Vertical bars represent standard errors (n=6).

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132 A B C Figure 5 5 Whole fruit firmness of Simmonds (A), Booth 7 (B) and Monroe (C) avocado untreated (control) or treated with aqueous 1MCP at 75 (M75) or 150 g L1 (M150) for 1 min at 20 C (Phase 2 experiments). Vertical bars represent standard errors (n=6). Dotted lines represent midripe stage (upper) and firmness threshold (15 N) for ripe fruit (lower).

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133 A B C D E F Figure 56. Respiration and ethylene producti on rates of Simmonds (A and B), Booth 7 (C and D) and Monroe (E and F) avocado untreated (control) or treated with aqueous 1MCP at 75 (M75) or 150 g L1 (M150) for 1 min at 20 C (Phase 2 experiments). Vertical bars represent standard errors (n=6). Dotted line represents firmness threshold (15 N) for ripe fruit.

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134 CHAPTER 6 USE OF AN ELECTRONIC NOSE TO CLASSIFY AVO CADO PULP BY RIPENESS STAGE Intr oduction The characteristic smell of a food is a result of the interaction of volatile compounds produced by the product with the human olfactory system. When sniffed by the human nose, volatiles interact with the olfactory epithelium, which generates a si gnal that will be interpreted by the brain as a smell. Although thousands of volatiles have been identified, humans only detect the aromatic compounds (odorants). Human sensitivity to odorants varies from person to person and is influenced by factors such as age and fatigue (Reineccius, 2006). The electronic nose (EN) is an instrument that attempts to mimic the human nose in an artificial olfactory system. Several commercial models are available, and they are composed of an array of sensors that are stimul ated when exposed to the volatile compou nds of a sample, and then generate a signal which is captured and interpreted by software (Rck et al., 2008). It is an alternative nondestructive evaluation of samples according to the volatile compounds generated by the product, identifying samples by means of pattern recognition (Deisingh et al., 2004; Li and Wang, 2006). Although it can detect nonaromatic volatile compounds, the electronic nose does not give information that leads to identification and quantification of individual compounds (Baldwin, 2004). T he EN needs to be trained first to create a library of patterns of the samples to be later analyzed. Then, the identification can take place by pattern comparison (Rck et al., 2008). The applications of the EN are diverse and include the use in the food industry (Ampuero and Bosset, 2003), environment analysis (Littarru, 2007) and in medicine, for

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135 disease diagnostics (Turner and Magan, 2004). The use of electronic noses in food control has been increasing over the years because they are cost effective and provide a short time analysis (Peris and Escuder Gilabert, 2009). Several studies demonstrate the ability of the EN to discriminate fruit ripeness stages and assess fruit ripeness, in the field or after harvest, for intact or processed products (Athamneh et al., 2008; Benedetti et al., 2008; Brezmes et al., 2005; Gmez et al., 2006 a ; Lebrun et al., 2008; Maul et al., 2000; Pathange et al., 2006; Solis Solis et al., 2007). During the last decade several studies reported successful use of 1 methylcyclopropene (1MCP) to delay ripening in fruits and vegetables (Huber, 2008; Watkins, 2008). Despite the efficiency of this product, it has been shown that it may change the volatile profile of a product ( Argenta et al ., 2003 ; Botondi et al., 2003 ; Herrera et al., 2007; Kondo et al., 2005; Moya Leon et al., 2006; Pechous et al., 2005). The purpose of this study was to evaluate the use of an electronic nose to classify avocado pulp by ripeness stage in fruit untreated or treated with aqueous 1MCP Material and Methods Experiment 1 Plant material Avocado (Persea americana Mill.) cv. Booth 7 fruit were used for this experiment This cultivar is a GuatemalanWest Indian hybrid and a major commercial cultivar in Florida har vested in midseason ( Tropical Research and Education Center 2008). Fruit were harvested from experimental plots at the Tropical Research Education Center, Homestead, FL, on date A (Sept. 2008), as established in the official shipping schedule for avocado ( Florida Avocado Administrative Committee, 2009). Fruit were harvested at the maturegreen stage early in the morning and immediately transported to the

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136 Postharvest Horticulture Laboratory in Gainesville, FL. Upon arrival fruit were held overnight at 20 C. The next day the fruit were sorted for absence of major defects and diseases and stored at 20 C prior to testing with the Electronic Nose (EN). The avocados were sorted into three ripeness stages: maturegreen, midripe and ripe ( Figure 61 ), based upon whole fruit firmness which was determined by a nondestructive compression test on whole, unpeeled fruit using an Instron Universal Testing Instrument (Model 4411, Canton, MA, USA) fitted with a flat plate probe (5 cm diameter) and 50kg load cell. Afte r establishing zero force contact between the probe and the equatorial region of the fruit, the probe was driven with a crosshead speed of 2 0 mm min1. The force was recorded at 2.5 mm deformation and was determined at two points on the equatorial region o f each fruit considering a 90 angle between points The same four fruit of each treatment were measured repeatedly every other day until they reached the full ripe stage. Fruit were considered com mercially ripe upon reaching 10 to 15 N firmness at the eq uator. The average firmness was 193 N for maturegreen, 98 N for midripe and 14 N for ripe fruit. Electronic n ose s et u p The EN (Cyranose 320; Smith Detections Pasadena, Inc., Pasadena, CA) is a portable instrument composed of 32 individual thinfilm carbonblack polymer composite chemiresistors configured into an array (Cyrano Sciences, 2001) ( Figure 62A ). An accessory apparatus was constructed to improve accuracy of the EN based on the apparatus developed by M.O. Balaban and Luis Martinez (Martinez, 2007) ( Figure 62B ). Compressed air was used during the analyses, purified by activated carbon and two moisture traps. An initial purge of the electronic nose was performed for 6 min prior to the first reading to avoid the influence of water desorbed from sensors due to long

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137 inactive time. The sequence of conditions for each reading was determined according to the time required for most sensors to have a constant signal level for the baseline, namely: 60 s baseline purge, 60 s sample draw, 30 s sample gas purge, 150 s air intake purge. Pump speed was set to medium (approximately 120 ml min1) during baseline purge and sample draw, then was set to high (approximately 180 ml min1) during sample gas and air intake purge. The apparatus was purged with air for 1 min between samples. All 32 sensors were active for the analyses. Other parameters of the method were: digital filtering on; substrate heater on (35 C); algorithm canonical; preprocessing autoscaling; normalization normalization 1. Sample p rep aration and s ampling c onditions Each fruit was peeled, halved and the pulp sliced into cubes of approximately 1 cm3. A sample of 100 g of pulp cubes was immediately placed in a 1.7L glass jar, which was immediately sealed for 5 min. Headspace samples wer e drawn inserting the 10.2cm needle on one of the two vents on the lid of the jar, keeping the other vent open to avoid vacuum ( Figure 62C ). Room conditions during analysis were 22 C and 56% R.H. The EN was first trained to establish a smellprint for each class ( ripeness stage) which is the pattern of the headspace in the jar containing the volatile compounds. The training session utilized six individual fruit for each ripeness stage. Later, i n the identification session, the EN was challenged to identify the ripeness stage of seven test samples 100 g of ripe avocado pulp sliced into 1cm3 cubes by comparing their smellprints with the known smellprints generated during the training session. The same set of conditions was used for both sessions.

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138 Exp eriment 2 Mature green Simmonds avocado, a major commercial West Indian cultivar in Florida harvested in early season ( Tropical Research and Education Center 2008) was purchased from a commercial grower in Homestead, FL, on date A, in July 2009. After transportation and overnight storage at 20 C, fruit were sorted and treated with aqueous 1MCP at 75 or 150 g L1 (1.39 or 2.77 mmol m3 a.i.) for 1 min and stored at 20 C. Untreated control was stored under the same conditions. Fruit were submitted to the electronic nose analysis at three ripeness stages determined by whole fruit firmness, as described for experiment 1 The average firmness was 118 N for maturegreen, 60 N for midripe and 13 N for ripe fruit. The electronic nose was trained to classify avocado pulp in different situations: all treatments and ripeness stages; ripe pulp for all treatments; and the three ripeness stages in each treatment separately. In all occasions 10 exposures of per class were used. Identification session was not performed. Although sampling preparation was the same as previously described, instrument set up and sampling conditions were slightly changed. A smaller 0.8 L glass jar, sealed for 3 min, was used. The sample draw was set to 30 s. Experiment 3 For this experiment Booth 7 fruit from the same location of Experiment 1 was used. Fruit were harvested on date D, in Octo ber 2009 and after transportation and overnight storage at 20 C, fruit were sorted and treated with aqueous 1MCP at 75 or 150 g L1 (1.39 or 2.77 mmol m3 a.i.) for 1 min and stored at 20 C. Untreated control was stored under the same conditions.

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139 Fruit were submitted to the electronic nose analysis at three ripeness stages determined by whole fruit firmness, as described for experiment 1. The average firmness was 190 N for maturegreen, 97 N for midripe and 13 N for ripe fruit. The same sampling preparation and conditions observed for experiment 1 were applied for this experiment. Only the training session was performed, as described for experiment 2. Experimental D esign and S tatistical A nalysis E xperiment 1 was conducted in a completely randomized design with six replicates per treatment C ross validation of the model s w as performed by the EN. Canonical Discriminant Analysis w as performed and the interclass Mahalanobis distances were generated by the software PCNose (Cyranose 320; Smith Detections Pasadena, Inc., Pasadena, CA). Experiments 2 and 3 were conducted in a completely randomized design with 10 replicates per treat ment. Real time sensor response data was recorded every 3 s and data analyzed by the Statistical Analysis Software SAS version 9.1 (SAS Institute Inc., Cary, NC) Stepwise discriminant analysis was used to select a pool of sensors (up to 10) to be used for class discrimination by canonical discriminant analysis Cross validation of the models and interclass Mahalanobis distances were also calculated for each training session. Results Experiment 1 The training session was considered successful as the EN accurately separated the three ripeness stages targeted in this study. The model with three ripeness stages was suitable for clear class discrimination. Cross validation of the model was 100%,

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140 meaning that all samples trained as one particular ripeness st age were classified as being of that ripeness stage. The Interclass Mahalanobis distance is the distance between two classes, and the ability of the model to successfully discriminate between classes increased as the Mahalanobis distance increased. A minim um of 5 is required for a good class separation (Cyrano Sciences, 2001) and therefore, there was a very good separation between midripe and ripe fruit ( Table 61) The Mahalanobis distance was much higher between maturegreen and midripe or maturegreen and ripe indicating that maturegreen fruit were more distinctively separated from other stages than midripe from ripe fruit. This was confirmed by Canonical Discriminant Analysis Plot, which shows three distinct clusters representing the three ripeness stages considered in this study ( Figure 63) In this test the EN had poor performance when challenged to classify the ripeness stage of test samples (ripe fruit) in the sample identification session ( Table 62 ). It correctly classified only 43% of the samples as ripe fruit. It did not misclassify the other 57% of the samples as maturegreen or midripe fruit, but rather as unknown, meaning that some samples were out of the range of training. Experiment 2 The training session of the EN was not completely successful when all treatments were in considered in the model The 10 sensors that most contributed to class discrimination were included in this order in the model by stepwise discrimination analysis: S23, S4, S11, S5, S31, S16, S6, S8, S10 and S12. Crossvalidation showed 85.7% correct classifications, with misclassification of 10 out of 70 samples, most of them involving those treated with 75 g L1 aqueous 1MCP (Table 6 3). Analysis of

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141 Mahalanobis distances showed that there were significant differences ( P <0.01) among all classes but some of the values were below the required threshold of 5 for a good class separation ( Table 64). Values were higher for separations of maturegreen fruit from the other treatments and other ripeness stages The closest classes, determined by the lowest Mahalanobis distance, were control (C MR) and 1MCP at 75 g L1 (M75 MR) at mid ripe stage. The canonical discriminant analysis plot ( Figure 64A) revealed that the first canonical variable explained most of the variation among ripeness stages while the second canonical variable helped to explain the variation among treatments within the same ripeness stage. The most distinct class was maturegreen fruit. Pulp from fruit treated with 1MCP at 150 g L1 was better separated from the other two treatments (control and 75 g L1) in either mid ripe or ripe fruit. The EN was successfully trained to separate ripe pulp of different treatments. Mahalanobis distances were lower from control to 1MCP at 75 g L1 (6.62) and to 1 MCP at 150 g L1 (7.09) than between the two 1MCP treatments (7.31). The canonical plot shows three distinct clusters confirming a good separation ( Figure 65A). The first canonical variable contributed to better separation of the two 1MCP treatments while the second canonical variable contributes to a better separation of untreated control pul p. When trained for separation of ripeness stages for each treatment separately, the EN was also successful. Mahalanobis distances ranged from 7.75 to 15.25, indicating a good separation of ripeness stages ( Table 65 ). Canonical plots show clear class separations for all treatments (Figures 66A, 6 6B and 66C).

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142 Experiment 3 The sensors that most contributed to class separation in Booth 7 were mostly different than those observed for Simmonds: S24, S32, S21, S8, S19, S10, S13, S22, S18 and S1. As observ ed for Simmonds the training of the EN was not successful to completely separate treatments of Booth 7 altogether. Cross validation showed 87.1% correct classifications (Table 66) Once again, most misclassifications were observed involving pulp from fruit treated with 75 g L1 aqueous 1MCP (Table 66 ). Mahalanobis distances for five classes comparisons were lower than 5, four of them involving pulp from fruit treated with 75 g L1 ( Table 67 ). The canonical plot revealed slightly better separation of clusters for Booth 7 than for Simmonds, with the first canonical variable explaining better separation of ripeness stages ( Figure 64B). However, 1MCP treatments had overlapped readings for a same ripeness stage. As observed for Simmonds, the EN was successfully trained to separate ripe Booth 7 pulp of different treatments. Higher Mahalanobis distances separated pulp from fruit treated with 1MCP at 150 g L1 from control and from 1MCP at 75 g L1 than between these two treatments. The canonical plot shows three distinct clusters confirming a good separation, with more distinct separation of 1MCP at 150 g L1 from control and 1MCP at 75 g L1 ( Figu re 6 5B). Once again, the first canonical variable contributed to better separation of the two 1MCP treatments while the second canonical variable contributes to a better separation of untreated control pulp. When trained for separation of ripeness stages for each treatment separately, the EN was also successful. Mahalanobis distances indicat ed a good separation of ripeness stages ( Table 65 ). Canonical plots show clear class separations for all treatments,

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143 mainly for 1MCP at 150 g L1 (Figures 6 6D, 6 6E and 66F). In each training, the first canonical variable was the responsible for separation of ripeness stages, mainly maturegreen from ripe. The second canonical variable helped on the separation of midripe fruit. Discussion The electronic nose ( EN ) analysis has proven to be a potential tool to classify avocado pulp by ripeness stage. Separation of ripeness stages of a single treatment was completely successful in all situations (Figures 63 and 66). However, the quality of separation was lower when the ripeness stages for more than one treatment were included in a full model during the training session ( Figure 64). In this case, some misclassifications were observed in the cross validation of the model, resulting in errors from 12.9% to 14.3%. However, separation of ripeness stages of a same treatment under this full model was still possible. Since the EN analysis is based on volatile compounds, it is likely that changes in volatile profile in each ripeness stage contributed to class separation. In tomato, sensor responses were positively correlated to the concentrations of 2 hexenal and negatively correlated to acetaldehyde, methanol and 2isobutylthiazole (Maul et al., 2000). Mango cultivars were separated in classes by an electronic nose due to their differences in volatile profile (Lebrun et al., 2008). Informal analysis of Booth 7 fruit aroma revealed that mature green fruit had a characteristic green, woody aroma, which is mostly lost during ripening. Ripe fruit had w eak greenish, nutty aroma. Terpenoids are the main volatile compounds in avocado (Pino et al., 2000; Pino et al., 2004; Sinyinda and

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144 Gramshaw, 1998) but almost non existent in ripe fruit, indicating that this group of volatiles may have influenced class sep aration in avocado using the EN The EN was also capable of detect ing differences for pulp from fruit treated with the ethylene inhibitor 1 MCP. This compound delays ripening and the onset of climacteric production in avocado and may reduce ethylene production (Adkins et al., 2005; Feng et al., 2000) and, therefore, it could have been a determinant factor for class separation. In this present study, separation by the EN of untreated control from 1MCP treatments was more evident for pulp from fruit treated with 1 MCP at 150 g L1 than for those treated with 75 g L1. This observation is consistent with results from previous experiments that higher concentrations promoted stronger effects and indicates that fruit treated with 75 g L1 were more similar to control than those treated with 150 g L1. Avocado produces ethylene at very low rates in maturegreen fruit but large amounts (> 100 L kg1 h1) during ripening (Eaks, 1978). It is possible that a higher concentration of 1 MCP may have had a stronger i nfluence in ethylene production and in the onset of climacteric. Therefore, higher differences from control may have been in part due to ethylene production. The capability of separation of ripe pulp from fruit of different treatments ( Figure 65) also cor roborates with the idea of the importance of ethylene for class separation. A lthough the EN was successfully trained to generate avocado pulp classification by ripeness stage, adjustments in the methodology are needed to increase the efficacy of sample id entification, as determined by the first experiment with Booth 7 The first corrective action would be to increase the number of fruit samples per ripeness stage during the training session. Use of a larger sample size would expose the EN to more

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145 variabi lity within each ripeness stage and therefore the patterns would be more representative of this variation, increasing the chances of correct identification. In both experiments with 1MCP, the number of replicates was increased from six to 10. Although identification session was not done in these two experiments, it is likely that the chances of a correct identification of the ripeness stage of a single treatment would be higher. A s econd alternative would be to reduce the headspace volume and increase the surface area to accelerate the accumulation of volatile compounds in the headspace. This was done for the experiment with Simmonds but the Mahalanobis distances ( Table 64) were comparable to the second experiment with Booth 7 ( Table 67 ), indicating t hat this action would not necessarily promote better separation. However, the use of a smaller container may reduce time of analysis. A third action would be to reduce identification quality in the EN software setup, since there is a very low possibility t hat a test sample will fall outside of the three ripeness stages established during the training session. Lastly, all 32 sensors were used in the present study for the method development; however, sensor responses vary and the contribution of each sensor to class discrimination is different. As a result of stepwise analysis, different pools of 10 sensors were identified for both cultivars as being most significant for class separation. The only two sensors in common were S8 and S10 and therefore these coul d be targeted for future studies with avocado pulp to improve the specificity of the analysis. The electronic nose, PEN2 (WMA Analytics Inc., Schwerin, Germany), is composed of an array of 10 metal oxide semiconductor type sensors, yet a single sensor was able to

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146 discriminate peaches according to their ripeness stage (Benedetti et al., 2008). A subset of sensors of the same electronic nose explained nearly all the variance during ripening of tomato (Gmez et al., 2006a) and mandarin (Gmez et al., 2006b). The sensors S5, S23 and S31 were in the selected pool of sensors for Simmonds. These sensors are sensitive to polar compounds such as water (Cyrano Sciences, Inc., 2001), but analysis of sensor data without these sensors did not alter final results (data not shown) as observed for apples (Pathange et al., 2006) For apples, f ew specific Cyranose 320 sensors (S14, S16, S17 and S32) responded more favorably to apple aroma than others (Pathange et al., 2006). Significant correlations were also found for B HN189 tomatoes between specific aroma compounds and individual sensors of the eNose 4000 (Neotronics Scientific, Flowery Branch, Ga.) (Maul et al., 2000). Future studies may target correlation of sensors with specific volatile compounds in avocado. Conc lusio ns T he electronic nose analysis was successful to classify avocado pulp by ripeness stage when the EN was trained for class separation of a single treatment, either untreated or treated with aqueous 1MCP at 75 or 150 g L1. Lower classification pow er was obtained when all treatments and ripeness stages were included in the same model during the training session, leading to higher chances of errors. Although different pools of 10 sensors were identified for both cultivars as being the most significa nt for class separation, sensors S8 and S10 were common in both pools and therefore these could be targeted for future studies with avocado pulp to improve the specificity of the analysis.

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147 Table 61 Interclass Mahalanobis distances between maturegreen mid ripe and ripe Booth 7 avocado pulp. (Training session) Mature green Mid ripe Ripe Mature green 55.74 68.84 Mid ripe 15.32 Table 62 Results from the identification session using ripe Booth 7 avocado pulp as test samples Teste d as Identified as Ripe ( fruit # ) Mature green Mid ripe Ripe Unknown 1 x 2 x 3 x 4 x 5 x 6 x 7 x Results (%) 0 0 43 57

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148 Table 63 Cross validation for the training session for Simmonds avocado pulp (n=10) from fruit either untreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis at three ripeness stages: maturegreen (MG), mid ripe (MR) and ripe (R). Identified as Cl assified as MG C MR M75 MR M150 MR C R M75 R M150 R MG 10 0 0 0 0 0 0 C MR 0 8 2 0 0 0 0 M75 MR 0 1 8 1 0 0 0 M150 MR 0 0 0 9 1 0 0 C R 0 0 0 1 8 1 0 M75 R 0 1 0 0 1 7 1 M150 R 0 0 0 0 0 0 10 Table 64 Interclass Mahalanobis distances for Simm onds avocado pulp (n=10) from fruit either untreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis at three ripeness stages: maturegreen (MG), mid ripe (MR) and ripe (R). MG C MR M75 MR M150 MR C R M75 R M150 R MG 7.44** 7.14** 6.99** 8.72** 10.84** 10.92** C MR 2.92** 5.79** 6.81** 7. 27** 9.50** M75 MR 4.75** 6.16** 6.39** 8.28** M150 MR 3.20** 5.02** 4.98** C R 3.20** 5.11** M75 R 5.36** M150 R ** Significant at P <0.01.

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149 Table 65 Interclass Mahalanobis distances for mature green (MG), mid ripe (MR ) and ripe (R) Simmonds or Booth 7 avocado pulp. Values represent class separation within each treatment separately: untreated (Control) or treated with 1 MCP at 75 g L1 (M75) or 150 g L1 (M150). Simmonds Booth 7 MR R MR R Control MG 9.93** 8.80** 6.65** 18.34** MR 9.44** 13.31** M75 MG 8.37** 14.84** 6.21** 9.26** MR 8.15** 6.64** M150 MG 9.02** 15.25** 9.53** 13.00** M R 7.75** 9.98** ** Significant at P <0.01.

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150 Table 66 Cross validation for the training session for Booth 7 avocado pulp (n=10) from fruit either untreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis at three ripeness stages: maturegreen (MG), mid ripe (MR) and ripe (R). Identified as Classified as MG C MR M75 MR M150 MR C R M75 R M150 R MG 8 1 1 0 0 0 0 C MR 1 9 0 0 0 0 0 M75 MR 0 1 7 1 1 0 0 M150 MR 0 0 1 9 0 0 0 C R 0 0 0 0 10 0 0 M75 R 0 0 0 0 1 9 0 M150 R 0 0 0 0 0 1 9 Table 67 Interclass Mahalanobis distances for Booth 7 avocado pulp (n=10) from fruit either untreated (C) or treated with 1MCP at 75 g L1 (M75) or 150 g L1 (M150) and assessed for analysis in three ripeness stages: maturegreen (MG), mid ripe (MR) and ripe (R). MG C MR M75 MR M150 MR C R M75 R M150 R MG 4.99** 7.10** 9.10** 11.32** 9.88** 13.12** C MR 3.91** 5.03** 8.53** 9. 02** 11.43** M75 MR 3.22** 4.98** 5.86** 8.67** M150 MR 6.08** 7.70** 9.73** C R 4.70** 7.06** M75 R 6.87** M150 R ** Significant at P <0.01.

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151 A B C Figure 61 Ripeness stages of Booth 7 avocado assessed for el ectronic nose analysis. A) mature green, B) midripe and C) ripe. A B C Figure 62 Details of the electronic nose analysis. (A) The electronic nose Cyranose 320. B) Apparatus constructed to improve the accuracy of the electronic nose, based on th e apparatus developed by M.O. Balaban and Luis Martinez (Martinez, 2007). Design used with permission. C ) Detail of headspace sample draw.

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152 Figure 63 Canonical Discriminant Analysis Plot of electronic nose readings (n=6) of maturegreen (MG), mid ripe (MR) and ripe (R) Booth 7 avocado pulp.

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153 A B Figure 64 Canonical Discriminant Analysis Plot of electronic nose readings (n= 10) of maturegreen (MatGr), mid ripe (MR) and ripe (R) avocado pulp. Fruit were either untreated (C) or treated with aqueous 1 MCP at 75 g L1 (M75) or 150 g L1 (M150) for 1 min at 20 C. A) Simmonds and B) Booth 7.

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154 A B Figure 65 Canonical Discriminant Analysis Plot of electronic nose readings (n= 10) of ripe (R) avocado pulp. Fruit were either untreated (C) or treated with aqueous 1 MCP at 75 g L1 (M75) or 150 g L1 (M150) for 1 min at 20 C. A) Simmonds and B) Booth 7.

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155 A D B E C F Figure 66. Canonical Discriminant Analysis Plot of electronic nose readings (n= 10) for maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds (left) or Booth 7 (right) avocado pulp. Fruit were either untreated (C) or treated with aqueous 1 MCP at 75 g L1 (M75) or 150 g L1 (M150) for 1 min at 20 C and trained separately for each treatment.

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156 CHAPTER 7 SENSORY ATTRIBUTES O F BOOTH 7 AVOCADO FOLLOWING ETHYLENE PRETREATMENT AND/OR EXPOSURE TO GASEOUS OR AQUEOUS 1 MCP Introduction Avocados are originally from Tropical America and three races of avocados are known: West Indian, Guatemalan and Mexican. Many existent cultivars are hybrids between two of these races. Mexican types are smaller and have higher oil content than West Indian or Guatemalan fruit (Crane et al., 2007b). The fruit is consumed fresh or to make guacamole and other dishes (Evans and Nala mpang, 2006). Consumer interest in avocados is growing mainly due to its nutritional and health benefits (Evans and Nalampang, 2009). The fruit is rich in monounsaturated lipids especially oleic acid (Kikuta and Erickson, 1968; Moreno et al., 2003; Pacett i et al., 2007) and in lipophilic antioxidants ( Wu et al., 2004) that can help on cancer prevention (Ding et al., 2007). Flavor quality of horticultural products is ultimately defined genetically and its description is very complex. Sensory evaluation of flavor by taste panels generally us es a large number of panelists ranking their perceptions on a hedonic scale (Baldwin, 2004). Texture, however, has no specific sensory receptors as do taste and aroma. The parameters of texture are perceived when the food is placed and deformed in the mouth and i t is an important indicative of produce freshness (Szczesniak, 2002). For avocado, generally the higher the fruit oil content, the richer the flavor (Storey et al., 1973; Lee et al., 1983; Woolf et al., 2004). In a sensory panel, 15 selections of avocado were evaluated for visible fibers, sweetness, firmness, texture, flavor and overall acceptability. Pulp texture had the highest correlation coefficient (r = 0.80) with overall acceptability, followed by flavor whi le v isible fibers was the most negative factor. Even though oil content was associated with flavor (r = 0.63), it had fairly low correlat ion

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157 with acceptability (r = 0.34) (Razeto et al., 2004). Similar results were found for nine selections and Hass and Bacon, where texture and flavor were positively correlated with acceptability, while fibers and mesocarp discoloration were negatively correlated with acceptability (Villa, 2005). Besides nutty flavor, some avocados have a slightly higher sugar content that contributes to a sweeter flavor (Shaw et al., 1980). Immature avocados have an unpleasant bitter flavor that leaves a distinctive long aftertaste on the palate (Brown, 1972). However, bitterness can also be detectable in some cases, even in ripe fruit s, and it is a negative factor for sensory acceptability (Shaw et al., 1980). Current research on postharvest has shifted to f ocus on maintaining optimal flavor quality while extending shelf life of a horticultural product (Kader, 2008). Concurrently, d ur ing the last decade the compound 1methylcyclopropene (1MCP) has been widely used in research to extend postharvest life of a wide range of horticultural products, including vegetables, flowers, climacteric and nonclimacteric fruits (Huber, 2008). The mo lecule 1methylcyclopropene (1MCP) is considered the most effective ethylene action inhibitor since it is active at extremely low concentrations, is already commercially available and it is considered to be nontoxic (Sisler, 2006; Environmental Protection Agency, 2008). Application of 1MCP technology may affect sensory acceptability of fruits and vegetables. This technology has been used commercially by apple industries around the world (Watkins, 2008). Anna apple treated with 1MCP was less aromatic, but preferred by panelists over nontreated fruit (Lurie et al., 2002). Similar results were reported later for the same cultivar, and added that the fruit is firmer and juicier than

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158 untreated, even though less aromatic (PreAymard et al., 2005). In a sens ory test performed for Gala apples, consumers distinguished between 1MCP treated and nontreated fruit, but there were no differences for overall liking scores. However, consumers that ate the fruit regularly preferred untreated fruit when compared to t hose that did not eat it (Marin et al., 2009). Informal sensory analysis of Florida 47 tomato treated with 1MCP revealed that aroma profiles were negatively and irreversibly affected, mainly when treatment was applied in early ripeness stages (Hurr et al., 2005). In Packhams Triumph pears, 1MCP treatment maintained textural characteristics of the fruit and was preferred by panelists over untreated fruit after 6 months of storage (MoyaLeon et al, 2006). Flavor and consumer liking of kiwi was not aff ected by 1MCP treatment (Harker et al., 2008a). Additionally, panelists did not notice differences in aroma, color and firmness of mango treated with 1MCP over the untreated fruit (Cocozza et al., 2004). Both gaseous and aqueous 1MCP delayed ripening o f avocado, with reports of significant suppression of ethylene production, softening, color changes and internal disorders (Feng et al., 2000; Jeong et al., 2002; Adkins et al., 2005; Woolf et al., 2005; Choi et al., 2008). However, despite the evidences o f changes of sensory attributes in several horticultural products, there are no reports of the influence of 1MCP in the sensory attributes of avocado. The objective of this study was to evaluate sensory attributes of Booth 7 avocado following ethylene pretreatment and/or exposure to gaseous or aqueous 1MCP.

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159 Material and Methods Plant M aterial E xperiments were carried out using 'Booth 7' avocado ( Persea americana Mill.), a GuatemalanWest Indian hybrid, and a major midseason commercial cultivar in Florida ( Tropical Research and Education Center 2008). All experiments used fruit harvested from the same grove (Tropical Research Education Center, Homestead, FL), on dates A and D of harvest (approximately 5 weeks difference) according to the official avocado shipping schedule for Florida ( Florida Avocado Administrative Committee 2009) In all experiments, f ruit were harvested at maturegreen stage early in the morning and immediately transported to the Postharvest Horticulture Laboratory in Gainesville, FL. Upon arrival fruit were held overnight at 20 C and then were selected for absence of major defects and diseases for treatment application. During the Season 1 (2008), fruit were harvested in October (early harvest EH) and November (late harvest LH). Initial quality analysis was completed 24 h after harvest, prior to ethylene treatment. All fruit were treated with ethylene (100 L L1) 24 h after harvest for 12 h at 20 C. Then the fruit were stored for an additional 12 h in an ethylenefree (<0.1 L L1 ethylene) cold room (20 C) (total=24 h) prior to treatment with gaseous or aqueous 1MCP Following 1 MCP t reat ment, treated a nd control fruit (not exposed to 1MCP) were maintained at 20 C / 92% R.H. until ripe. Fruit ripening was monitored and based on whole fruit firmness, respiration, ethylene production and weight loss. Texture Profile Analysis and sensory analysis was perf ormed on ripe fruit only (10 to 15 N whole fruit firmness).

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160 During Season 2 (2009), fruit harvested in October (early EH) and November (late LH) were not exposed to ethylene pretreatment but treated only with aqueous 1MCP 24 h after harvest, as descri bed below. Fruit were stored at 20 C / 90% R.H. until ripe (10 to 15 N whole fruit firmness) and assessed exclusively for sensory analysis. 1 MCP T reatment Fruit from Season 1 were treated with either aqueous 1MCP [ 75 or 150 g L1 a.i. (1.39 or 2.77 mm ol m3 a.i.)] immersed for 1 min at 20 C or gaseous 1MCP [ 75 or 150 nL L1 (3.15 or 6.31 mol m3 a.i.) for 12 h at 20 C prepared respectively from formulations AFxRD 300 (2% a.i., AgroFresh, Inc. Philadelphia, PA ) and Ethyblock (0.14% a.i., Floralife, Burr Ridge, IL). Aqueous treatments were labeled as A75 (75 g L1) and A150 (150 g L1) whereas gaseous treatments were labeled as G75 (75 nL L1) and G 150 (150 nL L1) Each aqueous solution was made by dissolving adequate amount of the powder in 20 L of distilled water. The solution was swirled gently for 1 min and applied 10 to 45 minutes after preparation (Choi et al., 2008). Groups of eight to 12 fruit were placed in mesh bags and completely immersed in the respective solution for the desired time. Bags were gently agitated to ensure full contact of solution with fruit surfaces. After removal from the solution, the fruit were allowed to drain excess solution and then dried with a paper towel. Gaseous 1MCP was produced by adding the measured amoun t of the powder to 40 mL distilled water in a 125mL Erlenmeyer flask that was immediately capped with a rubber septum and swirled gently until the powder was completely dissolved (Hurr et al., 2005) The flask was then placed in the center of a 174L metal chamber, fruit were

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161 placed for treatment, the septum was removed from the flask and the chamber was immediately sealed for the treatment duration. Fruit from Season 2 were only treated with aqueous 1MCP treatment as described above. Season 1: Ripening and Q uality P arameters Whole fruit firmness Firmness was determined nondestructively via compression test on whole, unpeeled fruit using an Instron Universal Testing Instrument (Model 4411, Canton, MA, USA) fitted with a flat plate probe (5 0 m m diameter) and 50kg load cell. After establishing zero force contact between the probe and the equatorial region of the fruit, the probe was driven with a crosshead speed of 20 mm min1. The force was recorded at 2.5 mm deformation and was determined at two points on the equatorial region of each fruit, with a 90 angle between points. Ten fruit of each treatment were measured repeatedly every other day until they reached the full ripe stage. Fruit were considered commercially ripe upon reaching 10 to 15 N firmness. Respiration and ethylene production For Season 1 experiments, r espiration (as CO2 evolution) and ethylene production rates were measured daily on four individual fruit for each treatment. Fruit were individually sealed for 20 min in 2 L plastic containers prior to sampling. A 1m L gas sample was withdrawn by a plastic syringe through a rubber septum for analysis. Carbon dioxide (CO2) was determined using a Gow Mac gas chromatograph (Series 580, Bridge Water, NJ, USA) equipped with a thermal conductivity detector (TCD). Ethylene was measured by injecting a 1.0 m L gas sample into a Tracor gas chromatograph (Tremetrics, Austin, TX, USA) equipped with a flame ionization detector.

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162 Weight loss Weight loss was calculated (fresh weight basis) using the Equation 7 1: (7 1) where: WL is the weight loss given in percentage, Wo is the initial weight and Wf is the final weight. Texture p rofile a nalysis TPA This analysis was performed using samples from ripe mesocarp tissue. F or each treatment a crosssection was removed from the middle third of the fruit (n=3 fruit), and three cylindrical samples (10 mm dia x 10 mm long) were withdrawn with a cork borer from the stem end to blossom end orientation. Each mesocarp sample was placed vertically (same sampling direction) o n a flat aluminum plate and submitted to the TPA, which was performed using an Instron Universal Testing Instrument (Model 4411, Canton, MA, USA) fitted with a flat cylindrical probe (13 mm dia.) and 2 kg load cell. After establishing zero force contact between the probe and the top surface of the cylindrical sample, the probe was driven with a crosshead speed of 200 mm min1 to 70% of original sample height There were two consecutive compressiondecompression cycles per test TPA parameters ( Table 71) were determined using the Blue Hill Software (Norwood, MA, USA) Seasons 1 and 2: S ensory A nalysis Ripe fruit (10 to 15 N whole fruit firmness) from EH and LH were used for consumer sensory acceptance test with 75 untrained consumer panelists in the sensory laboratory of the Food Science and Human Nutrition Department at the University of

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163 Florida. The gender, age and avocado eating habit of panelists were asked according to pre determined answers (Appendix B ). Each treatment was evaluated on the day fruit reached the ripe stage Fruit were sliced longitudinally in quarters, peeled and the pulp was sliced in pieces of approximately 2 cm x 2 cm x 1 cm. Panelists received two pieces of avocado pulp served in plastic cups ( Figure 71A) and were asked to evaluate texture, flavor and overall liking using a 9point hedonic scale where 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely. Panelists sat in individual booths ( Figure 71B) and scores and comments were recorded with the aid of a c omputer (Figure 71C) using the software Compusense five, version 4.8.8 (Compusense Inc., Guelph, Ontario, Canada) Due to an unexpected conflict with the academic schedule, sensory analysis of Season 1 late harvested fruit treated with A75 and G75 could not be performed when fruit reached full ripeness (1015 N). These fruit were evaluated 2 d after reaching the ripe stage Statistical A nalysis The experiments were conducted using a completely randomized design with five treatments Four replicates were us ed for ripening and quality parameters and 75 non trained panelists participated in each sensory analysis For each experiment, data from sensory analysis were analyzed by oneway ANOVA. Data from panelist gender and eating habit for both seasons were combined to determine correlations and to evaluate differences. Differences between means were determined using Duncans Multiple Range Test. Statistical procedures were performed using the Statistical Analysis Software SAS version 9.1 (SAS Institute Inc., Cary, NC).

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164 Results Season 1: Ripening and Quality Parameters Whole fruit firmness E arly harvested (EH) avocados had initial firmness of 193 N and untreated control fruit ripened (10 to 15 N) within 10 d of storage at 20 C ( Figure 72A ). Significant dif ferences in the rates of softening between 1MCP treated fruit and control began after 4 d of storage and were more evident after 6 d Among 1 MCP treatments, differences were more evident after 10 d, with A150 and G150 fruit firmer than A75 and G75. Both aqueous and gaseous 1MCP treatments delayed fruit softening, extending shelf life from 4 d with A75 to 10 d with A150 and G150, and fruit from all treatments softened to ripe stage. L ate harvested (LH) fruit had lower initial firmness (181 N) and control fruit ripened 2 d faster than EH fruit ( Figure 72B) Differences in softening rates between 1MCP treated fruit and control were again more evident after 4 d of storage. Delay of ripening by A75 and G75 was 4 d Fruit treated with A150 and G150 had ripeni ng prolonged for 6 d beyond control, shorter than the delay observed for EH fruit. Respiration rate and ethylene production Control EH fruit reached the climacteric peak respiration rate within 8 d of storage at 168 mg CO2 kg1 h1 ( Figure 73 A). 1 MCP tr eatments delayed the onset of the climacteric and reduced peak respiration rate from 7.7% (A75) to 26.0% (G75) as compared with control fruit The initial respiration rate of LH untreated fruit (87 mg CO2 kg1 h1) (Figure 73B ) was higher than that of EH fruit (57 mg CO2 kg1 h1), consistent with a more advanced stage of maturity. The climacteric rise began earlier in every treatment in LH than in EH

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165 fruit and peak respiration rates for the former were higher. Gaseous 1MCP treatments significantly suppressed peak respiration rates by 20.2% (G150) and 23.4% (G75) as compared with control. Peak ethylene rate of EH control fruit was reached o n the same day as peak respiration, at 147 L C2H4 kg1 h1 ( Figure 74A ). However, there were no significant differences for peak ethylene production due to treatment ( data not shown). The longest delays were observed for fruit treated with A150 and G150 achieving maximum production 9 to 11 d aft er the untreated control fruit. Ethylene climacteric peak in LH fruit was delayed by 4 to 6 d ( Figure 74B ) however there were no significant differences in peak ethylene production. Weight loss Untreated EH fruit had 4.8% weight loss, similar to A75 fruit, but significantly lower than t he weight loss of 1MCP treated fruit (8.5% to 10.2%) ( Figure 75) Weight loss of LH fruit was overall higher to that of EH and highest weight loss levels were observed for A150 and G150 treatments a result of extended r ipening time. Considering that LH fruit ripened faster, the final weight loss of these fruit indicated faster rates of weight loss than EH fruit. However, no evident signs of shriveling were noticed in the present study. Texture p rofile a nalysis TPA Exp osure to 1MCP resulted in s ignificant differences in several ripe mesocarp texture parameters ( Table 72 ). Hardness 1st cycle was significantly higher than control for A150 and G150 in EH fruit, but no differences among treatments were observed for LH fru it. The hardness 2nd cycle was significantly lower than control for A150 and G150. Springiness gumminess and chewiness were not affected by 1MCP treatments in

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166 either EH or LH fruit. Cohesiveness was lower for gaseous treatments in LH fruit and significan tly lower than EH fruit. Adhesiveness for A150 and G150 treatments was significantly higher than for other treatments in EH. In LH, there were no differences among treatments for this parameter, but all 1 MCP treatments had lower adhesiveness in LH when co mpared with EH. Sensory A nalysis Season 1 Sensory analysis revealed significant differences for texture and flavor among treatments in both EH and LH ( Table 73 ). EH fruit treated with A150 or G150 had significantly higher texture scores than control fruit, representing opinions from like slightly to like moderately. Treatments of A75 or G75 did not differ from control and between them, but the later treatment was slightly preferred, not differing from the other two highest 1MCP concentrations. Treat ment A150 was rated significantly superior than untreated control for overall liking, while the other treatments had intermediate average scores. For LH fruit, treatments A75 and G75 (analyzed 2 d after reaching ripefruit firmness) were least preferred for texture, while there were no significant differences between control and the fruit treated with highest concentrations of 1 MCP. However, there were no significant differences for overall liking among treatments. Season 2 For fruit from Season 2, treated only with aqueous 1MCP, sensory analysis revealed no differences among treatments for all parameters in EH fruit ( Table 74 ). In LH fruit, both flavor and overall liking for the treatment A150 were least preferred, but scores for texture did not differ.

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167 Correlations and effects of gender and avocado eating habit Based on combined scores from the sensory analyses for both seasons and harvest times, positive correlations were observed for panelists gender and avocado eating habit with texture, flavor and overall liking; weakest correlations were for gender ( Table 75 ). Texture was also highly correlated with flavor and overall liking, but the strongest correlation was observed for flavor and overall liking. Males constituted 46.6% of all panelists and fem ales, 53.4% ( Figure 76A). Confirming the weak correlation, only slightly higher overall scores were observed for females for all three sensory parameters ( Figure 76B). On average, 6.9% of the panelists eat avocado once a week, 18.4% two to three times per month, 30.3% once per month, 20% once per year and 24.4% had never eaten fresh avocado ( Figure 77A). Scores for texture, flavor and overall liking were consistently higher with increased frequency of consumption ( Figure 77B). Overall scores given by the panelists that never eaten fresh avocado before were lower, near 4, meaning dislike slightly. For panelists that eat once a year or once a month, the scores were, on average, close to 6, meaning like slightly. Those that eat avocado more frequently g ave scores near to 7, like moderately. The age of panelists was not considered in these analyses since 85% of the group was in the 18 to 29 year old category. Discussion In the present study, the pretreat ment with ethylene (100 L L1 for 12 h) used in Season 1 experiments, was applied 24 h after harvest, prior to 1MCP treatments. Control, lateharvested (LH) fruit ripened faster (8 d ) than early harvested (EH) fruit (10 d ), indicating that LH fruit were more physiologically mature and, therefore, more sensitive to ethylene. Avocados do not ripen while attached to the tree (Bower and

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168 Cutting, 1988) and exposure to ethylene is commercially used to promote uniform ripening of harvested fruit (Eaks, 1966). The sensitivity of avocados to ethylene increases with time after harvest (Gazit and Blumenfeld, 1970) and treatments up to 12 h after harvest are not enough to accelerate the ripening process when applied soon after harvest (Eaks, 1966; Zauberman et al., 1988). These results are consiste nt with other studies which report that lateharvested avocados ripened faster than early harvested fruit when treated with ethylene (Eaks, 1966; Jeong et al., 2002b). The concentrations used in the present study, 75 or 150 g L1 aqueous 1MCP and 75 or 150 nL L1 gaseous 1 MCP, show the efficacy of this product in low concentrations. Additionally, treatments from both 1MCP formulations effectively inhibited ethylene action even when applied within 48h delay after harvest, 12 h after a 12h pretreat ment with ethylene. Z hang et al. (2009 b ) has shown the capacity of 1MCP to delay ripening in tomato when applied 6 h after an ethylene exposure at 100 L L1 for 6 h. The capacity of 1 MCP to overcome the effects of an ethylene pretreat ment and significantly delay ripening suggests that the fruit requires a longer exposure to ethylene not only to trigger, but to fully develop ethylenedependent ripening processes as suggested by Zauberman et al. (1988) Also, since e thylene remains bound at the binding site for a few minutes (Sisler, 2006) and receptor degradation may occur after ethylene binding (Kevany et al., 2007; Binder, 2008) it is likely that new receptors were available for 1MCP at the time of application. In this study higher concentrations of 1MCP were more effective in EH than in LH fruit, possibly due to the more advanced maturity of LH fruit. While 1MCP can be effective at advanced stag es of fruit ripening (Huber, 2008), climacteric fruit like

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169 avocado have been reported to be more sensitive to 1 MCP at early stages of ripening (Jeong and Huber, 2004; Hurr et al., 2005; M a nenoi et al., 2007). The differential response of fruit to 1MCP treatments has important practical implications, indicating that, the longer the fruit stays on the tree, the shorter will be the days to ripe and the shorter the contribution of 1MCP for shelf life extension. The effect of harvest maturity at the time of treatment may be correlated to endogenous ethylene. In McIntosh apples, fruits with higher internal ethylene concentrations of 50 to 100 L L1 responded to 1 MCP, but ripening delays were shorter than those observed for fruit treated having lower internal ethylene concentration (Watkins et al., 2007). Retreatment of Bartlett pears with 1MCP after 4 weeks of storage was more effective than retreatment after 6 weeks of storage when fruit had higher ethylene production (Ekman et al., 2004). In tomato, higher levels of endogenous ethylene reduced influence of 1 MCP on ripening (Zhang et al., 2009 b ). In the present study, at the time of 1MCP application, ethylene production was 3 and 6 L kg1 h1 for EH and LH fruit, respectively and t he onset of the climacteric rise occurred earlier in the latter fruit. Since 1MCP inhibits ethylene action, it is possible that internal levels of ethylene prior to 1MCP treatments were sufficient to in duce advances in some ethylenedependent ripening processes and reduce the influence of 1MCP Softening is one the most affected ripening parameters in avocado, as shown in several studies ( Feng et al., 2000; Jeong and Huber, 2004; Adkins et al., 2005; Wo olf et al., 2005 ; Choi et al., 2008) The texture profile analysis performed in this study showed EH fruit treated with A150 or G150 had higher values for hardness of the 1st cycle ( Table 72), indicating that they were firmer than fruit from other treatment. An increase

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170 in food hardness increases the number of necessary chews and muscular work of the masticatory apparatus (Foster et al., 2006). This is consistent with the significant ly lower values observed for the second bite simulation for both A150 and G150, since the hardness of the first and the second bite simulations were negatively correlated ( data not shown). These two treatments also increased adhesiveness in EH fruit, indicating that the pulp of this fruit would adhered more in the mouth (Szczesniak, 2002). Interestingly, LH fruit treated with gaseous 1MCP were less cohesive and less gummy than EH fruit, indicating a pulp that breaks and disintegrates more easily (Szczesniak, 2002). Additionally, G150treated LH fruit were also less chewy and les s adhesive than EH fruit. Except for lower adhesiveness, these effects were not observed in LH fruit treated with aqueous formulation. Altogether, these results indicate differential effects of gaseous and aqueous formulations in texture parameters of LH avocado pulp and potential differences in texture due to harvest date. In the present study, both gaseous and aqueous formulations were similar in terms of delaying avocado ripening. Uniform application of treatments was carefully observed. Sorption of 1MC P by avocado tissues occurs quickly (Choi and Huber, 2009), but diffusion of 1MCP in tissues may be limited (Choi et al., 2008), involving tissue composition (Choi and Huber, 2009) and enzymic metabolism of 1MCP (Huber et al., 2010). In addition, the mor e advanced maturity of late harvested fruit may have influenced the results. The interaction of these factors and their effects on texture and sensory properties of avocado requires further investigation. Although 1MCP is very effective on delaying ripeni ng of several horticultural products, it has been reported to affect sensory acceptability of many horticultural crops (Watkins, 2008). However, t he results from these sensory analys e s show promise for

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171 commercial avocado packers and shippers since a single treatment with low concentrations of 1MCP extended shelf life without compromising sensory attributes. In this study, scores of Season 1EH fruit for texture, flavor and overall liking for all 1MCP treatments were similar or higher than control fruit. I n Anna apple, although 1MCP reduced aroma production, the fruit was still preferred over untreated control (Lurie et al., 2002; Pre Aymard et al., 2005). For Gala apples, overall liking scores of 1MCP treated fruit were not different from control (Marin et al., 2009). Application of 1MCP did not affected overall liking scores for Gala apple (Marin et al., 2009) and kiwi (Harker et al., 2008a), and aroma, color and firmness of mango (Cocozza et al., 2004). In this study, LH fruit in Season 1 had si gnificantly lower scores for texture when treated with A75 and G75, and for flavor when treated with A75. These differences were attributed to the fact that these fruit were overripe due to the 2day delay to conduct the sensory analysis. For EH fruit tre ated only with 1MCP (Season 2), scores of sensory attributes revealed that panelists did not distinguish between these and untreated control fruit. In LH fruit, A75 had no difference from control and only A150 in LH fruit was underscored for flavor and ov erall liking ( Table 74 ). According to panelists comments, only 5% complained about firm texture and bitterness, which can be perceived even in untreated fruit and can be a regular characteristic aftertaste of some avocado cultivars (Brown, 1972; Shaw et al., 1980). Additional sensory analysis conducted with lateharvested Simmonds (West Indian type) and Beta (GuatemalanWest Indian hybrid) avocado treated with aqueous 1 MCP showed no differences between control and treated fruit (Appendix C ).

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172 The use of the correct ripeness for consumption is of great importance for consumers perception of texture and flavor. According to the correlations performed on the combined data set from all experiments, these two attributes were highly positively correlated (0.70). Panelists that mostly disliked texture complained of pulp being mushy, while the preferred texture was referred to as creamy and often times as not too hard, not too soft. For flavor, many panelists complained about pulp as being bland, while positive comments emphasized the buttery and nutty flavor of avocado as being determinant. As with dairy products, in which fat content is a determinant for creaminess (Tournier et al., 2007), the perception of the higher oil content of avocado seemed to be a determinant for higher scores of texture and flavor. However, flavor was more strongly correlated to overall likeness (0.91) than texture (0.74). This result was contrary to that reported by Razeto et al. (2004) where acceptance of Hass avocado and 15 Hass x Bacon breeding program selections was better correlated with texture (0.80) than flavor (0.59) and oil content (0.34). This information is important to determine future research and marketing strategies. For example, the acceptability of blueberries was reported to be more correlated with flavor than texture (Saftner et al., 2008), but for apple (PreAymard et al., 2005; Harker et al., 2008b) and kiwi (Harker et al., 2008a), texture had a higher impact than flavor. Liking and purchase int ent of Hass avocados are strongly dependent on higher dry matter accumulation (Gamble et al., 2010), which is highly correlated with oil content (Lee et al., 1983). T he higher the fruit oil content, the richer the flavor (Storey et al., 1973; Lee et al., 1983; Woolf et al., 2004).

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173 T he differences in sensory scores due to gender were relatively weak but m ore determinant to final scores was how often the panelists eat avocado. The opposite result was reported for apples (Pneau et al., 2006), in which gender had a significant effect on scores for taste, aroma and freshness, but there were no differences in these attributes due to frequency of consumption. In that study, females gave higher scores than males in those attributes, as observed in the present study for avocado. However, for consumers that do not eat Gala, Fuji or Red Delicious apples, there was a preference for untreated rather than 1MCP treated fruit (Marin et al., 2009). For the present study, the subsets of data with the scores of each of the most representative groups of panelists those that never at e avocado (18 panelists) and those that eat once a month (23 panelists) were also analyzed separately to test their influence on final results. In all four experiments the means did not differ significantly among treatments for texture, flavor and overal l liking (data not shown). The only exception was for panelists who eat avocado once a month for scores given to LH fruit treated with 1MCP only (Season 2), where A150 had significantly lower scores than control for flavor and overall liking. These result s were similar to what was observed with the set of 75 panelists. The average scores of these subsets were similar to the overall average scores observed for the combined data set (Figure87B). Another test, made with subsets of panelists that eat avocado at least once a month (45.6%) and those that never ate or eat once a year (44.4%), also gave similar results to the first test of subsets (data not shown). Altogether, these tests indicate that there are no significant differences for avocados treated with 1 MCP for texture, flavor and overall liking when evaluated by panelists of same avocado eating habit.

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174 Conclusions Treatment with either aqueous (75 or 150 g L1) or gaseous (75 or 150 nL L1) 1 MCP effective ly delayed ripening by 4 to 10 d for EH and by 4 to 6 d for LH fruit without significant loss of visual quality or sensory attributes. Females gave slightly higher scores than males. Scores for texture, flavor and overall liking were higher with increased consumption of avocado by panelists, with m uch stronger correlation with sensory attributes than gender. However, tests indicated no significant differences among 1MCP treatments when evaluated by panelists with the same frequency of consumption of avocados. Postharvest application of gaseous or aqueous 1 MCP did not have negative effects on avocado fruit quality and, therefore, are suitable for use on early and lateharvested Booth 7 avocado.

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175 Table 71 Definitionz of Texture Profile Analysis TPA parameters analyzed for ripe mesocarp tiss ue withdrawn from mid segment of early (EH) and late harvested (LH) Booth 7 avocado fruit pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). Parameter Definition z Hardness 1 st cycle Force necessary to attain a given deformation in the 1 st bite simulation. Hardness 2 nd cycle Force necessary to attain a given deformation in the 2 nd bite simulation. Springiness R ate of return of the deformed tissue to the initial, undeformed condition Cohesiveness Extent to which a material can be deformed before it ruptures. Gumminess Energy required to disintegrate a semi solid food to a state ready for swallowing. Adhesiveness Work necessary to overcome the attractive forces between the surface of the food and the surface of other materials with which the food comes in contact. Chewiness E nergy required to masticate a solid food to a state ready for swallowing zAdapted from Szczesniak (2002) and Bourne (2004).

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176 Table 72 Texture profile analysis o f ripe mesocarp tissue taken from mid segment of early (EH) and lateharvested (LH) Booth 7 avocado fruit pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). Parameter Harvest Treatment z y Control A75 A150 G75 G150 Hardness 1st cycle (N) EH 1.80 bC 1.95 bC 2.90aA 2.24 aBC 2.62aAB LH 2.70 aA 2.67aA 3.16aA 2.77aA 2.56aA Hardness 2nd cycle (N) EH 0.46 aA 0.32aAB 0.21aB 0.46aA 0.17bB LH 0.40 aA 0.41aA 0.43aA 0.26aA 0.54aA Springiness EH 1.38 aA 0.61aA 0.07bA 0.52aA 0.35aA LH 1.19aA 0.35aA 0.32aA 0.22aA 0.37aA Cohesiveness EH 0.015 aA 0.005 aA 0.014aA 0.049aA 0.040aA LH 0.024aAB 0.015aA 0.040aAB 0.088bB 0.091bB Gumminess (N) EH 0.038aA 0.012aA 0.037aA 0.123aA 0.129aA LH 0.021aA 0.024aA 0.179aA 0.220bA 0.222bA Adhesiveness (mJ) EH 0. 58aB 0. 60aB 0. 26aA 0. 61aB 0. 25aA LH 0. 68aA 1.06bA 0. 95bA 0. 90bA 0. 94bA Chewiness (N*mm) EH 0 .267aA 0.024aA 0.005aA 0.122aA 0.112aA LH 0.495aA 0.008aA 0.005aA 0.035aA 0.075bA ZFor each parameter, v alues followed by the same small letter in a column or by the same capital letter in a row do not differ significantly according to Duncans Mul tiple Range Test ( P < 0.05). yU ntreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L-1 (A150) for 1 min at 20 C or exposed to gaseous 1MCP at 75 (G75) or 150 nL L-1 (G150) for 12 h at 20 C and stored at 20 C.

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177 Table 73 Average t exture, flavor and overall liking scores from 75 untrained panelists for early (EH) and late harvested (LH) Booth 7 avocado fruit pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). Treatment z Texture y x Flavor Overall Liking EH LH EH LH EH LH Control 5.63c 5.96 a 5.35b 5.77 a 5.27 b 5.68 a A 75 5.85bc 4.97 b 5.61ab 5.04 b 5.71ab 5.13a A 150 6.53a 5.64 a 6.01a 5.57 ab 5.95a 5.43 a G75 6.09abc 4.92 b 5.83ab 5.23 ab 5.84ab 5.09 a G150 6.29ab 5.64 a 5.72ab 5.47 ab 5.83ab 5.37 a zU ntreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L-1 (A150) for 1 min at 20 C or exposed to gaseous 1MCP at 75 (G75) or 150 nL L-1 (G150) for 12 h at 20 C and stored at 20 C. y9 point hedonic scale where 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely xValues followed by the same letter in a column do not differ significantly according to Duncans Multiple Range Test (P < 0.05). Table 74 Average t exture, flavor and overall liking scores from 75 untrained panelists for early (EH) and late harvested (LH) Booth 7 avocado fruit treated with 1 MCP only (Season 2). Treatment z Texture y x Flavor Overall Liking EH LH EH LH EH LH Control 5.64a 6.03a 5.12a 6.13a 5.35a 5.99a A 75 5.91a 6.29a 5.50a 5.93a 5.70a 6.09a A 150 5.76a 5.63a 5.42a 5.27b 5.62a 5.32b zUntreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L1 (A150) for 1 min at 20 C and stored at 20 C. y9 point hedonic scale where 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely xValues followed by the same small letter in a column do not differ significantly according to Duncans Multiple Range Test (P < 0.05). Table 75 Overall Pearson correlation coefficients between parameters of sensory analysis o f ripe Booth 7 avocado fruit based on combined scores for both seasons (2008 and 2009) and harvest times (early and lateharvest). Parameter z AEH y LIK TEX F LA GEN 0.05* 0.11** 0.07* 0.12** OFT 0.36** 0.29** 0.34** LIK 0.74** 0.91** TEX 0.70** zGender (GEN); avocado eating habit (AEH); Liking (LIK); Texture (TEX); Flavor (FLA). yNS, *, ** are, respectively, nonsignificant, P <0.05 and P <0.01.

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178 A B C Figure 71 Details of sensory analysis. A) Sample served to panelists; B) Individual booths; C) Recording scores in the computer. Pictures by Lorenzo A. Puentes.

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179 A B Figure 72 Whole fruit firmness in early (A) and lateharvested (B) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). U ntreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L1 (A150) for 1 min at 20 C or exposed to gaseous 1MCP at 75 (G75) or 15 0 nL L1 (G150) for 12 h at 20 C and stored at 20 C. Vertical bars represent standard error (n=4). Dotted line represents firmness threshold (15 N) for ripe fruit.

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180 A B Figure 73 Respiration in early (A) and lateharvested (B) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). U ntreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L1 (A150) for 1 min at 20 C or exposed to gaseous 1MCP at 75 (G75) or 150 nL L1 (G150) for 12 h at 20 C and stored at 20 C. Vertical bars r epresent standard error (n=4).

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181 A B Figure 74 Ethylene production in early (A) and lateharvested (B) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). U ntreated (control) or immersed in aqueous soluti on of 1 MCP at 75 (A75) or 150 g L1 (A150) for 1 min at 20 C or exposed to gaseous 1MCP at 75 (G75) or 150 nL L1 (G150) for 12 h at 20 C and stored at 20 C. Vertical bars represent standard error (n=4).

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182 A Figure 75 Weight loss in early (EH) a nd lateharvested (LH) Booth 7 avocado pretreated with ethylene 24 h after harvest at 100 L L1 for 12 h (Season 1). U ntreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L1 (A150) for 1 min at 20 C or exposed to gaseous 1MCP at 75 (G75) or 150 nL L1 (G150) for 12 h at 20 C and stored at 20 C. Vertical bars r epresent standard error (n=4).

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183 A B Figure 76 Overall frequency (A) and scores (B) given by panelists according to gender on texture, flavor and overall liking of ripe Booth 7 avocado fruit. Values are combined frequency and scores for both seasons (2008 and 2009) and harvest times (early and lateharvest). Each parameter was scored according to a 9 point hedonic scale where 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely.

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184 A B Figure 77 Overall frequency of eati ng avocado (A) and scores (B) given by panelists according to how often they eat avocado on texture, flavor and overall liking of ripe Booth 7 avocado fruit. Values are combined frequency and scores for both seasons (2008 and 2009) and harvest times (ear ly and lateharvest). Once a week (1/W); two to three times a month (23/M); once a month (1/M); once a year (1/Y); never. Each parameter was scored according to a 9 point hedonic scale where 1 = dislike extremely, 5 = neither like nor dislike and 9 = lik e extremely.

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185 CHAPTER 8 FINAL CONCLUSIONS Fruit ripening was strongly affected by postharvest application of aqueous 1MCP. 1 min treatments at 200 g L1 or above exaggerated the ripening asynchrony (stem end firmer than blossom end) of the fruit and hig her concentrations also led to significant shriveled appearance of the fruit and high decay severity, limiting shelf life. H owever, a 4 day post treatment with ethylene at 100 g L1 effectively promoted complete recovery from the strong ripening asynchrony caused by aqueous 1MCP treatment at 900 g L1 and might be an alternative to overcome this limitation. Pretreatment with ethylene to promote uniform ripening was more effective with longer delay to ethylene treatment (24 h) and longer exposure time of the fruit (24 h). However, aqueous 1MCP treatment of fruit pretreated with ethylene may increase variability of ripening within fruit. Fruit volatile profile was mostly composed of sesquiterpenes in maturegreen and mid ripe fruit, with caryophyllene as the most abundant compound, and total emissions are greatly reduced in ripe fruit. Hexanal was a major contributor in Simmonds and alkanes (decane, undecane, dodecane and tridecane) were an important group in Booth 7 and Monroe. The results of this study suggest that ethylene is involved in the regulation of esters in Simmonds and the sesquiterpene copaene in all cultivars studied, both reduced during ripening. Aqueous 1MCP at 75 or 150 g L1 for 1 min significantly increased the emission of alkanes in Booth 7 and Monroe, indicating that it may have favored this pathway in these two cultivars Total volatiles emission was not affected by ethylene or 1MCP in ripe fruit in Simmonds, but was higher in 1MCP treated ripe Booth 7 and Monroe due to higher emission of

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186 alkanes. Electronic nose analysis was successful to classify avocado pulp by ripeness stage of a single treatment, either untreated or treated with aqueous 1MCP at 75 or 150 g L1. However, discrimination of classes was not completely possible when the electronic nose was trained under a full model including all treatments and ripeness stages, increasing the chances of errors. Sensory attributes (flavor, texture and overall liking) of fruit were not significantly affected by either aqueous (75 or 150 g L1) or gaseous (75 or 150 nL L1) 1 MCP In summary, a single postharvest immersion in aqueous 1MCP (75 or 150 g L1 for 1 min ) effective ly extended postharvest quality and shelf life of West Indian and GuatemalanWest Indian hybrid avocados by 20% to 100% and did not affect ripe fruit quality Overall, stronger effects were observed for Guatemalan West Indian hybrids (Booth 7 and Monroe). 1MCP treatments were more ef fective in early harvested fruit or at 150 g L1. However, specific conditions must be developed and the commercial benefits of postharvest quality and shelf life extension must be evaluated for each cultivar. Besides the risk of exaggerated ripening asynchrony, t he use of high concentrations may excessively extend the ripening period and shelf life be limited by excessive weight loss or severe decay

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187 APPENDIX A VOLATILE COMPOUNDS D ETECTED IN SIMMONDS BOOTH 7 AND MO NROE

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188 Table A 1. Volatile compounds emitted by maturegreen (MG), mid ripe (MR) and ripe (R) Simmonds, Booth 7 and Monroe avocado (n=6) stored at 20 C, and respective descriptor s, odor threshold and relative abundance (%) in each ripeness stage for each cultivar (Phase 2 experiments). Relative abundance during ripening ( %) s Volatile compound Descriptorz Odor Threshold y Simmonds Booth 7 Monroe MG MR R MG MR R MG MR R ALCOHOLS 1 hexanol Flower F green F Cut grass R 500 x 2500 w 88.6 8.2 3.2 95.6 0.0 4.4 2 Methyl 1 butanol Wine F onion F green R ethe real R 300v 0.0 0.0 100.0 3 Methyl 1 butanol Whiskey F malt F,R burn F 250 x 0.0 0.0 100.0 0.0 0.0 100.0 3 Methyl 3 buten 1 ol Sweet 8.6 1.2 90.3 Cis 3 hexen 1 ol Fresh R green R grassy F,R 70 x 100 w 20.4 3.3 76.2 ALDEHY DES 2 Methyl 2 butenal Green F fruit F 0.0 0.0 100.0 Cis 3 hexenal Green F,R apple F banana R 0.25 x 24.9 10.3 64.7 70.0 17.7 12.3 Hexanal Green F,R fatty F,R grassy R 4.5 x 72.6 19.8 7.7 77.9 17.7 4.5 65.8 19.8 14.4 ALKA NES Decane Alkane F 48.1 39.5 12.3 40.6 32.8 26.6 Dodecane Alkane F 39.8 55.0 5.2 41.4 48.2 10.4 38.9 39.4 21.7 Tridecane Alkane F 39.7 50.8 9.5 40.4 42.2 17.5 Undecane Alkane F 51.5 38.1 10.5 41.6 33.8 24.5 ESTERS Ethyl crotonate 0.0 0.0 100.0 Ethyl isovalerate 0.0 75.8 24.2 Ethyl tiglate 65 u 0.0 0.0 100.0 Farnesyl acetate Green R floral R 67.9 32.1 0.0 61.0 39.0 0.0 Hexyl acetate fruity F,R herb F sweet R 10 w 92.8 7.2 0. 0 100.0 0.0 0.0 Hexyl butyrate Apple peel F 250 v 79.3 20.7 0.0 Methyl butyrate Ether F fruit F ,R sweet F,R 43 w 0.0 0.0 100.0 Methyl isovalerate 32.5 28.8 38.7 50.9 49.1 0.0 82.7 17.3 0.0

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189 Table A 1. Continued. Relative a bundance during ripening (%) s Volatile compound Descriptorz Odor Threshold y Simmonds Booth 7 Monroe MG MR R MG MR R MG MR R KETONES 3 pentanone Ether F 12.6 4.8 82.5 0.0 0.0 100.0 MONOTERPENES Limonene Citrus R fresh R 10 t 200 w 79.5 20.5 0.0 63.1 36.9 0.0 myrcene Balsamic F must F,R wet soil R 13 t 15 w 80.2 19.8 0.0 61.1 38.9 0.0 61.4 38.6 0.0 Cis ocimene Citrus F herb F ,R flower F sweet R 34 w 76.4 23.6 0.0 SESQUITERPENES Allo aromadendrene Wood F 44.5 55.5 0.0 52.2 47. 8 0.0 bergamotene Wood F warm F tea F 70.6 29.4 0.0 57.8 42.2 0.0 54.4 45.6 0.0 Cis bisabolene 53.4 46.6 0.0 bisabolene Balsamic F 71.6 28.4 0.0 54.1 45.9 0.0 cadinene Thyme F medicine F wood F 67.5 32.5 0.0 53.3 46.7 0.0 54.2 45 .8 0.0 caryophyllene 120 t 63.8 36.2 0.0 60.3 39.7 0.0 50.6 49.4 0.0 caryophyllene Wood F,R spice F,R 64 t 65.6 34.2 0.2 57.6 42.4 0.1 50.6 49.2 0.2 copaene Wood F spice F 75.9 24.1 0.0 58.8 41.2 0.0 54.5 45.5 0.1 cubebene Citrus F fruit F 60.1 39.9 0.0 47.0 48.8 4.2 cubebene Citrus F fruit F 62.7 37.2 0.1 53.6 46.4 0.0 45.6 54.4 0.0 elemene Wood F 61.2 38.8 0.0 elemene 59.1 40.9 0.0 39.7 60.3 0.0 farnesene Wood F citrus F sweet F 69.4 30.6 0.0 52.5 47.5 0.0 54.6 45 .4 0.0 Cis farnesene Citrus F green F 70.8 29.2 0.0 50.5 49.5 0.0 57.8 42.2 0.0 Germacrene B 57.5 42.5 0.0 54.7 45.3 0.0 muurolene Herb F wood F spice F 64.0 36.0 0.0 58.7 41.3 0.0 53.5 46.5 0.0 trans nerolidol Wood F,R flower F,R wax F 69 .1 30.9 0.0 selinene 58.3 39.2 2.4 sesquiphellandrene Wood F 68.7 31.3 0.0 55.5 44.5 0.0 51.1 48.9 0.0 Valencene Green F oil F woody R citrusy R 43.4 40.8 15.8 Ylangene 53.9 46.1 0.0

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190 Table A 1. Continued. Rela tive abundance during ripening (%) s Volatile compound Descriptorz Odor Threshold y Simmonds Booth 7 Monroe MG MR R MG MR R MG MR R Total alcohols (ALC) 62.1 6.3 31.7 3 4 4 0.0 65.6 0.0 0.0 100.0 Total aldehydes (ALD) 61.9 17.7 20.4 7 6.0 17.5 6.5 65.8 19.8 14.4 Total alkanes (ALK) 39.8 55.0 5.2 43.5 46.3 10.2 40.1 38.8 21.1 Total esters (EST) 86.7 12.0 1.3 45.3 31.8 22.9 11.4 2.4 86.2 Total ketones (KET) 12.6 4.8 82.5 0.0 0.0 100.0 0.0 0.0 0.0 Total monoterpenes (MOT) 78.5 21.5 0.0 61.6 38.4 0.0 61.4 38.6 0.0 Total sesquiterpenes (SQT) 69.0 30.9 0.1 56.3 43.7 0.0 52.1 47.6 0.3 TOTAL volatiles (TOV) 64.7 25.4 9.9 55.0 43.0 2.0 46.9 43.2 9.9 zAccording to Flavornet Database (F) (Acree and Arn 20 04) and the Citrus Research Educ ation Center Database (R) (Rouseff, 2010). yOdor threshold in water ( g kg-1) according to Buttery et al. (1971)(x), Du et al. (2010)(w) Takeoka et al. (1990)(v), Takeoka et al. (1992)(u) and Buttery et al. (1987)(t). sValues represent % of total emission rate observed for each ripeness stage during ripening of the cultivar

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191 APPENDIX B SEQUENCE OF QUESTION S ASKED TO PANELISTS DURING SENSORY ANALYSIS OF FRESH AVOCADO Question # 1. Please indicate your gender. Male Female Question # 2. Male: Please indicate your age range. Under 18 1829 3044 4565 Over 65 Question # 3. Female: Please indicate your age range. Under 18 1829 3044 4565 Over 65 Take a bite of cracker and a sip of water to rinse your mouth. Remember to do this before you taste each sample. WHEN ANSWERING A NY QUESTION, MAKE SURE THE NUMBER ON THE CUP MATCHES THE NUMBER ON THE MONITOR. Please click on the 'Continue' button below.

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192 Question # 4. Not including guacamole, how often do you eat avocados? Never Once a day 2 3 times a week Once a w eek 2 3 times a month Once a month Once a year Question # 5 Please indicate how much you like or dislike sample Overall Liking Dislike extremely Dislike very much Dislike moderately Dislike slightly Neither like nor dislike Like slight ly Like moderately Like very much Like extremely 1 2 3 4 5 6 7 8 9 Question # 6 Please indicate how much you like or dislike the Texture of sample Texture Dislike extremely Dislike very much Dislike mo derately Dislike slightly Neither like nor dislike Like slightly Like moderately Like very much Like extremely 1 2 3 4 5 6 7 8 9 Question # 7 Please indicate how much you like or dislike the Flavor of sa mple Flavor Dislike extremely Dislike very much Dislike moderately Dislike slightly Neither like nor dislike Like slightly Like moderately Like very much Like extremely 1 2 3 4 5 6 7 8 9 Question # 8 Using the keyboard located in the tray under the counter, please describe what you liked and/or disliked about the sample (please be specific)

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193 APPENDIX C SENSORY ANALYSIS OF SIMMONDS AND BETA Table C 1. Average texture, flavor and overall liking sc ores from 75 untrained panelists for Simmonds avocado fruit untreated (control) or immersed in aqueous solution of 1MCP at 75 (A75) or 150 g L1 (A150) for 1 min at 20 C and stored at 20 C. Treatment Texture z, y Flavor Overall liking Control 5.85a 5 .93a 5.84a A 75 6.15a 5.81a 5.79a A 150 6.12a 6.01a 5.99a z9 point hedonic scale where 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely yValues followed by the same small letter in a column do not differ significantly according to Duncans Multiple Range Test (P < 0.05). Table C 2. Average texture, flavor and overall liking scores from 75 untrained panelists for Beta avocado fruit untreated (control) or immersed in aqueous solution of 1 MCP at 150 g L1 (A150) for 1 min at 20 C and stored at 20 C. Treatment Texture z, y Flavor Overall liking Control 5.01a 4.71a 4.66a A 150 4.69a 4.57a 4.52a z9 point hedonic scale where 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely yValues followed by the same small letter in a column do not differ significantly according to Duncans Multiple Range Test (P < 0.05).

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213 BIOGRAPHICAL SKETCH Marcio Eduardo Canto Pereira was born in 1973 and raised in Rio de Janeiro, Brazil. He g raduated with a B.S. in agronomy in 1996 and received his masters degree in Plant Science in 1999, both at the Federal University of Vi osa, Brazil In October 2002, Marcio was hired by the Brazilian Agricultural Research Corporation Embrapa and started his professional car e er as a researcher in postharvest physiology and technology of tropical fruits at Embrapa Cassava & Tropical Fruit Crops, in Cruz das Almas, Bahia, Brazil. In 2006, Marcio started his doctoral program at the Horticultural Sciences Department of the University of Florida under the supervision of Steven A. Sargent. Upon graduation, Marcio plans to return to Brazil to continue his work as a researcher for Embrapa.