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Characterization of Citrus Peel Maturation and the Effect of Water Stress, Growth Regulators and Date of Harvest

Permanent Link: http://ufdc.ufl.edu/UFE0042512/00001

Material Information

Title: Characterization of Citrus Peel Maturation and the Effect of Water Stress, Growth Regulators and Date of Harvest
Physical Description: 1 online resource (472 p.)
Language: english
Creator: Alam-Eldein, Shamel Mohamed
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: citrus -- growth -- harvest -- maturity -- peel -- postharvest -- regulators -- senescence -- stress -- water
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 research is a different approach to understand the role of peel maturation as it relates to postharvest handling and keeping quality. Currently, citrus is harvested based on juice TSS: acid. Peel changes have not been related to best harvest time. Peel maturity is the physiological state of the peel that relates to maximum peel health and quality retention during handling, storage, and marketing. "Mature" peel would be expected to maintain longest postharvest peel quality, whereas "immature" or "senescent" peel would develop decay or disorders much sooner. Peel color, firmness, FDF, sugars, glycosidases, ABA, volatile components and juice TSS: acid were measured over harvest times to determine if peel maturation and senescence can be monitored by some combination of factors to minimize fruit disorders resulting from immature or senescent peel. WS and GR were studied to induce differences in maturity to see if any measurements show corresponding changes, so that they could be used to indicate stage of peel development. PCA, MSR and SR were used to obtain a broad picture about the peel maturity window, which related to common postharvest problems, weight loss, decay and CI that limit the storage life of fruit. Weight loss, decay and CI significantly related to days to harvest from bloom date, TSS: acid, FDF, firmness and color, which are practical candidates to predict optimal harvest time to reduce postharvest loss. TSS: acid was synchronized with peel storage characteristics, suggesting that pulp and peel maturity may be synchronized. Data suggested that the harvest window of ?Marsh? grapefruit for 40oF storage should start in December (265 DFB) at 8.7Kg FDF, and end by early April (383 DFB) at 6.4Kg FDF, while for 70oF storage, harvest should start by late November (256 DFB) and end by March (355 DFB). ?Valencia? orange harvest window was March (357 DFB) at 9.71Kg firmness for 40oF and 70oF storage and end by late May (443 DFB) for 40oF and by late April (412 DFB) for 70oF at 7.85Kg firmness. Thes harvest windows minimized unmarketable fruit. To extend marketing, fruit should be harvested, handeled, shipped and sold quickly with no storage.
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 Shamel Mohamed Alam-Eldein.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Albrigo, Leo G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042512:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042512/00001

Material Information

Title: Characterization of Citrus Peel Maturation and the Effect of Water Stress, Growth Regulators and Date of Harvest
Physical Description: 1 online resource (472 p.)
Language: english
Creator: Alam-Eldein, Shamel Mohamed
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: citrus -- growth -- harvest -- maturity -- peel -- postharvest -- regulators -- senescence -- stress -- water
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 research is a different approach to understand the role of peel maturation as it relates to postharvest handling and keeping quality. Currently, citrus is harvested based on juice TSS: acid. Peel changes have not been related to best harvest time. Peel maturity is the physiological state of the peel that relates to maximum peel health and quality retention during handling, storage, and marketing. "Mature" peel would be expected to maintain longest postharvest peel quality, whereas "immature" or "senescent" peel would develop decay or disorders much sooner. Peel color, firmness, FDF, sugars, glycosidases, ABA, volatile components and juice TSS: acid were measured over harvest times to determine if peel maturation and senescence can be monitored by some combination of factors to minimize fruit disorders resulting from immature or senescent peel. WS and GR were studied to induce differences in maturity to see if any measurements show corresponding changes, so that they could be used to indicate stage of peel development. PCA, MSR and SR were used to obtain a broad picture about the peel maturity window, which related to common postharvest problems, weight loss, decay and CI that limit the storage life of fruit. Weight loss, decay and CI significantly related to days to harvest from bloom date, TSS: acid, FDF, firmness and color, which are practical candidates to predict optimal harvest time to reduce postharvest loss. TSS: acid was synchronized with peel storage characteristics, suggesting that pulp and peel maturity may be synchronized. Data suggested that the harvest window of ?Marsh? grapefruit for 40oF storage should start in December (265 DFB) at 8.7Kg FDF, and end by early April (383 DFB) at 6.4Kg FDF, while for 70oF storage, harvest should start by late November (256 DFB) and end by March (355 DFB). ?Valencia? orange harvest window was March (357 DFB) at 9.71Kg firmness for 40oF and 70oF storage and end by late May (443 DFB) for 40oF and by late April (412 DFB) for 70oF at 7.85Kg firmness. Thes harvest windows minimized unmarketable fruit. To extend marketing, fruit should be harvested, handeled, shipped and sold quickly with no storage.
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 Shamel Mohamed Alam-Eldein.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Albrigo, Leo G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042512:00001


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1 CHAR A CTERIZATION OF CITRUS PEEL MATURATION AND THE EFFECT OF WATER STRESS, GROWTH REGULATORS AND DATE OF HARVEST By SHAMEL MOHAMED ALAM ELDEIN 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 2011

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2 2011 by Shamel Mohamed Alam -Eldein

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3 To my late father To my mother To my wife To my sons To my daughter To my brother To my sist er

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4 ACKNOWLEDGMENTS I would like to thank all persons who enriched my academic and personal experience through my program. I extend my appreciation to all friends at Citrus R esearch and Education Center in Lake Alfred, Florida for their friendship and support. I would like also to thank members of my supervisory committee during my master s study, who taught me what good scientist and teacher are. My great thanks to my beloved family, as well. My lovely late father, mother, brother, sister, wife, son s daughter, father in law and mother -in -law always encouraged me to follow my dream, and without their love and support, I w ould never be who I am today. My appreciation also goes to the Egyptian government for granting me a scholarship to obtain my Ph.D. f rom the University of Florida.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 9 L IST OF FIGURES ............................................................................................................................ 14 CHAPTER 1 LITERATURE REVIEW ........................................................................................................... 29 Fruit Development and Maturation ............................................................................................ 30 Maturity and Grade Standards .................................................................................................... 32 Maturity Standards (Internal Qualities) .............................................................................. 33 TSS, acidity, and TSS: acid rati o ................................................................................ 33 Grade Standards (External Qualities) ................................................................................. 34 Weight loss ................................................................................................................... 34 Deca y ............................................................................................................................ 36 Chilling injury (CI) ...................................................................................................... 37 Firmness / peel turgidity / turgor pressure .................................................................. 39 Fruit detachment force (FDF) ...................................................................................... 40 Peel color ...................................................................................................................... 41 Harvest Date ................................................................................................................................ 42 Storage Conditions ...................................................................................................................... 44 Water Stress ................................................................................................................................. 46 Growth Regulators ...................................................................................................................... 49 Sucrose and Reducing Sugars .................................................................................................... 55 Glycosidases ................................................................................................................................ 58 Abscisic Acid (ABA) .................................................................................................................. 61 Volatile Components ................................................................................................................... 64 2 CHARACTERIZATION OF CITRUS PEEL MATURATION AND THE EFFECT OF WATER STRESS, GROWTH REGULATORS AND DATE OF HARVEST ON POSTHARVEST LIFE OF THE FRUIT DURING STO RAGE ............................................. 70 Materials and Methods ................................................................................................................ 72 Field Experiment (Season 2004/2005), Effect of Harvest Date ....................................... 72 Field Experiment (Season 2005/2006), Effect of Harvest Date, Water Stress and Growth Regulators ........................................................................................................... 73 Field Experiment (Season 2006/2007), Effect of Harvest Date, Water Stress and Growth Regulators ........................................................................................................... 74 Storage Experiment (Season 2004/2005 and Season 2005/2006) .................................... 75 TSS, acidity, and TSS: acid ratio ................................................................................ 75 Peel color ...................................................................................................................... 75 Firmness / peel turgidity / turgor pressure .................................................................. 76

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6 Fruit detachment force (FDF) ...................................................................................... 76 Weight loss ................................................................................................................... 76 Decay & chilling injury (CI) ....................................................................................... 76 Statistical Analysis ............................................................................................................... 77 Results and Discussion ............................................................................................................... 77 Effect of Harvest Date and Storage Conditions ................................................................. 77 TSS, acidity, and TSS: acid ratio ................................................................................ 77 Peel color ...................................................................................................................... 78 Firmness / peel turgi dity / turgor pressure .................................................................. 79 Fruit detachment force (FDF) ...................................................................................... 79 Weight loss ................................................................................................................... 80 Decay ............................................................................................................................ 81 Chilling injury (CI) ...................................................................................................... 83 Effect of Water Stress and Growth Regulators .................................................................. 84 TSS, acidity, and TSS: acid ratio ................................................................................ 84 Peel color ...................................................................................................................... 86 Firmness / peel turgidity / turgor pressure .................................................................. 89 Fruit detachment force (FDF) ...................................................................................... 91 Weight loss ................................................................................................................... 94 Decay ............................................................................................................................ 96 Chilling injury (CI) ...................................................................................................... 99 Conclusion ................................................................................................................................. 101 3 CHARACTERIZATION OF CITRUS PEEL MAT URATION AND THE EFFECT OF WATER STRESS, GROWTH REGULATORS AND DATE OF HARVEST ON SUGAR CONTENT ................................................................................................................. 156 Materials and Methods .............................................................................................................. 157 Fie ld Experiments .............................................................................................................. 157 Storage Experiment (Season 2005/2006) ......................................................................... 157 Tissue Preparation for Chemical Analyses ...................................................................... 157 Soluble Sugars Extraction and Determination ................................................................. 157 Statistical Analysis ............................................................................................................. 158 Results and D iscussion ............................................................................................................. 158 Effect of Harvest Date ....................................................................................................... 158 Effect of Water Stress and Growth Regulators ................................................................ 161 Effect of Storage Conditions ............................................................................................. 167 Conclusion ................................................................................................................................. 170 4 CHARACTERIZATION OF CITRUS PEEL MATURATION AND THE E FFECT OF WATER STRESS, GROWTH REGULATORS AND DATE OF HARVEST ON INTERNAL LEVEL OF GLYCOSIDASES IN GRAPEFRUIT RIND ............................... 191 Materials and Methods .............................................................................................................. 192 Field Experiments .............................................................................................................. 192 Tissue preparation for chemical analyses ......................................................................... 192 Glycosidases Extraction .................................................................................................... 192

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7 Protein Analysis ................................................................................................................. 193 Galactosidase Activity ................................................................. 194 Enzymatic As -Mannosidase Activity .................................................................. 194 Statistical Analysis ............................................................................................................. 195 Results and Discussion ............................................................................................................. 195 Effect of Harvest Date ....................................................................................................... 195 Effect of Water Stress and Growth Regulators ................................................................ 198 Conclusion ................................................................................................................................. 202 5 CHANGES IN ABSCISIC ACID LEVELS IN MARSH GRAPEFRUIT PEEL IN RELATION TO MATURITY AND THE EFFECT OF WATER STRESS, GROWTH REGULATORS AND DATE OF HARVEST ........................................................................ 210 Materials and Methods .............................................................................................................. 211 Field Experiments .............................................................................................................. 211 Tissue Preparation for Chemical Analyses ...................................................................... 211 Abscisic Acid Extraction ................................................................................................... 212 ELISA Test Quantification ............................................................................................... 213 Statistical Analysis ............................................................................................................. 213 Results and Discussion ............................................................................................................. 214 Effect of Harvest Date ....................................................................................................... 214 Effect of Water Stress and Growth Regulators ................................................................ 216 Conclusion ................................................................................................................................. 220 6 CHARACTERIZATION OF CITRUS PEEL MATURATION AND THE EFFECT OF WATER STRE SS, GROWTH REGULATORS AND DATE OF HARVEST ON CITRUS OIL COMPONENTS ................................................................................................ 229 Materials and Methods .............................................................................................................. 230 Field Experiments .............................................................................................................. 230 Storage Experiment (Season 2005/2006) ......................................................................... 230 Oil Extraction ..................................................................................................................... 230 Determinatio n of Oil Components .................................................................................... 231 Statistical Analysis ............................................................................................................. 231 Results and Discussion ............................................................................................................. 231 Ketones ............................................................................................................................... 231 Harvest date ................................................................................................................ 232 Water stress and growth regulators ........................................................................... 233 Storage conditions ...................................................................................................... 235 Aldehydes ........................................................................................................................... 235 Harvest date ................................................................................................................ 235 Water stress and growth regulators ........................................................................... 236 Storage conditions ...................................................................................................... 237 Esters .................................................................................................................................. 238 Harvest date ................................................................................................................ 238 Water stress and growth regulators ........................................................................... 239 Storage conditions ...................................................................................................... 241

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8 Alcohols .............................................................................................................................. 242 Harvest date ................................................................................................................ 242 Water stress and growth regulators ........................................................................... 243 Storage conditions ...................................................................................................... 244 Terpenes ............................................................................................................................. 245 Harvest date ................................................................................................................ 245 Water stress and growth regulators ........................................................................... 247 Storage conditions ...................................................................................................... 251 Conclusion ................................................................................................................................. 252 7 CHARACTERIZING CITRUS PEEL MATURATION AND HANDLING PROBLEMS MULTIVARIATE AND REGRESSION ANALYSES .................................. 336 Materials and Methods .............................................................................................................. 338 Results and Discussion ............................................................................................................. 340 Marsh Grapefruit at 40oF (4.5oC) ................................................................................... 340 Marsh Grapefruit at 70oF (21oC) .................................................................................... 351 Valencia Orange at 40oF (4.5oC) .................................................................................... 360 Valencia Oranges at 70oF (21oC) ................................................................................... 366 Simple Regression vs. Multiple Regressions of Marsh Grapefruit and Valencia Orange ............................................................................................................................. 372 Marsh grapefruit and Valencia orange stored at 40oF ....................................... 373 Marsh grapefruit and Valencia orange stored at 70oF ....................................... 376 Combination of MSR and SR .................................................................................... 379 Conclusion ................................................................................................................................. 379 8 CONCLUSIONS ....................................................................................................................... 408 APPENDIX A TABLES .................................................................................................................................... 413 B FIGURES ................................................................................................................................... 419 LIST OF REFERENCES ................................................................................................................. 441 BIOGRAPHICAL SKETCH ........................................................................................................... 472

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9 LIST OF TABLES Table page 2 1 Changes in Marsh grapefruits TSS: acid ratio with harvest date over three seasons.. ................................................................................................................................ 105 2 2 Changes in Valencia ora nges TSS: acid ratio with harvest date over two seasons. .... 105 2 3 Changes in Marsh grapefruits color index with harvest date over three seasons ......... 105 2 4 Changes in Valencia oranges color index with harvest date over two seasons. .......... 106 2 5 Changes in Marsh grapefruits tissue turgidity (Kg) with harvest date over t hree seasons. ................................................................................................................................. 106 2 6 Changes in Valencia oranges tissue turgidity (Kg) with harvest date over two seasons. ................................................................................................................................. 106 2 7 Changes i n Marsh grapefruits detachment force (Kg) with harvest date over three seasons. ................................................................................................................................. 107 2 8 Changes in Valencia oranges detachment force (Kg) with harvest date over two seasons.. ................................................................................................................................ 107 2 9 Percentage weight loss of control Marsh grapefruit stored for 12 weeks at two temperatures over two seasons. ........................................................................................... 107 2 10 Percentage weight loss of control Valencia orange stored for 12 weeks at two temperatures over one season .............................................................................................. 108 2 11 Percentage decay of control Marsh grapefruit stored for 12 weeks at two temperatures over two seasons ............................................................................................ 108 2 12 Percentage decay of control Valencia orange stored for 12 weeks at two temperatures over one season .............................................................................................. 108 2 13 Percentage chilling injury of control Marsh grapefruit (two seasons) and Valencia orange (one season) stored for 12 weeks at 40oF .............................................................. 109 3 1 Effect of harvest date on the amount of of Marsh grapefruit peel during three seasons. ................................................................ 172 3 2 of Vale ncia orange peel during two seasons. ................................................................... 173 3 3 D.W.) of Marsh grapefruit peel during two seasons. ...................................................... 174

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10 3 4 D.W.) of Valencia orange peel during two seasons.. ...................................................... 175 3 5 / mg D.W.) of Valencia orange peel during two seasons. ............................................... 176 3 6 Effect of water stress, g rowth regulators and storage temperature on the amount of storage for 12 weeks at 40oF and 70oF in 2005/2006 season. ........................................... 177 3 7 Effect of water stress, growth regulators and storage temperature on the amount of for 12 weeks at 40oF and 70oF in 2005/2006 season. ........................................................ 178 3 8 Effect of soil coverage with Tyvek and storage temperature on the amount of sucrose weeks at 40oF and 70oF in 2005/2006 season.. .................................................................. 179 4 1 Effect of harvest date on the activity of glycosidases (units / g D.W.) of Marsh grapefruit peel during three seasons. ................................................................................... 203 4 2 Effect of water stress and growth regulators on the activity of glycosidases (units / g D.W.) of Marsh grapefruit peel during two seasons. ...................................................... 204 5 1 Effect of harvest date on ABA cont ent (mg / g D.W.) of March grapefruit peel during three seasons. ............................................................................................................ 221 5 2 Effect of water stress and growth regulators on ABA content (mg / g D. W.) of March grapefruit peel during two seas ons.. ..................................................................... 222 6 1 Changes of Marsh grapefruits nootkatone peak area (millions) with harvest date over three years. ................................................................................................................... 256 6 2 Effect of water stress and growth regulators on March grapefruits nootkatone peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.. ................................................................................................................... 257 6 3 Cha nges of Valencia oranges aldehydes peak area (millions) with harvest date over two years. ..................................................................................................................... 257 6 4 Effect of water stress and growth regulators on Valencia oranges aldehydes peak area (mi llions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.. ................................................................................................................... 258 6 5 Effect of soil coverage with Tyvek on Valencia oranges aldehydes peak area (millions) a t harvest over two years and during storage for 12 weeks during the 2005/06 season. .................................................................................................................... 259

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11 6 6 Changes of Marsh grapefruits geranyl acetate peak area (millions) with harvest date over three years. ........................................................................................................... 259 6 7 Changes of Valencia oranges geranyl acetate peak area (millions) with harvest date over two years. .............................................................................................................. 260 6 8 Effec t of water stress and growth regulators on March grapefruits geranyl acetate peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.. ................................................................................................. 260 6 9 Effect of water stress and growth regulators on Valencia oranges geranyl acetate peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.. ................................................................................................. 261 6 10 Effect of soil coverage with Tyvek on Valencia oranges geranyl acetate peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.. ................................................................................................................... 262 6 11 Changes of Marsh grapefruits linalool peak area (millions) with harvest date over three years. ............................................................................................................................ 262 6 12 Changes of Valencia oranges linalool peak area (millions) with harvest da te over two years. .............................................................................................................................. 263 6 13 Effect of water stress and growth regulators on March grapefruits linalool peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. .................................................................................................................... 263 6 14 Effect of water stress and growth regulators on Valencia oranges linalool peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.. ................................................................................................................... 264 6 15 Effect of soil coverage with Tyvek on Valencia oranges linalool peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/ 06 season. .................................................................................................................... 265 6 16 Changes of Marsh grapefruits terpenes peak area (millions) with harvest date over three years. ............................................................................................................................ 265 6 17 Changes of Valencia oranges terpenes peak area (millions) with harvest date over two years. .............................................................................................................................. 266 6 18 Effect of water stress and growth regulators on March grapefruits terpenes peak area ( millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. ` .................................................................................................................. 266

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12 6 19 Effect of water stress and growth regulators on Valencia oranges terpenes peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.. ................................................................................................................... 267 6 20 Effect of soil coverage with Tyvek on Valencia oranges terpenes peak area (million s) at harvest over two years and during storage for 12 weeks during the 2005/06 season. .................................................................................................................... 269 7 1 Stepwise regression of physical characteristics and chemical characteristics (except volatile co mponents) of Marsh grapefruit (3 rep. / treat.) harvested and stored at 40oF for 12 weeks during two different seasons.. .............................................................. 383 7 2 Stepwise regression of physical characteristics and chemical characteristics (with volatile components) of Marsh grapefruit (2 rep. / treat.) harvested and stored at 40oF for 12 weeks during two different seasons ................................................................ 384 7 3 Stepwise regression of physica l characteristics and chemical characteristics (except volatile components) of Marsh grapefruit (3 rep. / treat.) harvested and stored at 70oF for 12 weeks during two different seasons. ............................................................... 385 7 4 Stepwise regression of physical characteristics and chemical characteristics (with volatile components) of Marsh grapefruit (2 rep. / treat.) harvested and stored at 70oF for 12 weeks during two different seasons. ............................................................... 386 7 5 Stepwise regression of physical characteristics and chemical characteristics (with and without volatile components) of Valencia orange harvested and stored at 40oF and 70oF for 12 weeks during 2005/2006 season. ..................................................................... 387 7 6 Summary of physical and chemical characteristics that showed significant relationship with weight loss, decay and chilling injury in MSR of Marsh grapefruit and Valencia orange for storag e at 40oF and 70oF. ......................................................... 388 7 7 Comparison of simple regression (r and P value) of grapefruit, orange and combined data in regards to physical and chemical characteristics (with volatile component s) at 40oF for 12 weeks ............................................................................................................... 389 7 8 Comparison of simple regression (r and P value) of grapefruit, orange and combined data in regards to physical and chemical characteristics (with volatile com ponents) at 70oF for 12 weeks ................................................................................................................. 390 7 9 Summary of physical and chemical characteristics that showed significant relationship with post storage variables in MSR & SR and may be used to determine harvest date of Marsh grapefruit and Valencia orange for storage at 40oF and 70oF. ...................................................................................................................................... 391 A 1 Effect of water stress and growth regulators on chemical and physical characteristics of Marsh grapefruit during 2005/06 and 2006/07 seasons.. ........................................... 414

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13 A 2 Effect of water stress and growth regulators on chemical and physical characteristics of Valencia orange during 2005/06 and 2006/07 s easons.. ............................................ 415 A 3 Effect of soil coverage with Tyvek on chemical and physical characteristics of Valencia orange during 2005/2006 and 2006/2007 seasons.. ......................................... 417 A 4 Days between bloom date and harvest dates of Marsh grapefruit and Valencia orange in three seasons ......4 18

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14 LIST OF FIGURES Figure page 2 1 Changes in Marsh grapefruits TSS: acid ratio with harvest date over three seasons. 109 2 2 Changes in Valencia oranges TSS: acid ratio with harvest date over two seasons.. ... 110 2 3 Changes in Marsh grapefruits color index with harvest date over three seasons. ....... 110 2 4 Changes in Valencia oranges color index with har vest date over two seasons. .......... 111 2 5 Changes in Marsh grapefruits tissue turgidity (Kg) with harvest date over three seasons.. ................................................................................................................................ 111 2 6 Changes in Valencia oranges tissue turgidity (Kg) with harvest date over two seasons. ................................................................................................................................. 112 2 7 Changes in Marsh grapefruits fruit detachment force (FDF) (Kg) with harvest date ov er three seasons.. .............................................................................................................. 112 2 8 Changes in Valencia oranges fruit detachment force (FDF) (Kg) with harvest date over two seasons. .................................................................................................................. 113 2 9 Percentage weight loss of control Marsh grapefruit stored for 12 weeks at two temperatures over two seasons ............................................................................................ 113 2 10 Percentage weight loss of Marsh grapefruit during storage at 40oF for 12 weeks in 2004/2005 season. ................................................................................................................ 114 2 11 Percentage weight loss of Marsh grapefruit during storage at 70oF for 12 weeks in 2004/2005 season ................................................................................................................. 115 2 12 Percentage weight loss of Marsh grapefruit during storage at 40oF for 12 weeks in 2005/2006 season. ................................................................................................................ 116 2 13 Percentage weight loss of Marsh grapefruit during s torage at 70oF for 12 weeks in 2005/2006 season. ................................................................................................................ 117 2 14 Percentage weight loss of Valencia orange during storage at 40oF and 70oF for 12 weeks in 2005/2006 season ................................................................................................. 118 2 15 Percentage weight loss of Valencia orange during storage at 40oF for 12 weeks in 2005/2006 season. ................................................................................................................ 118 2 16 Percentage weight loss of Valenci a orange during storage at 70oF for 12 weeks in 2005/2006 season. ................................................................................................................ 119

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15 2 17 Percentage decay of control Marsh grapefruit stored for 12 weeks at two temperatures over two seasons ............................................................................................ 119 2 18 Percentage decay of Marsh grapefruit during storage at 40oF for 12 weeks in 2004/2005 season. ................................................................................................................ 120 2 19 Percentage decay of Marsh grapefruit during storage at 70oF for 12 weeks in 2004/2005 season. ................................................................................................................ 121 2 20 Percentage decay of Marsh grapefruit during storage at 40oF for 12 weeks in 2005/2006 season. ................................................................................................................ 122 2 21 Percentage decay of Marsh grapefruit during storage at 70oF for 12 weeks in 2005/2006 season. ................................................................................................................ 123 2 22 Percentage decay of Valencia orange during storage at 40oF and 70oF for 12 weeks in 2005/2006 season. ............................................................................................................ 124 2 23 Percentage decay of Valencia orange during storage at 40oF for 12 weeks in 2005/2006 season. ................................................................................................................ 124 2 24. Percentage decay of Valencia orange during storage at 70oF for 12 weeks in 2005/2006 season ............................................................................................................... 125 2 25 Percentage chilling injury o f control Marsh grapefruit (two seasons) and Valencia orange (one season) stored for 12 weeks at 40oF. .............................................................. 125 2 26 Percentage chilling injury of Marsh grapefruit during storage at 40oF for 12 weeks in 2004/2005 season ........................................................................................................... 126 2 27 Percentage chilling injury of Marsh grapefruit during storage at 40oF for 12 weeks in 2005/2006 season. ............................................................................................................ 127 2 28 Percentage chilling injury of Valencia orange during storage at 40oF for 12 weeks in 2005/2006 season ............................................................................................................. 128 2 29 Effect of field water stress and growth regulators t reatments on TSS: acid ratio of Marsh grapefruit at harvest during the 2005/2006 and 2006/2007 season. ................... 129 2 30. Effect of field water stress and growth regulators treatments on TSS: acid ra tio of Valencia orange at harvest during the 2005/2006 and 2006/2007 season. .................... 130 2 31 Effect of soil coverage with Tyvek on TSS: acid ratio of Valencia orange at harvest during the 2005/2006 and 2006/2007 season. .................................................................... 131 2 32 Effect of field water stress and growth regulators treatments on color index of Marsh grapefruit at harvest during the 2005/2006 and 2006/2007 seasons. ................. 132

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16 2 33 Effect of field water stress and growth regulators treatments on color index of Valencia orange at harvest during the 2005/2006 and 2006/2007 seasons. .................. 133 2 34 Effect of soil coverage with Tyvek on color index of Valencia orange during at harvest during the 2005/2006 and 2006/2007 seasons. ..................................................... 134 2 35 Effect of f ield water stress and growth regulators treatments on Marsh grapefruits tissue turgidity at harvest during the 2005/2006 and 2006/2007 seasons. ....................... 135 2 36 Effect of field water stress and gro wth regulators treatments on Valencia oranges tissue turgidity at harvest during the 2005/2006 and 2006/2007 seasons. ....................... 136 2 37 Effect of soil coverage with Tyvek on Valencia oranges tiss ue turgidity at harvest during the 2005/2006 and 2006/2007 seasons. .................................................................. 137 2 38 Effect of field water stress and growth regulators treatments on fruit detachment force of Marsh grapefruit at ha rvest during the 2005/2006 and 2006/2007 seasons. ... 138 2 39 Effect of field water stress and growth regulators treatments on fruit detachment force of Valencia orange at harvest during the 20 05/2006 and 2006/2007 seasons. .... 139 2 40 Effect of soil coverage with Tyvek on fruit detachment force of Valencia orange at harvest during the 2005/2006 and 2006/2007 seasons. ..................................................... 140 2 41 Changes in cumulative percentage weight loss of water stress and growth regulators treated Marsh grapefruit during storage at 70oF during the 2005/2006 season. ............ 141 2 42 Changes in cumulative percentage weight loss of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season. ............ 142 2 43 Changes in cumulative percentage weight loss of water stress and growth regulators treated Valencia orange during storage at 70oF during the 2005/2006 season. ............. 143 2 44 Change s in cumulative percentage weight loss of water stress and growth regulators treated Valencia orange during storage at 40oF during the 2005/2006 season. ............. 144 2 45 Effect of soil coverage with Tyvek on cumulative percentage weight loss of Valencia orange during storage at 70oF during the 2005/2006 season. ......................... 145 2 46 Effect of soil coverage with Tyvek on cumulative percentage weight loss of Valencia orange during storage at 40oF during the 2005/2006 season. ......................... 146 2 47 Changes in cumulative percentage decay of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season. ............ 147 2 48 Changes in cumulative percentage decay of water stress and growth regulators treated Marsh grapefruit during storage at 70oF duri ng the 2005/2006 season. ............ 148

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17 2 49 Changes in cumulative percentage decay of water stress and growth regulators treated Valencia orange during storage at 40oF during the 2005/2006 season. ............. 149 2 50 Changes in cumulative percentage decay of water stress and growth regulators treated Valencia orange during storage at 70oF during the 2005/2006 season. ............. 150 2 51 Effect of soil coverage with Tyvek on cumulative percentage decay of Valencia orange during storage at 40oF during the 2005/2006 season. ............................................ 151 2 52 Ef fect of soil coverage with Tyvek on cumulative percentage decay of Valencia orange during storage at 70oF during the 2005/2006 season. ............................................ 152 2 53 Changes in cumulative percentage chilling inju ry of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season. ................................................................................................................................... 153 2 54 Changes in cumulative percentage chilling injury of water stres s and growth regulators treated Valencia orange during storage at 40oF during the 2005/2006 season. ................................................................................................................................... 154 2 55 Effect of soil coverage with Tyvek on cumulative percentage chilling injury of V alencia orange during storage at 40oF during the 2005/2006 season. ......................... 155 3 1 Effect of harvest date of Marsh grapefruit on sucrose and reducing sugars content during 2004/2005 season.. ................................................................................................... 180 3 2 Effect of harvest date of Marsh grapefruit on sucrose and reducing sugars content during 2005/2006 season.. ................................................................................................... 181 3 3 Effect o f harvest date of Marsh grapefruit on sucrose and reducing sugars content during 2006/2007 season.. ................................................................................................... 182 3 4 Effect of harvest date of Valencia orange on sucrose and reducing sugars content during 2005/2006 season.. ................................................................................................... 183 3 5 Effect of harvest date of Valencia orange on sucrose and reducing sugars content during 2006/2007 season.. ................................................................................................... 184 3 6 Effect of field water stress and growth regulators treatments on sucrose and reducing sugars content of Marsh grapefruit peel during the 2005/2006 season. ......................... 185 3 7 Eff ect of field water stress and growth regulators treatments on sucrose and reducing sugars content of Marsh grapefruit peel during the 2006/2007 season. ......................... 186 3 8 Effect of field water stress a nd growth regulators treatments on sucrose and reducing sugars content of Valencia orange peel during the 2005/2006 season. .......................... 187

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18 3 9 Effect of field water stress and growth regulators treatme nts on sucrose and reducing sugars content of Valencia orange peel during the 2006/2007 season. .......................... 188 3 10 Effect of soil coverage with Tyvek on sucrose and reducing sugars content of Valenci a orange peel during the 2005/2006 season.. ...................................................... 189 3 11 Effect of soil coverage with Tyvek on sucrose and reducing sugars content of Valencia orange peel during the 2006/2007 season.. ...................................................... 190 4 1 -mannosidase activity during the 2004/2005 season. ................................................................................ 205 4 2 -galactosidase -mannosidase activity during the 2005/2006 season. ................................................................................ 206 4 3 -mannosidase activity during the 2006/2007 season. ................................................................................ 207 4 4 -mannosidase activity of Marsh grapefruit peel at harvest during the 2005/2006 season. ................... 208 4 5 galactosidase and mannosidase activity of Marsh grapefruit peel at harvest during the 2006/2007 season. ................................................................................................................................... 209 5 1 Effect of harvest date of Marsh grapefruit on ABA level of flavedo during three seasons.. ................................................................................................................................ 223 5 2 Effect of harvest date of Marsh grapefruit on ABA level of Al bedo during three seasons.. ................................................................................................................................ 224 5 3 Effect of field water stress and growth regulators treatments on ABA level in flavedo of Marsh grapefruit peel at harvest during the 2005/2006 season. .............................. 225 5 4 Effect of field water stress and growth regulators treatments on ABA level in albedo of Marsh grapefruit peel at harvest during the 2005/2006 season. ................................ 226 5 5 Effect of field water stress and growth regulators treatments on ABA level in flavedo of Marsh grapefruit peel at harvest during the 2006/2007 season. ................................ 227 5 6 Effect of field water stress and growth regulators treatments on ABA level in albedo of Marsh grapefruit peel at harvest during the 2006/2007 season. ................................ 228 6 1 Effect of harvest date o f Marsh grapefruit on nootkatone peak area (millions) during three seasons ............................................................................................................ 270 6 2 Effect of field water stress and growth regulators treatments on nootkatone of Marsh grapefruit peel dur ing the 2005/2006 season. ...................................................... 271

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19 6 3 Effect of field water stress and growth regulators treatments on nootkatone of Marsh grapefruit peel during the 2006/2007 season. ...................................................... 272 6 4 Changes in nootkatone of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season. ....................................... 273 6 5 Changes in nootkatone of water stress and growth regulators treated Marsh grapefruit during storage at 70oF during the 2005/2006 season. ....................................... 274 6 6 Effect of harvest date of Valencia orange on aldehydes during two seasons ............... 275 6 7 Effect of field water stress and growth regulators treatments on aldehydes of Valencia orange peel during the 2005/2006 season. ....................................................... 276 6 8 Effect of field water stress and growth regulators treatments on aldehydes of Valencia orange peel during the 2006/2007 season. ....................................................... 277 6 9 Eff ect of soil coverage with Tyvek on aldehydes of Valencia orange during the 2005/06 and 2006/07 seasons.. ............................................................................................ 278 6 10 Changes in aldehydes of water stress and growth regulators treated Valenci a orange during storage at 40oF during the 2005/2006 season.. ........................................... 279 6 11 Changes in aldehydes of water stress and growth regulators treated Valencia orange during storage at 70oF during the 2005/2006 season. ............................................ 280 6 12 Effect of soil coverage with Tyvek on aldehydes of Valencia orange during storage at 40oF and 70oF during the 2005/2006 season.. ................................................................ 281 6 13 Effect of harvest date of Marsh grapefruit on geranyl acetate during three seasons .. .. 282 6 14 Effect of harvest date of Valencia orange on geranyl acetate during two seasons.. ..... 283 6 15 Effect of field water stress and growth regulator treatments on levels of geranyl acetate of Marsh grapefruit at harvest during the 2005/2006 season. ............................ 2 84 6 16 Effect of field water stress and growth regulator treatment on levels of geranyl acetate of Marsh grapefruit at harvest during 2006/2007 season. .................................. 285 6 17 Effect of field water stress and growth regulator treatments on levels of geranyl acetate of Valencia orange at harvest during 2005/2006 season. ................................... 286 6 18 Eff ect of filed water stress and growth regulator treatments on levels of geranyl acetate of Valencia orange at harvest during 2006/2007 season. ................................... 287 6 19 Effect of soil coverage with Tyvek on geranyl acetate of Valencia orange during 2005/06 and 2006/07 seasons.. ............................................................................................ 288

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20 6 20 Changes in geranyl acetate of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season. ....................................... 289 6 21 Changes in geranyl acetate of water stress and growth regulators treated Marsh grapefruit during storage at 70oF during the 2005/2006 season. ....................................... 290 6 22 Changes in geranyl acetate of water stress and growth regulators treated Valencia oranges during storage at 40oF during the 2005/2006 season. .......................................... 291 6 23 Changes in geranyl acetate of water stress and growth regulators treated Valencia oranges during storage at 70oF during the 2005/2006 season. .......................................... 292 6 24 Changes in geranyl acetate of Tyvek treated Valencia oranges during storage at 40oF and 70oF in 2005/2006 season.. .................................................................................. 293 6 25 Effect of harvest date of Marsh grapefruit on linalool during three season s .. ............... 294 6 26 Effect of harvest date of Valencia orange on linalool during two seasons .. .................. 295 6 27 Effect of field water stress and growth regulators treatments on linalool of Marsh grapefruit peel during the 2005/2006 season. ..................................................................... 296 6 28 Effect of field water stress and growth regulators treatments on linalool of Mars h grapefruit peel during the 2006/2007 season. ..................................................................... 297 6 29 Effect of field water stress and growth regulators treatments on linalool of Valencia orange peel during the 2005/2006 season. .......................................................................... 298 6 30 Effect of field water stress and growth regulators treatments on linalool of Valencia orange peel during the 2006/2007 season. .......................................................................... 299 6 31 Effect of soil coverage with Tyvek on linalool of Valencia orange peel during the 2005/06 and 2006/07 seasons.. ............................................................................................ 300 6 32 Changes in linalool of water stress and growth regulator s treated Marsh grapefruit after 12 weeks storage at 40oF during the 2005/2006 season. ........................................... 301 6 33 Changes in linalool of water stress and growth regulators treated Marsh grapefruit after 12 weeks storage at 70oF during the 2005/2006 season. ........................................... 302 6 34 Changes in linalool of water stress and growth regulators treated Valencia oranges after 12 weeks storage at 40oF during the 2005/ 2006 season. ........................................... 303 6 35 Changes in linalool of water stress and growth regulators treated Valencia oranges after 12 weeks storage at 70oF during the 2005/2006 season.. .......................................... 304 6 36 Effect of soil coverage with Tyvek on linalool of Valencia orange peel after storage at 40oF and 70oF during the 2005/2006 season.. ................................................................ 305

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21 6 37 -pinene in Marsh grapefruits peel oil during three seasons .. ................................................................................................................................ 306 6 38 Effect of harvest date on myrcene in Marsh grapefruits peel oil during three seasons .. ................................................................................................................................ 307 6 39 Effect of harvest date on terpenes in Valencia oranges peel oil during the 2005/06 season.. .................................................................................................................................. 308 6 40 Effect of harvest date on terpenes in Valencia oranges peel oil during the 2006/07 season.. .................................................................................................................................. 309 6 41 -pinene of Marsh grapefruit peel oil during the 2005/2006 season. ............................................................... 310 6 42 Effect of field water stress and growth regulator s treatments on myrcene of Marsh grapefruit peel oil during the 2005/2006 season.. .............................................................. 311 6 -pinene of Marsh grapefruit p eel oil during the 2006/2007 season. ............................................................... 312 6 44 Effect of field water stress and growth regulators treatments on myrcene of Marsh grapefruit peel oil during the 2006/2007 season.. .............................................................. 313 6 45 Effect of field water stress and growth regulators treatments on a -pinene of Valencia orange peel oil during the 2005/2006 season.. ................................................. 314 6 46 Effect of field water stress and growth regulators treatments on myrcene of Valencia orange peel oil during the 2005/2006 season.. ................................................. 315 6 47 Effect of field water stress and growt h regulators treatments on valencene of Valencia orange peel oil during the 2005/2006 season. .................................................. 316 6 48 -pinene of Valencia orange peel oil during the 2006/2007 season. .................................................. 317 6 49 Effect of field water stress and growth regulators treatments on myrcene of Valencia orange peel oil during the 2006/2007 season. .................................................. 318 6 50 Effect of field water stress and growth regulators treatments on valencene of Valencia orange peel oil during the 2006/2007 season. .................................................. 319 6 51 -pinene of Valencia orange peel oil during the 2005/06 and 2006/07 seasons.. ...................................................................................... 320 6 52 Effect of soil coverage with Tyvek on myrcene of Valencia orang e peel oil during the 2005/06 and 2006/07 seasons.. ...................................................................................... 321

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22 6 53 Effect of soil coverage with Tyvek on valencene of Valencia orange peel oil during the 2005/06 and 2006/07 seasons. ....................................................................................... 322 6 54 -pinene of water stress and growth regulators treated Marsh grapefruit during storage at 70oF for 12 weeks during the 2005/2006 season. ................................. 323 6 55 Changes in myrcene of water stres s and growth regulators treated Marsh grapefruit during storage at 70oF for 12 weeks during the 2005/2006 season. ................................. 324 6 56 -pinene of water stress and growth regulators treated Marsh grapefruit during storage at 40oF for 12 weeks during the 2005/2006 season. ................................. 325 6 57 Changes in myrcene of water stress and growth regulators treated Marsh grapefruit during storage a t 40oF for 12 weeks during the 2005/2006 season. ................................. 326 6 58 -pinene of water stress and growth regulators treated Valencia oranges during storage at 40oF for 12 weeks during the 2005/2006 season. ................................. 327 6 59 Changes in myrcene of water stres s and growth regulators treated Valencia oranges during storage at 40oF for 12 weeks during the 2005/2006 season. ................................. 328 6 60 Changes in valencene of water stress and growth regulators treated Valencia oranges during storage at 40oF for 12 weeks during the 2005/2006 season. ................... 329 6 61 -pinene of water stress and growth regulators treated Valencia oranges during storage at 70oF for 12 weeks during the 2005/2006 season. ................................. 330 6 62 Changes in myrcene of water stres s and growth regulators treated Valencia oranges during storage at 70oF for 12 weeks during the 2005/2006 season. ................................. 331 6 63 Changes in valencene of water stress and growth regulators treated Valencia oranges during storage at 70oF for 12 weeks during the 2005/2006 season. ................... 332 6 64 -pinene of Valencia orange peel during storage at 40oF and 70oF for 12 weeks during the 2005/2006 season.. ............................ 333 6 65 Effect of soil coverage with Tyvek on myrcene of Valencia orange peel during storage at 40oF and 70oF for 12 weeks during the 2005/2006 season. ............................. 334 6 66 Effect of soil coverage with Tyvek on valencene of Valencia orange pee l during storage at 40oF and 70oF for 12 weeks during the 2005/2006 season. ............................. 335 7 1 Principal component analysis of Marsh grapefruit control fruit harvested in 2004/2005 season and stored fo r 12 weeks at 40 F including all variables except volatile components.. ........................................................................................................... 392 7 2 Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 40 F including all variables except volatile components.. ... 393

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23 7 3 Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 40 F including all variable s except volatile components. ........... 394 7 5 Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 40 F including all variables. ................................................... 396 7 6 Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 40 F including all variables. .......................................................... 397 7 7 Principal component analysis of Marsh grapefruit control fruit harvested in 2004/2005 season and stored for 12 weeks at 70 F including all variables except volatile components. ............................................................................................................ 398 7 8 Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 70 F including all variabl es except volatile components. ... 399 7 9 Principal componen t analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 70 F including all variables except volatile components. ........... 400 7 10 Principal component analysis of Marsh g rapefruit control fruit harvested in 2004/2005 season and stored for 12 weeks at 70 F including all variables. .................... 401 7 11 Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 70 F including all variables.. .................................................. 402 7 12 Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 70 F including all variables. .......................................................... 403 7 13 Principal component analysis of Valencia orange harvested in 2005/2006 season and stored for 12 weeks at 40 F including all variables except volatile components.. ... 404 7 14 Principal component analysis of Valencia orange harvested in 2005/2006 season and stored for 12 weeks at 40 F including all variables. ................................................... 405 7 15 Principal component analysis of Valencia orange harvested in 2005/2006 season and stored for 12 weeks at 70 F including all variables except volatile components. .... 406 7 16 Principal component analysis of valencia orange harvested in 2005/2006 season and stored for 12 weeks at 70 F including all variables. ................................................. 407 B1 Monthly Temperatures (F) of winter ha ven area from Sept 2003 to Sept 2007. ............ 420 B2 Monthly Precipitation (inches) of Winter Haven area from Sept 2003 to Sept 2007. ..... 420 B3 Changes in peel sugar content of water stress and growth regulators treated Marsh grapefruit at harvest and after 12 weeks storage at 40oF and 70oF during the 2005/2006 season.. ............................................................................................................... 421

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24 B4 Changes in peel sugar content of water stress and growth regulators treated Valencia orange at harvest and after 12 weeks storage at 40oF and 70oF during the 2005/2006 season.. ............................................................................................................... 422 B5 Ef fect of soil coverage with Tyvek on sugar content of Valencia orange peel at harvest and after 12 weeks storage at 40oF and 70oF during the 2005/2006 season.. ..... 423 B6 Effect of water stress and growth regulators on ABA level of Marsh grapefruit peel during 2005/2006 and 2006/2007 seasons.. ........................................................................ 424 B7 Principal component analysis of Marsh grapefruit harvested in 2004/2005 season and stored for 12 weeks at 40OF using 17 physical and chemical charcteristcs (without volatile components). ............................................................................................ 425 B8 Principal component analysis of Marsh grapefruit harvested in 2005/2006 se ason and stored for 12 weeks at 40OF using 17 physical and chemical charcteristcs (without volatile components). ............................................................................................ 426 B9 Principal component analysis of Marsh grapefruit harvested over 2 sea sons and stored for 12 weeks at 40OF using 17 physical and chemical charcteristcs (without volatile components). ........................................................................................................... 427 B10 Principal component analysis of Marsh grapefruit harvested in 2004/2005 season and stored for 12 weeks at 40OF using 22 physical and chemical charcteristcs (with volatile components). ........................................................................................................... 428 B11 Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 40OF using 22 physical and chemical charcteristcs (with volatile components). ........................................................................................................... 429 B12 Principal component analysis of Marsh grapefruit harvested ove r 2 seasons and stored for 12 weeks at 40OF using 22 physical and chemical charcteristcs (with volatile components). ........................................................................................................... 430 B13 Principal component analysis of Marsh grapefruit harvested in 2004/2005 season and stored for 12 weeks at 70OF using 16 physical and chemical charcteristcs (without volatile components) ............................................................................................. 431 B14 Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 70OF using 16 physical and chemical charcteristcs (without volatile components) ............................................................................................. 432 B15 Principal component analysis of Marsh grapefruit harve sted over 2 seasons and stored for 12 weeks at 70OF using 16 physical and chemical charcteristcs (without volatile components) ............................................................................................................ 433

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25 B16 Principal component analysis of Marsh grapefruit harve sted in 2004/2005 season and stored for 12 weeks at 70OF using 21 physical and chemical charcteristcs (with volatile components) ............................................................................................................ 434 B17 Principal component analysis of Marsh grapefruit har vested in 2005/2006 season and stored for 12 weeks at 70OF using 21 physical and chemical charcteristcs (with volatile components) ............................................................................................................ 435 B18 Principal component analysis of Marsh grapefruit h arvested over 2 seasons and stored for 12 weeks at 70OF using 21 physical and chemical charcteristcs (with volatile components) ............................................................................................................ 436 B19 Principal component analysis of Valencia orange harvested over in 2005/2006 season and stored for 12 weeks at 40OF using 11 physical and chemical charcteristcs (without volatile components) ............................................................................................. 437 B20 Principal component analysis of Valencia ora nge harvested over in 2005/2006 season and stored for 12 weeks at 40OF using 17 physical and chemical charcteristcs(with volatile components). ............................................................................ 438 B21 Principal component analysis of Valenci a orange harvested over in 2005/2006 season and stored for 12 weeks at 70OF using 10 physical and chemical charcteristcs (without volatile components). ............................................................................................ 439 B22 Principal component analysis of Valencia orange harvested over in 2005/2006 season and stored for 12 weeks at 70OF using 16 physical and chemical charcteristcs (with volatile components) .................................................................................................. 440

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26 LIST OF ABBREVIATIONS ABA Abscisic A cid ANOVA Analysis of Variances AOAC Association of Official Analytical Chemists CI Chilling Injury CONT Control CREC Citrus Research and Education Center DFB Days from Bloom DW Dry Weight FDF Fruit Detachment Force GA3 Gibberellic Acid GC -MS Gas Chromatography Mass Spectro metry GR Growth Regulators GR*WS Growth Regulators and Water Stress MSR Multiple Stepwise Regression PCA Pricncipal Component Analysis RH Relative Humidity SAS Statistical Analysis System SPME Solid Phase Micro Extraction SR Simple Regression TSS Total Soluble Solids TSS: acid ratio Total Soluble Solids to total acids ratio WS Water Stress 2, 4 D 2 4 Dichlorophenoxyacetic acid w Water potential

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27 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillme nt of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF CITRUS PEEL MATURATION AND THE EFFECT OF WATER STRESS, GROWTH REGULATORS AND DATE OF HARVEST By Shamel Mohamed Alam Eldein Decmber 2011 Chair: Leo Gene Albrigo Major: Hor ticultural Sciences This research is a different approach to understand the role of peel maturation as it relates to postharvest handling and keeping quality. Currently, citrus is harvested based on juice TSS: acid Peel changes have not been related to b est harvest time. P eel maturity is the physiological state of the peel that relates to maximum peel health and quality retention during handling, storage, and marketing M ature peel would be expected to maintain longest post harvest peel quality whereas immature or senescent peel would develop decay or disorders much sooner. Peel color, firmness F DF sugars, glycosidases, ABA, volatile components and juice TSS: acid were measured over harvest times to determine if peel maturation and senescence can be monitored by some combination of factors to minimize fruit disorders resulting from immature or senescent peel. WS and GR were studied to induce differences in maturity to see if any measurements show corresponding changes, so that they could be used to indicate stage of peel development. PCA, MSR and SR were used to obtain a broad picture about the peel maturity window which related to common postharvest problems, weight loss, decay and CI that limit the storage life of fruit W eight loss, decay and CI significant ly related to days to harvest from bloom date, TSS: acid FDF, firmness and color which are practical candidates to predict optimal harvest time to

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28 reduce postharvest loss TSS: acid was synchronize d with peel storage characteristics, suggest ing that pulp and peel maturity may be synchronized. Data suggested that the harvest window of Marsh grapefruit for 40oF storage should start in December ( 265 DFB ) at 8.7Kg FDF, and end by early April ( 383 DFB ) at 6.4Kg FDF, while for 70oF storage harve st should start by l ate November (256 DFB ) and end by March (35 5 DFB ). Valencia orange harvest window was March ( 357 DFB ) at 9.71Kg firmness for 40oF and 70oF storage and end by late May ( 443 DFB ) for 40oF and by late April ( 412 DFB ) for 70oF at 7.85Kg firmness Thes harvest window s minimized unmarketable fruit T o extend marketing fruit sho u ld be harvested, handeled, shipped and sold quickly with no storage.

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29 CHAPTER 1 LITERATURE REVIEW The word citrus is derived from a reference to the citron in Pliny s Natural History. The Latin cedrus comes from the Greek kedrus which refers to the kedromelon or cedar apples (citron) used by Greek Palestinians during the Jewish Feast of the Tabernacles (Jahoda, 1976). Citrus fruits are members of the Rue family, Rutaceae, and include orange, grapefruit, lemon, lime, tangerine and mandarin, pummelo, and kumquat. It is generally accepted that pummelo, mandarin, and c itron are the founding true species of all citrus varieties existing today ( Spiegel Roy and Goldschmidt, 1996). The two targeted cultivars in this study were Marsh grapefruit Citrus paradisi Macfadyen, and Valencia orange Citrus sinensis (L.) Osbeck (Hodgson, 1967) which are common two cultivars in Florida citrus industry that represent long harvest season and short harvest season, respectively Marsh grapefruit was brought to Florida from the West Indies in 1810 and Valencia orange was brought from Europe (mainly Spain) in 1800 (Webber et al., 1967). Citrus fruit rank first in their contribution of Vitamin C to human nutrition, worldwide (Nagy, 1980) Botanically, citrus fruit are classified as a hesperidium a particular kind of berry with a leathery rind and divided internally into segments. The rind has two components: the pigmented part, called the flavedo (cuticle -covered epidermis contains plastids and several colored subepidermal layers contain oil glands), and the whitish sp ongy part, called the albedo (thin -walled parenchyma cells with numerous large air spaces) The segments, internal to rind, are filled with juice sacs and form the juicy, edible part of the fruit ( Swingle, 1943). Citrus are second only to grapes in planting and production of perennial fruits worldwide (Spiegel Roy and Goldschmidt, 1996). The importance of citrus in world commerce has increased t remendously since the early 1900s. Modern communications, consumer awareness of

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30 high quality citrus, and improved produce transportation methods have also resulted in expansion of production from origins in Southeastern Asia to most of the worlds subtropi cal and tropical areas in countries with suitable climates for citrus culture (Braddock, 1999). Citrus is commercially grown primarily in regions with mild winters located between 20o and 40o of latitude in both hemispheres. More than 70% of the world total production of citrus for processing is in humid subtropical climates in Brazil, mostly S ao Paulo and the United States, mostly Florida, which are characterized by cool and dry winters (McKnight and Hess, 2000 ). Brazil and the United States are the largest orange, processing producers with over 85% of world production. The United States is the largest grapefruit, mostly Florida, and lemon producers. China produces the most tangerines (Braddock, 1999). Commercial production of citrus fruits in the United States is limited to Arizona, California, Florida Texas, Louisiana and Alabama. The fresh fruit production in the United States is about 1 0% of the worlds production of lemon, 14% of its oranges and 21% of its grapefruit. Florida is the leading U. S. producer of citrus fruit, most of which (about 95%) is processed (FAO, 2010; USDA NASS, 2010) Most of California citrus fruit are marketed fr esh. Minimum maturity requirements of citrus fruit are based on juice content (lemon and lime) or soluble solids content, titratable acidity, and the ratio of the two (orange, grapefruit and tangerine) ( Kader and Arpaia, 2002). Fruit Development and Maturation The peculiar structure of a citrus fruit, a hesperidium with clear differentiation between a thin colored exocarp and oute r mesocarp, a thick spongy inner mesocarp, and a juicy endocarp composed of juice sacs enclosed in a membrane to form the segments (i.e. not tissues in the accepted meaning of the term), have a special interest for scientists to study fruit growth, maturat ion, and postharvest senescence. There are three fairly well -defined periods of growth and development; stage 1, a period of cell division; stage 2, a period of cell enlargement; and

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31 stage 3, a period of compositional changes, mainly increase in sugars and aromatic compounds with color changes and a decrease in organic acid (Bain, 1958). The growth pattern of the peel is differ ent from that of the pulp, and the first stage of cell division can continue in the epidermal cells until close to fruit maturation ( Scott and Baker, 1947). Contrarily, Coggins (1969b ) stated that peel senescence sometimes begins before fruit has attained internal maturity. Also, it has been found that respiration rate of the citrus peel is considerably higher than that of the pulp, but tends to decrease after harvest while that of the rest of the fruit remains constant ( Vakis et al., 1970). Citrus fruits are non climacteric: they do not ripen further once they have been removed from the tree Thus, it is important that they are picked at the right stage of maturity. Orderly marketing requires legal standards for maturity (internal qualities) and grade standards (external qualities) of fruit in the various citrus production areas of th e world which are necessary to prevent the harvest and shipment of immature fruit and shipment of unwholesome fruit (Grierson, 2006b ). The m aturity of citrus fruit is measured primarily by different internal characteristics such as total soluble solids (TSS), acidity, TSS: acid ratio, and juice content (UNCTAD, 2004), plus a color break requirement particularly for early maturing cultivars (USDA, 1957; USDA, 1969b ). Like other nonclimacteric fruit, citrus fruit do not possess a well -defined ripening mechanism as do apple, pear, banana, mango and avocado, which are climacteric fruits (Grierson, 1973). Citrus also lacks a well -defined abscission period T hus they are typically harvested a s a matter of human discretion rather than a naturally imposed physiological ne cessity (Grierson and Ting, 1978). So, using the term ripening with citrus is erroneous because there is no starch, oil, etc, present in citrus fruit during maturation capable of being converted to sugars

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32 or other soluble products. Instead, they gradually become edible and mature to good eating quality, and remain so on the tree over a period that may last 1 2 months for tangerines, 45 months for Valencia orange and up to 6 7 months for grapefruit. The dates and duration of citrus harvest are related to the tim ing of onset of physiological changes, principally increase in sugar, decrease in acid, and the amount of juice. These changes are expressed most sharply in the TSS: acid ratio, which is commonly called a maturity index (Grierson, 2006a ). The inedible portion of the fruit, the peel or exocarp, also matures. Although many compositional changes occur in the peel, maturity in this tissue often refers to susceptibility of the peel to injury, which will be reflected i n the storability and marketability of the fruit. Th e point at which citrus peel passes from an immature to a mature state, as well as from mature to senescent ha s been difficult to define, in part, because of the gradual and slower nature of physiological changes in this tissue H owever, physiological chan ges (e.g. enzyme activity, polyamines, volatile components etc) that occur during maturation and mature to senescent transition periods could be used to define peel maturity more accurately ( Burns and Baldwin, 1994). So far, no maturity indices for citrus peel exist because no one has sufficiently defined t he changes of the citrus peel might serve as maturity indices. The purpose of this chapter is to review some peel changes that characterize citrus peel m aturation and senescence, and some factors or conditions that might affect citrus peel maturity. Maturity and Grade Standards Maturity standards usually refer to the internal quality of the fruits: TSS, acidity, TSS: acid ratio, and juice volume, which pr ovide an objective measure of palatability. Grade standards refer to the external quality, such as color, texture, firmness, varietal characteristics and be free dom from bruises, scars, un-healed cuts, decay, C hilling injury, freezing injury, rind staining, pitting, and growth cracks that provide an objective measure of appearance. Appearance is the

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33 chief quality influencing consumers initial selection of fruits, and palatability is important in determining subsequent sales. So, the only real basis of maturity and grade standards is clear to be economics (Grierson, 2006b; Taylor et al., 1994) Geographical influences, seasonal weather conditions, rootstocks, cultural practices, and tree age all have profound effects on external and internal fruit qualities wherever citrus is grown (Grierson, 2006b ). Maturity Standards (Internal Qualities) The purpose of this sub-section is to review only the maturity parameters related to this work, which are TSS, a cidity and TSS: acid ratio. TSS, acidity, and TSS: acid ratio TSS generally increases slightly from mo nth to month as the acidity decreases during the earlier stages of maturation. After maturation, there is a tendency for the TSS to remain more or less constant, but very late in the season it frequently decreases (Harding and Fisher, 1945). These changes in juice composition take place more slowly in grapefruit than in oranges ( Chace and Church, 1924 ). The warmer climates of tropical and humid subtropical areas produce earlier maturi ng fruit with lower acidity than in cooler cold winter subtropical area (Rouse and Zekri, 2006). Fruits from the upper and exterior ca nopy sections exposed to the sun, especially the southern exposure in the northern hemisphere, have more soluble solids and lower acid content than that from other canopy sectors ( Fallahi et al., 1989 ; Kender and Hartmond, 1999; Syvertsen and Albrigo, 1980), because leaves from the canopy exterior hav e higher CO2 assimilation rates than interior leaves (Fallahi and Moon, 1988). During postharvest storage, TSS: acid ratio of citrus fruit increases very slightly because the fruit decrease in acidity using acids as substrates in respiration (T ri carboxyl ic acids cycle) (Rana and Sing, 1992; Samson, 1986). The higher the percentage of acid content in lemons at harvest the more keeping quality of the fruit (Schiffman Nadel and Cohen, 1973).

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34 About 75 80% of the TSS in the juice of nonacid citrus is sugar, mainly sucrose with glucose and fructose in roughly equal, but smaller amounts than sucrose. The sucrose and sugar proportions are high enough and sufficiently consistent that TSS can be expressed in terms of percentage sucrose. Acidity, represented by organic acids, of which citric acid is the major component, are equal to roughly a tenth or somewhat less of the TSS in the juice of nonacid citrus, and up to about 80% in that of acid fruits (Grierson, 2006b ). TSS: acid ratio of 8:1 and 7:1 were stipulated for oranges and grapefruit, respectively, as acceptable for interstate commerce (Collison, 1913). The current standards in Florida are 8.5:1 and 7.5:1 for oranges and grapefruits, respectively (Wardowski et al., 1995) Grade Standards (External Qualities) The purpose of this sub-section is to review parameters related to this work, which are weight loss, decay, chilling injury, firmness, fruit detach ment force, and peel color. Weight loss Postharvest fruit weight los s is essentially due to water loss ( Albrigo and Ismail, 1981) through transpiration, which accounts for 90% of total weight loss ( Ben Yehoshua, 1969), and initially comes from the peel ( Kaufmann, 1970). Water loss is also closely related to field and postharvest environments to which the fruits are exposed (McCornack, 1975) Water loss is a serious factor that adversely affect the quality of the fruit resulting in deformation of grapefruit (Ben Yehoshua et al., 1979) and stem -end rind breakdown of oranges (Hopkins and McCornack, 1958), limiting the economic postharvest life of fruits ( Kawada and Albrigo, 1979). Oranges start showing shrinkage at weight loss of 2.5%, and become unsalable at 5% of the original weight under normal handling conditions (Grierson and Wardowski, 1978). Maintaining citrus fruit in a humid environment and low temperature is an important factor in reducing the rate of water loss during storage (McCornack, 1975) because the rate of water

PAGE 35

35 loss is influenced by the relative pressure exerted by water vapor within and outside of the fruit (vapor pressure gradient) (Grierson and Wardowski, 1975). The rate of water loss is high immediately after picking and the next few days in storage, then decline afterwards because of low temperature and high surrounding humidity that influenc e stomatal behavior (Levy, 1980a ). Both internal water potential and external evaporative demand can cause decline in transpiration (Syvertsen et al., 1981). High temperature cause shrivelin g and drying of the peel, which immediately become visible on the peel, causing a dull appearance, softening, and peel senescence ( Ben Yehoshua, 1969; El Otmani, 2006). The loss of wei ght involves mainly the peel, not the pulp of the fruit. Thus, after 2 months storage at 20C a nd 5075% RH, the peel of Valencia oranges lost 9.5% of its weight, whereas the pulp lost only 2.1% (Ben Yehoshua, 1969). Fruit size affects wate r loss, because transpiration rate is greater in smaller fruits, apparently due to larger surface area : volume ratio in orange (Haas, 1927), but this surface to volume effect is not as important a factor in grapefruit weight loss (McCornack, 1975) Natural wax quantity, morphology and composition play a role in restricting water vapor loss. Wax accumulates gradually as the fruit mature s, and s enescing (late season) peel has greater epicuticular wax accumulation with significant structural changes than immature (early season) peel (El Otmani and Coggins, 1985a ; El Otmani and Coggins, 1985b ). This natural wax is either removed ( Kaplan, 1986) or redistributed ( Albrigo, 1973) after washing in packinghouse, so waxing treatment of fruits, plus humidity control in storage is important to control water loss (Burns and Albrigo, 1988) especi ally if the fruit will be kept in corrugated boxes, which are known to absorb water of about 10 15% of the carton weight (Emond and Nunes, 2006). The disadvantage of waxing treatment is the restriction of gas exchange through the peel surface and thus often causes anaerobic condition in the fruit internal

PAGE 36

36 atmosphere that may leads to the development of off -flavor due to increase in ethanol and acetaldehyde (Davis et al., 1967; Hagenmaier and Baker, 1993; Hagenmaier and Shaw, 2002; Porat et al., 2005 ), especially if oxygen is low when the fruit is not under refrigeration For citrus fruit with shellac/resin coatings stored near room temperature the interior oxygen is often less than 4% and sometimes less than 0.5%, which is low enough to cause off -flavor. With wax coatings, oxygen content is generally several percentage points higher (Hagenmaier and Baker, 1993). The disadvantage of less gas exchange is magnitude, especially if the peel is naturally less permeable (e.g. grapefru it is less permeable than mandarins) (Shi et al., 2007) Also, Shi et al. (2005) found that mandarins, but not grapefruit, respond to the anaerobic conditions by increasi ng the production of the stress hormone ethylene that accelerate senescence process. Decay As fruits become older, susceptibility to postharvest diseases increases, because peel becomes less firm and hence less force is required to puncture the peel by the pathogens (Coggins et al., 1969a ). Flavedo is the first line of resistance to invasion by decay pathogens. Mechanical injuries to the peel inflicted during harvesting and handling are the principal sites of infection by the wound -invading pathogens Penicillium and Geotrichum (El Otmani, 2006). Pos tharvest Penicillium decay is the main problem during transportation and the typically brief period of storage ( Kinay et al., 2005). Storage at low temperature and high humidity is beneficial to the maintenance of the natural resistance of both the peel and the button (fruit calyx) of the frui t to infection (Eckert and Eaks, 1989), but the most obvious effect of low temperature is to retard the growth of pathogens in infected fruits H owever, disease symptoms will appear a few days after the infected fruit are transferred to the ambient temperature, so washing fruits with a soap and sanitizer such as sodium ortho -phenylphenate (SOPP) or chlorine is an effective method to remove or inactivate spores on the surface of the fruit. Also, application of chemical

PAGE 37

37 fungicides during postharvest treatment of fruits can be used to reduce decay losses by inhibiting the development of latent fungi (Smilanick et al., 2006). To eliminate the need for synthetic fungicides complying with consumer preferences (orga nic requirements and reducing environmental pollution while maintaining fruit quality), a new approach to the control of postharvest pathogens has been implemented by the application of essential oil amended coatings to citrus. (Du Plooya et al., 2009) In warm and humid climates, such as Florida and Brazil, decay is more important in postharvest storage than that in arid cli mates, such as California, where postharvest shrinkage and softening play larger roles (Grierson and Miller, 2006c ). Common diseases that appear during storage are; green mold ( penicillium digitatum ) and blue mold ( Penicillium i talicum ), which develop from airborne spores infecting lesions incurred during harvesting and packinghouse handling, and brown rot (phytophthora citrophthora), sour rot ( Geotrichum candidum ), diplodia stem -end rot ( Lasiodiplodia theobromae ), and phomopsis stem end rot (Phomopsis citri ), which are quiescent infections of the fruit initiated during the growing season and become active after picking ( Arpaia and Kader, 2002; Brooks, 1942; Grierson and Miller, 2006c ). Chilling injury (CI) This is a physiological disorders induced by low, not freezing, tempera tures (33 40oF) that affect both trees and fruit of tropical and subtropical origins. It is most often characterized by areas of the peel that collapse and darken to form pits, which become more pronounced after fruit are warmed to room temperature followi ng exposure to chilling temperature. Symptoms generally require at least 3 to 6 weeks to develop at low storage temperature (35 40oF) (Ritenour et al., 2003) The primary cause of CI may be the result of a loss of cellular integrity caused by damage to cell membranes (McCollum and McDonald, 1991). Chilling injury is a time by

PAGE 38

38 temperature problem. If the produce is stored below the threshold temperature for short periods, the cell can repair t he damage. If exposure is prolonged, irreversible damage occurs and visible symptoms often result. Injury occurs sooner and severer, the lower the temperature is below the threshold temperature (Skog, 1 998). CI may depend upon climate (Young 1961), cultural practices (Eaks, 1991), fruit position in the canopy ( Purvis, 1980), exposure to sun (Nordby and McDonald, 1995), wi nter field temperature ( Kawada et al., 1978 ), picking date ( Pantastico et al., 1968), and temperature before refrigeration (Purvis and Yelenosky, 1993). Lafuente et al (1997) found that the susceptibility of Fortune m andarins to CI increased concomitantly with color change from green to orange, which indicates that chlorophyll may play an important role in CI resistance. It is interesting to note that chilling has been related to oxidative stress ( Harayadi and Punkin, 1991; Wise and Taylor, 1987), and chlorophyll has been demonstrated to protect plants against oxidative damage ( Larson, 1988). Usually immature oranges are susceptible to CI when stored at temperature below 35oF (Skog, 1998), but no CI was noticed at 3032oF for mature orange frui t in Florida ( Albrigo and Brown, 1973 ). Grapefruit is more susceptible than oranges to CI caused by storage temperature below 50oF for mid and late season fruit and 60oF for early season fruit. Waxed fruits can be stored at lower temperatures (but above the freezing point of the tissue) because they are less susceptible to CI because of the wax protection (Pantastico et al., 1968; Ritenour et al., 2003) CI symptoms are rind pitting, which prevails near 40oF, and brown -staining of the rind, common near 32oF (Hatton and Cubbedge, 1983). Such injuries are often accompanied by an increase in susceptibility to decay and the appearance of off -flavors (Cohen et al., 1990). It is also known that CI de creases with higher ABA levels (Serrano et al., 1997) The severity of damage can be alleviated by storag e at 50 60oF, however, because decay increases as the temperature increases,

PAGE 39

39 a proper temperature balance must be maintained, and fruit must be picked at proper maturity and handled carefully both in the field and in the packinghouse to avoid CI ( Am ador, 2004; Grierson, 1974; Pantastico et al., 1968 ) Firmness / peel turgidity / turgor pressure Fruit peel turgidity is positively cor related with fruit firmness, and can be used as an index of firmness and maturity of many fruit crops (Reid, 2002), but in citrus, there is a negative correlation between peel turgidity and water content of the peel and pulp. For this reason, peel turgidity has never been used as an index of fruit firmness, instead, it has been used as an index of tissue turgidity (Oberbacher, 1965). So, in citrus, the wor d firmness is associated with fruit turgidity and peel thickness. Preharvest, fruit becomes less firm with maturation, as the segments tend to pull away from the peel late in the season (most noticeably in mandarins) and with the excessive development o f the albedo typical of late-bloom fruit (Grierson, 2006a ). Mild winter nights are necessary for thin -skinned fruit, and cold winter nights result in thick -skinned fruit (Wutscher, 1976). Fruit softening is attributed to the dissolution of cell wall components duri ng development and maturation (Huber, 1983), which is a dominant feature of ripening in most climacteric fruits, but does not play a significant role in the maturation of citrus fruit. Although, peel and pulp of citrus fruit are ri ch in pectins, their decomposition into soluble fragments with maturation is very slow, with the exception of certain mandarin cultivars. Generally, mat ure rind contained lower concentrations of cellulose, hemicellulose, and pectic substances (Eaks and Sinclair, 1980) than immature rind. Also, polysaccharide fractions of flavedo tissue decrease as maturation proceed (Muramatsu et al., 1999). Senescing peel has an albedo layer with small cells of low cytoplasmic

PAGE 40

40 content with low metabolic activity, larger intercellular spaces and weakened cell wall, which bre ak easily (Coggins, 1969b ). Postharvest reduction in citrus fruit firmness is largely due to loss of water, mainly from the peel ( Spiegel Roy and Goldschmidt, 1996), causing fruit deformation, which increase with fruit maturation and senescence ( Kawada and Albrigo, 1979), and to some extend is associated with invasion of the peel by several pathogenic postharvest diseases, which secret e large amounts of ce ll -wall degrading enzymes (Eckert and Eaks, 1989). Any treatment that retards moisture loss, such as lo w temperature storage, high humidity and proper waxing will reduce water loss and retain peel turgidity and fruit firmness ( Kawada and Albrigo, 1979). Fruit detachment force (FDF) Detachment force is the force required to separate the fruit from the stem/peduncle, and it is an indicator of fruit maturity and senescence, as it decreases with fruit maturation (Ladaniya, 2008). In harvesting system based on pulling citrus fruits, losses from rupture of the peel at the stem e nd (most noticeably in mandarins and tangerines) is excessive if the natural abscission layer is not yet developed (Ladaniya, 2008 ), and this is the reason of using torsion force (rotation detachment) for fresh fruit market, because the calyx is not detached in it and losses are less (Juste et al., 1988). Tree fruit moisture level has been shown to affect FDF. Less force is required to remove oranges following rains and early in the morning when the fruit is in a turgid condition (Coppock, 1961). Detachment force and percent of plugged Marsh grapefruit decreased with greater detachment angles (Coppock et al., 1969) Also, FDF of Navel orange increased with increasing fruit size and larger stem/peduncle diameter ( Hield et al., 1967; Kend er and Hartmond, 1999). At maturity, citrus fruit remain firmly attached to the stem, and FDF is extremely variable within the tree and generally higher in the top and exterior parts of the canopy, where fruits are more exposed to the sun and develop str onger stems ( Kender and

PAGE 41

41 Hartmond, 1999). In general, wounding or mechanical injuries of the peel reduce FDF due to triggering of internal ethylene and initiation of abscission zone. The closer the location of the wound to the calyx abscission zone, the larger the reduction in FDF (Kostenyuk and Burns, 2004) Fruit detachment force is positively correlated with the ratio of endogenou s ratio of IAA to ABA or endogenous IAA only but negatively to endogenous ABA in the fruit abscission zone. This hormonal balance is important to determine the sensitivity of citrus fruit to abscission chemicals (Yuan et al., 2001). Peel color Color is considered to be one of the most important external factors of fruit q uality, as the appearance of the fruit greatly influences consumer preference (Singh and Reddy, 2006) The color of citrus fruit is largely determined by prevailing weather conditions during fruit maturation. Areas with hot dry summer and cool humid winter, like Californ ia (Mediterranean climate) generally produce fruit with better color, but thicker and coarser peel than areas having more humid growing season and warmer winter nights, like Florida (humid subtropical climate). As air and soil temperatures fall below 13oC (55.5oF), chlorophyll degradation takes place, revealing the underlying carotenoids and giving fruit a bright yellow to orange color (Young and Erickson, 1961). This is called Color break which is a change produced by nature converting the dark green color to the extent that a tinge of yellow or orange color is appare nt (Grierson, 2006b ), coinciding with the onset of peel maturity (Erickson, 1960) but is not related to pulp maturity. The change in fruit color during postharvest storage under refrigerated condition is less than that under ambient condition (Singh and Reddy, 2006) and light green -colored fruit have longer storage life than well -colored fruit ( Schiffman Nadel and Cohen, 1973). The water -insoluble pigments, carotenoids, are the main color pigments in cit rus fruits (Coggins, 1986). Not all citrus species, however, accumulate the same types or amounts of

PAGE 42

42 carotenoids. -citraurin myrcitate ester is the major carotenoid in mature Marsh grapefruit (Philip, 1973) whereas 9 cis -violaxanthin is the major carotenoid in mature Valencia orange fruits (Stewart and Whitaker, 1972). There are many ways to evaluate fruit color, but many researchers publish their data in either the CIELAB (L*, a*, b*) or in the Hunter (L, a, b) scale (McGuire, 1992). Both of these are based on the Opponent -Colors Theory, which assumes that the receptors in the human eye perceive color as the followin g pairs of opposites; light & dark, red & green, and yellow & blue. The (L) value indicates the level of color lightness (chroma) (light; L=0 and dark; L=100), the (a) value indicates the hue gradation or tint (redness; +a, and greenness; a), and the (b ) value indicates the density intensity or saturation (yellowness; +b and blueness; -b). All three values are required to completely describe the color of an object in three -dimensional L, a, b color space, where (L) represent X axis, (a) represent Y axi s, and (b) represent Z axis. The formula for Hunter (L, a, b) scale is calculated using square roots of X, Y and Z, whereas, the formula of CIELAB (L*, a*, b*) scale is calculated using the cube roots of X, Y and Z. The Hunter scale is preferred in the foo d industry, especially when measuring light colors (Hunter Associates Laboratory, 2008) and this is the scale that was used to measure citrus fruit color in this study. Harvest Date Problems in postharvest storage are often related to the time of picking fruit within the season ( Burns and Albrigo, 1997). For long storage, fruit must be picked when it still has considerable vitality H owever, determining the optimal harvest time is a matter of judgment, since there is no well -define physiological indication as in climacteric fruits (Grierson and Miller, 2006c ). Lack of a well defined maturity mechanism results in a more or less prolonged period of edibility during which the fruit of a given cultivar may be marketed (Grierson, 2006b ).

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43 Marsh grapefruit has a long harvest season (September May) and Valencia orange has a short harvest season (February June) ( USDA, 2004). Under certain climatic conditions, the internal fruit qualit y of early maturing varieties reach maturity standards while the peel is still green, and under some other climatic condition, some varieties obtain optimum peel color and other characteristics suitable for marketing before juice characteristics reach mark et quality. In those areas, fruit peel enters the senescent stage while the internal juice characteristics are still of good market quality (Rouse and Zekri, 2006). Wwn et al (2001) found that Ponkan mandarin for storage should be harvested several times from the same orchard or same tree, and that only those half -yellow fruits are suitable for storage. Ponkan f ru it which are yellow at harvest are unfit for long term storage. Green fruits should be left on the tree until their color turn to half -yellow before harvest. The yellow fruits showed significantly higher storage decay than half yellow and green fruits, whe reas green fruits had slightly lower TSS than others. Half -yellow fruits harvested early had higher acidity at harvest than similar fruits harvested at later dates, such a difference, however, disappeared after storage, due to consumption of acids in respi ration. They concluded this work saying that percentage peel coloration seemed to be a better maturity index than the calendar date for Ponkan mandarin. On the other hand, delaying time of Washington Navel fruit harvest, from mid December to mid -April, increased TSS and peel thickness, and decreased juice percentage, acidity and fruit detached force (El -Hammady et al., 2000). Fruit firmness also was reduced with advancing picking date of Mars h grapefruit (Rivero et al., 1979), but susceptibility to CI, after storage at temperature below 46oF, was greatest in early harvested Tarocco blood orange, less in mid -season harves t, and negligible in late -season harvest (Schirra et al., 1997 ). Burns and Albrigo (1997) found that early in the season, grapefruit were more sensitive to oleocellosis and

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44 other harvest induced blemishes but were more resistant to decay. As the season progresses and the fruit senesce, they become more susceptible to decay and granulation and the fruit taste becomes isipidily sweet Also, Schiffmann Nadel and Cohen (1973) stated that lemon fruit picked in summer changed color in storage more quickly and had shorter storage life than fruit picked in winter. Storage Conditions Unlike many climacteric fruits, most citrus fruit have good keeping quality. Successful storage of citrus fruits is conditional on prior accurate evaluation of whatever is the major hazard liable to end stor age life (e.g. weight loss, softening, decay, CI, off -flavors), and the correct procedures being followed in the grove or in the packinghouse prior to storage to minimize the major problem (Grierson and Miller, 2006c ). Although storage temperature is important the response of citrus fruits to temperature is much less dramatic than that of climacteric fruits, because of the low respiration rate of citrus fruits (Biale, 1961). Fruit response is proportional to optimum temperature in the range of 32 60oF (0 15.5oC) (Grierson, 1976) depending on; cultivar, geographical ar ea, maturity stage at harvest storage period, susceptibility to CI, preharvest treatments, and prestorage treatment (Arpa ia and Kader, 2002; Grierson and Miller, 2006c ). High temperature during storage favors fungal decay, rapid water loss, increased softening, and enhanced decrease in Vitamin C, but very low temper ature causes CI (Grierson, 1974; Grierson and Miller, 2006c ). Yuan et al (2001) found that cold weather (5 10oC) in January increased ABA level in Valencia oranges peel. ABA level is correlated with the storage temperature, for instance, fruit stored at 12oC showed an increase of ABA which did not occur at 2 5oC, but this could be partially related to the higher rate of water loss at higher temperature. Changes in ABA level with storage temperature depended on the stage of maturity, but not on their susceptibility to CI (Lafuente et al., 1997)

PAGE 45

45 Storage time and temperature also effect fruit aroma, for example, the sesquiterpene, nootkatone, which is a characteristic compound o f grapefruit increased with storage time but decreased from wax application and cold storage ( Sun and Petracek, 1999). Ting and Attaway (1971) found that volatile flavor components were lost during prolonged storage Petracek et al. (1998) found that storing waxed fruit at high temperature increases fruit respiration rate, and as a result, the fruit internal atmosphere changes due to s tress associated physiology and biochemical processes. These changes were characterized by low O2 and high CO2 and ethanol partial pressure, which affect the volatile compositions of the fruit (Dou, 2003). Controlling relative humidity (RH) in a range of 8595% (Grierson, 1976) is important during storage to avoid weight loss, shriveling, softening and fruit deformation if humidity is low, but if humidity is too high decay increases (Ben Yehoshua, 1987). It is also important to remember that corrugated boxes can lose more tha n 50% of their strength if they are exposed to high humidity and absorb water from the fruit thus increasing water loss (Emond and Nunes, 2006). In Florida, recommended storage conditions of grapefruit are 50 60oF (10.815.5oC) and 9095% RH for estimated storage life of 5 8 weeks (Arpaia and Kader, 1996a; Grierson, 1976) but for oranges recommended conditions are 32 34oF (0 1oC) and 85 90% RH for estimated storage life of 8 weeks (Arpaia and Kader, 1996b; Grierson, 1976) In general, ventilation of stored citrus fruit is necessary (Baier, 1945) to eliminate carbon dioxide and various volatile components from storage rooms (Norman and Houck, 1977). Some remarkable decreases in decay losses have been attributed to adequate ventilation (Tanaka et al., 1957). Also, inappropriate storage conditions, such as high temperature and inadequate ventilation, that cause a reduction in oxygen level, lead to the perception of off -flavor (Waks et al., 1985) by enhanced accumulation of ethanol, acetaldehyde and other volatile compounds ( Ke

PAGE 46

46 and Kader, 1990). Ventilation can be done by opening the doors of storage rooms and using fans for air circulation, vents with fans or by using some forms of air purification as alternative to ventilation, such as activate d carbon (scrubbed with potassium permanganate absorbed into suitable medium) that absorb most of volatiles components ( Smock, 1979). Water Stress Water plays a crucial role in the life of the plant. It typically constitutes 8095% of the mass of growing plant tissue. Plants absorb approximately 500g of water to build 1g of organic matter. Water stress occurs when the demand for water exceeds the available amount in the soil due or to poor draining or excessive irrigation with the soil becoming flooded or waterlogged. Water then fills the pores and blocks the diffusion of oxygen in the gaseous phase, resulting in anaerobic condition and lack of oxygen supply to the roots, anoxia. This is so-called flood stress (Taiz and Zeiger, 2002). This study is only concerned with water stress from water demand exceeding water supply, drought stress. During water deficiency; too little water is available in a suitable thermo -dynamic state and the rate of transpiration exceeds the rate of water uptake. The reasons of this may be; soil dryness, inadequate water uptake by plants in shallow soils, osmotic binding of water in sali ne soils, high evaporation from soil, and high transpiration from the plant due to excessive evaporative demand of the air. Drought stress does not occur suddenly, but rather develops slowly and intensity increases with time ( Munne Bosch and Alegre, 2004; Munns, 2002). Plant response to drought stress may also be influenced by plant genotype, soil type, climate, possible acclimation to previous exposure to stress, phase of growth, and the part of the plant that is exposed to the stress ( Kozlowski a nd Pallardy, 2001). Water stress is a main limiting factor in obtaining high yield or better quality of citrus fruits (Levy, 1980b ), especially in high -yielding cultivars such as grapefruit (Levy e t al., 1978). Photosynthetic rate and stomatal conductance of severely drought stressed Satsuma mandarin

PAGE 47

47 trees were significantly lower than those of well -watered trees (Syvertsen, 1982; Vu and Yelenosky, 1989; Yakushiji et al., 1998). Water stress has many effects on plant physiology and productivity involving reactions ranging from subce llular levels to whole trees and interaction between trees in the orchard ( Marsh, 1973). Water stress induces the process of cell senescence through specific changes in cell ultrastructure (e.g. chromatin condensation, thylakoid swelling, plastoglobuli accumulation, and chlorophyll degradation), metabolism (e.g. protein degradation, lipid peroxidation) and gene expression ( Munne -Bosch and Alegre, 2004). Fruit growth is considered as one of the most sensitive indicators of water stress in citrus (Cohen and Goell, 1984; Hilgeman, 1977). Water stress affects fruit size negatively and the growth stage, during which w ater stress occurs, determines the magnitude and irreversibility of such damage ( Ginestar and Castel, 1996). Phase 1 (cell division) is the most sensitive to water stress in grapefruit, but phase 2 (cell enlargement) is the most sensitive to water stress in Valencia orange, and the fruit recovery after alleviation of water stress is better in Valencia than grapefruit (Mostert and Van Zyl, 2000). It has been reported that drought stress during the period of fruit rapid growth (June December) should be avoided because it decreases fruit size (Boman et al., 1999) and stress for a short period following June drop increase acidity and decrease TSS (Hilgeman, 1977). Studie s have shown that cultural practices, such as fertilization, weed control, pest control, and pruning do not improve yield or fruit size if irrigation is not satisfactory. However, a moderate drought stress in late summer and fall can be desirable to mainta in high juice quality (Boman et al., 1999) better than fruit juice from regularly irrigate d trees (Goell and Levy, 1970 ). The high juice soluble solids: acid ratio may result from; enhanced metabolic processes ( Yelenosky, 1978), accumulation of dry matter during water stress period, which was not completely utilized for v olume growth of the fruit even after irrigation was resumed ( Cohen

PAGE 48

48 and Goell, 1988), or sugar accumulation by active osmoregulation to maintain cell turgor and minimize the detrimental effect of water stress ( Yakushiji et al., 1998; Yakushiji et al., 1996). In general, acidity and soluble sugars content in juice and peel, peel thickness, and peel to -pulp ratio increase in drought -stressed trees (Boman et al., 1999; Hockema and Etxeberria, 2001; Huang et al., 2000; Mostert and Van Zyl, 2000) but there is a reduction in the inci dence of green mold (Ritenour et al., 2002) and fruit turgor pressure (Huang et al., 2000). Acidity increased more rapidly than TSS un der dry conditions. Consequently, TSS: acid ratio decreased (Wittwer, 1995). Irrigation usually reduces TSS and acidity, and it has a greater effect on acidity in grapefruit than in orange (Barbera and Carimi, 1988; Boman et al., 1999) Peel color break is usually delayed when irrigation is increased, because water availability imp roves nitrogen nutrition, which delays color break and increases regreening (Boman et al., 1999) They also mentioned that in humid Japan, color break was accelerated in Satsuma mandarin when soil was covered by plastic sheet to induce drought stress. Water stress stimulates the activity of glycosidases causing deterioration of cell walls, resu lted in fruit softening (Konno et al., 1986) It also increases the level of endogeno us ABA (Lafuente and Sala, 2002) IAA (Koshita and Takahara, 2004) GA3 (Sardo and Germana, 1988), and ethylene formation ( Ben Yehoshua and Aloni, 1974). ABA promotes the closure of stomata to minimize transpirational water loss (Bray, 2002; Finkelstein et al., 2002 ), and increase fruit resistance to chilling injury (Kawada et al., 1979). Coppock (1961) reported that tree moisture level has been shown to affect FDF negatively; less force is required to remove oranges when the fruit is in a turgid condition. Drought stress advanced maturity and senescence of the citrus peel and effected volatile compounds, which consisted mostly of monoand sesquiterpenes (the major components of citrus oil) ( Sharon -Asa et al., 2003). Drought stress can be a factor affec ting the overall emission

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49 of terpenes from citrus tree. Severe and mild stress reduce the amount of emitted sesquiterpenes, but no effect was noticed from slight stress (Hansen and Seufert, 1999) The plant water status can be expressed by water potential ( w), whi ch measures how hydrated a plant is and thus provides a relative index of how water stress the plant is experiencing. Plant can only take up water when the plant w is less than the soil w. As soil becomes drier, the plant similarly becomes less hydrated and attains lower w. Leaf water potential (w) of well -watered plant ranges from 0.2 to 1.0 Mpa, whereas it can range from 2.0 to 5.0 Mpa in stressed plant (Taiz and Zeiger, 2002). Levy et al. (1978) stated that a soil w of about 0.1 Mpa was reached 20 days after irrigation, and it was 0.7 to 0.8 Mpa after 40 days after irrigation. Growth Regulators Citrus growth and development are complex processes governed by several factors, such as, culti var, climate, soil type, hormonal balance, and cultural practices including plant growth regulators, which can provide significant economic advantages to citrus growers when used appropriately (El Otmani, 2006). Plant growth regulators are organic compounds either natural or synthetic that control one or more specific physiological processes within the plant to accelerate or retard the rate of growth or maturation, and they are characterized by their low rate of application; high application rates of some of these compounds often are considered herbicides. They are classified into five groups; auxin, gibberellins, cytokinins, ethylene, and abscisic acid (Lemaux, 1999; Stewart, 1954). Among this group, this study is mainly related to auxins and gibberellins applied exogenously to citrus fruit. The endogenous level of both hormones has been studied in lemon fruit. Josan et al. (1999) stated that after one month of fruit set, the activity of both hormones was low, then increased gradually with fruit development and declining again toward maturity. Generally, their activity was greater in the pulp than in the peel.

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50 Similar findings have been reported by Takahashi et al. (1975), Goldschmidt (1976) Kuraoka et al. (1977), and Dhillon (1986), suggesting that these hormones are contributing to cell division (growth stage I; mainly auxin) and cell enlargement (growth stage II; mainly gibberellins) (Taiz and Zeiger, 2002 ). The use of plant growth regulators in agricultural production within the United States began in the 1930s (Fishel, 2006) Three growth regulators are in common use on citrus. These are 2,4 dichloro phenoxyacetic acid (2,4 D), gibberellic acid (GA3), and ethylene (Coggins, 1981). They were used to promote citrus seedling growth, to control the vegetative growth in mature trees and to improve cold hardiness of citrus trees. They have been used to control citrus fruit production by influencing flowering, fruit set, fruit thinning, preharvest control of fruit drop, and in the control of fruit disease. They have also been used to influence fruit quality factors such as rind quality, rind color, fruit size, to decrease juice acidity, and to increase juice soluble solids (Wilson, 1983). Depending on cultivar and timing, growth regulators can also be used to extend the harvest seaso n by delaying rind aging, and reduce preharvest fruit drop (Davies and Albrigo, 1994). Gibberellic acid (GA3) is used pri marily as a preharvest treatment to delay certain aspects of rind senescence of navel orange and seedless grapefruit (Coggins, 1981). It has been linked with citriculture since the late 1950s, when it was first used in navel oranges to influence several fruit quality factors (Coggins and Hield, 1958). Since then, it has been used to modify color development by delaying chlorophyll degradation (Coggins and Henning, 1988; Coggins an d Hield, 1968; El Zeftawi, 1980a ; Garcia Luis et al., 1992; Goldschmidt et a l., 1970; Porat et al., 2001) via inhibition of chlorophyllase synthesis (Trebitsh et al., 1993) and inhibition of carotenoid biosynthesis (Jun et al., 2002), and to reduce several physiological blemishes and

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51 diseases of the peel (Coggins, 1969b ), as well a s reduction in preharvest and postharvest physiological disorders (Coggins, 1981). The efficacy of GA3 with respect to peel senescence is related to the physiological age of the fruit and to the amount of time between application and harvest (Greenberg et al., 1992). GA3 is typically not applied unless the grove is scheduled for late harvest. Autumn application of GA3 to citrus fruit approaching maturity (just prior to color break) enhances peel firmness, delays color change, and peel senescence and the subsequent peel disorders and decay (Chitzanidis et al., 1988; Coggins, 1981; McDonald et al., 1997; Pozo et al., 2000; Stover, 2000) allowing a late harvest ( Agusti et al., 1981), however, this may interfere with peel coloration (Greenberg et al., 1992), enhancing regreening (Thomson et al., 1967) of grapefruit in California, but not in Florida (Dinar et al., 1976). Also, this application may cause a reduction or delay of subsequent flow ering and next years yield ( Monselise and Halevy, 1964). Summer application has no effect on subsequent flowering or color variation, but autumn application is more effective for other fruit quality factors (Greenberg et al., 1992). It has also been reported that spraying oranges with GA3 at color break reduced preharvest fruit drop (Cameiro de Medeiros et al., 2000), postharvest decay ( El Otmani and Cogg ins, 1991), chilling injury of fruit stored at 1 5oC and prolonged storage life of fruit stored at 20oC ( Arpaia and Eaks, 1990). Fucik (1981) found that GA3 reduced water loss from grapefruit d uring postharvest storage. GA3-treated fruits have peel with a structure and wax characteristics similar to that of young fruits ( Coggins, 1969b ), which means less wax accumulation and retarding wax ultrastructure changes (El -Otmani and Coggins, 1985a ), but this reduction in wax quantity has no significant effect on fruit weight loss (El Otmani et al., 1986). This is an ind ication that not only wax quantity, but also morphology and composition play a role in restricting water vapor and gas diffusion (El -Otmani and Coggins, 1985a ; El Otmani and Coggins, 1985b ). The role of GA3 in

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52 delaying senescence is observed in preservation of more functional mitochondrial and plasma membranes in the form of a higher rate of O2 uptake, a higher rate of respiration, a lag in sugar accumulation, a preservative effect on sol ute efflux, and the survival of protoplasts (Nolte et al., 1990). Exogenously applied GA3 delays loss of chlorophyll, RNA and proteins ( Fletcher and Osborne, 1965a ; Fletcher and Osborne, 1965b ) in flavedo and maintains a compact structure of the albedo ( Monselise, 1979). Insufficient irrigation decreased the effectiveness of GA3 in South Africa (Gilfillan et al., 1973 ), whereas it has little ef fect on GA3 efficacy for mature Marsh grapefruit trees in Florida (Ferguson et al., 1983). Generally, GA3 does not affect juice quality when it is used to delay peel senescence and extend harvest season (Cog gins, 1981), however, it can show some slight effect when applied near color break, and this effect is variable based on cultivar. A preharvest spray of GA3 increased juice soluble solids and reduced acidity of grapefruit at harvest (El -Zeftawi, 1980b ), however, McDonald et al. (1997) found no effect on soluble solids or acidity of Marsh grapefruit, and no effect was noticed on navel orange (Coggins, 1969b ), but when applied to Valencia orange, it increased soluble solids and acids in the juice. No effect was noticed on soluble solids and acids of Satsuma mandarin ( Kuraoka et al., 1977 ) or Sunburst mandarin (Pozo et al., 2000) GA3 is not considered a primary growth regulator in controlling fruit abscission (Gomez -cadenas et al., 2000 ). Rasmussen (1981) stated that in Valencia orange, a high level of GA3 in the flavedo of the fruit at the regreening stage may help to prevent ABA from inducing ethylene and promote abscission, however, the effect of GA3 on ABA content was reported to be negligible (Jun et al., 2002; Zacarias et al., 1995 ). Coggins et al. (1969a ) stated that change in oil composition using GA3 is less likely since the senescence index based on the evaluation of oil constituents of GA3-treated fruit had changed

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53 less than that of non -treated fruit. Later, Wilson et al. (1990) and McDonald et al. (1997) stated that treatment by GA3 generally increased peel oil concentration. The phenoxy group of synthetic auxins, among which 2,4 D is one of the most active, caused formative effects when studied during the early period of World War II, and for a time the research on this auxin was carried on under military secrecy (Zimmerman and Hitchcock, 1942). Later, 2,4 D was identified as an excellent herbicide (in high concentrations) and a modifier of fruit growth and ripening (in low concentrations) (Leopold, 1955; Leopold, 1958). Acting like the natural auxins in plant, 2,4 D was used primarily as a preharvest treatment to delay and reduce abscission of mature citrus fruit (Coggins, 1981) and delay harvesting (M onselise, 1977) by inhibiting ethylene effects that promote the activity of the hydrolytic enzymes; endo -beta glucan and polygalacturonase at the abscission zone (Burns and Lewandowski, 2000; Goren, 1993) thus reducing fruit drop, as demonstrated by highe r fruit detachment force (Chitzanidis et al., 1988; Goren et a l., 2000). It also provided the secondary benefits of increasing fruit size ( Stewart, 1954) of the new crop. This effect was more noticeable in Valencia orange than grapefruit. Some of this response may have occurred from increasing the size of fruit peduncle (El Otmani et al., 1993 ), more vascular differentiation (Aloni, 1995), and increased sink capacity of en docarp and thus of fruit resulting in greater mobilization activity of water, carbohydrates and minerals to the fruit leading to faster growth and good juice quality (Agusti et al., 1992) represented by higher oBrix at harvest (El -Zeftawi, 19 80b ). In general smaller fruit have substantially higher percent acid and lower total soluble solids than larger fruit (Hutton, 1989). Preharvest application of 2,4 D to oranges and lemon have been reported to delay color development (Stewart, 1954), and delay rind softening (Erner et al., 1993) on the tree and in cold room storage, but this effect is very small compared with that of GA3 (El Otmani et

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54 al., 1990). Treatment with 2,4 D did not gave any control of black rot in orange ( Albrigo and Brown, 1973), but it prevent stem -end rot in lemon (Hall et al., 1973 ). Also, postharvest dipping of Valencia orange in 100 500ppm 2,4 D retained the fruit buttons and reduced water loss during ethylene degreening. It also delayed the development of Alternaria rot and prolonged the storage life of lemon (Stewart, 1954). For long -distance shipme nt and prolonged storage, 2,4 D is recommended to control stem -end rots of oranges and grapefruit ( Schiffman Nadel et al., 1972). The plant growth regulators 2,4 D and GA3 are excellent companion growth regulators for reducing drop of mature fruit and delaying rind senescence, respectively (Coggins, 1981). A combination of GA3 and 2,4 D enhance peel firmness, red uce penicillium decay, delay color development, prevent fruit drop, and extend the harvest season of grapefruit and oranges (Chitzanidis et al., 1988 ; El Otmani et al., 1990; Ferguson et al., 1982; Kinay et al., 2005; Medeiros et al., 2000; Sen et al., 2001). Apart from the delay in rind coloration, there is no delay in juice legal maturity (Rouse and Zekri, 2006). A combination of 10 ppm GA 3 and 20 ppm 2,4 D prolonged storage life of Marsh grapefruit on tree and in cold storage ( El Otmani, 2006), and significantly reduced water loss of Ruby Red grapefruit during cold storage compared to GA 3 or 2,4 D alone (Fucik, 1981). Use of 2,4 D alone or combined with GA3 depends on the circumstances. If the harvest season is going to be short, and mature fruit drop is not severe, both growth regulators can be applied in the same solution, otherwise, 2,4 D can be applied separately latter in the season ( Coggins, 1981). Preharvest application of 6 ppm 2,4D and/or 10 ppm GA3 to Clementine mandarin, Marsh grapefruit, or Valencia orange delayed peel aging and prolong harvest season, but had no effect on fruit weight loss in storage, acidity, TS S, or TSS: acid ratio (El Otmani and Coggins, 1991; Gilfillan et al ., 1973).

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55 Sucrose and Reducing Sugars Over 400 different constituents have been isolated from citrus fruit. The principle components of nonacid citrus fruit are carbohydrates, which include mainly sucrose, glucose, fructose and traces of several other s ugars (Nagy and Attaway, 1980; Nagy et al., 1977; Ting and Attaway, 1971; USDA -ARS, 1960). These sugars are potential effectors of juice quality (Ting, 1969), and peel color (Huff, 1984). Sugar also have a role in plant response to water stresses (Price et al., 2004) Sugars constitute about 76% of the TSS in orange juice (Ting and Attaway, 1971). Large fruit always have lower TSS (% by weight) than small fruit of the same cultivar and tree (Harding and Lewis, 1941). Frui t grown in warm winter (i.e. Florida) have higher sugar content than those in cold winter (i.e. California) (Tucker and Reuther, 1967). Sugars increase gradually during maturation, until reaching their peak then start declining gradually while the fruit remain on the tree (Bartholomew and Sinclair, 1951). Datio and Sato (1985) found in Satsuma mandarin that sucrose and glucose are the predominant sugars in early stages of peel maturation, then increase gradually with fructose as fruit maturation progress, and all three sugars become predominant in both peel and juice. Albedo tissue work as a transit sink or reservoir for sucrose enroute by symplastic pathway via plasmodesmata (Garcia Luis et al., 1991) to juice sacs during late -season fruit development (Koch, 1984; Koch and Avigne, 1990; Yen and Koch, 1990) Biochemical ch anges after harvest remain more or less constant, and any increase in sugar: acid ratio during postharvest storage is mainly because acids tend to decrease faster than sugars due to respiration (Ting and Attaway, 1971). Sinclair and Crandall (1949) reported that physiological breakdown of the fruit while on the tree and in storage, and the deleterious effects caused by the invasion of fungi, result in chemical changes in the carbohydrate constituents of grapefruit peel.

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56 Peel sugars have long been implicated as potential effectors of color break. Soluble sugars in the peel were initially found to correspond with color break in Valencia orange by Huff (1984) who noticed that regreening of Valencia orange in spring is preceded by a decrease in peel soluble sugars. Color break is stimulated by low winter temperature (Erickson, 1968) and level of peel sugar (Goldschmidt and Koch, 1996; Huff, 1984; Iglesias et al., 2001) Low temperature (5oC) can increase invertase activity and level of r educing sugars in flavedo of Marsh grapefruit on tree and during storage at 5oC (Purvis and Rice, 1983). The time of color break can be advanced if sugar level in the peel is increased by stem injections of sucrose, which stimulate the conversion of chloroplast to chromoplast (Iglesias et al., 2001) The inverse correlation between the extent of green color retention and hexoses (e.g. glucose and fructose), but not sucrose, in flavedo is also well known ( Fidelibus et al., 2008). Availability of significant hexoses cause downregulation of genes encoding enzymes for chlorophyll synthesis, photosynthesis, and plastid conversion (Fidelibus et al., 2008; Pourtau et al., 2006; Price et al., 2004; Rolland et al., 2006) Also, hexoses (mainly glucose) have influence on gene expression for biosynthesis and perception of ABA (Rognoni et al., 2007; Rolland et al., 2006) and its interaction with ethylene initiating fruit color in response to stress (Price et al., 2004; Rognoni et al., 2007) indicates a role of sugar in environmental responses (Price et al., 2004) Soluble sugars could be contributing effectors to the GA3-mediated delay in chloroplast chromoplast conversions by flavedo (Fidelibus et al., 2008). The role of GA3 in retarding color break may be through the involvement of ethylene (presumably ethylene would increase conversion) and sugars. There is evidence in S atsuma mandarin that sucrose stimulate and nitrogen repress chloroplast to chromoplast conversion in flavedo, and this conversion requires initial nitrogen depletion and subsequent sucrose accumulation (Iglesias et al., 2001) They also

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57 found that sucrose stimulation operates v ia ethylene, whereas GA3 acts as repressor of the sucrose -ethylene stimulation. Also, GA3 can alter sucrose metabolism in plant ( Cheikh et al., 1992) and alter expression of photosynthetic genes in plant ( Fujii et al., 2008 ). The reduction of glucose and fructose levels in flavedo after application of GA3 was previously confirmed in Shamouti orange (Goldschmidt et al., 1977; Monselise and Goren, 1965) and Satsuma mandarin ( Kuraoka et al., 1977). Fidelibus et al. (2008) found in Hamlin orange that GA3 treatment maintained a descending sucrose gradient from the albedo to the flavedo typical of young, photosynthetically active fruit, reducing flavedo glucose and fructose level below those of nontreated fruit. They also found that this gradient dissipated during peel color change of non treated fruit. Water str ess triggers the physiological function of osmoregulation, which involve solute (i.e. sugars, organic cations, organic acids and amino acids) accumulation in cells sufficient to lower osmotic potential of cells and allow them to absorb water to maintain ce ll turgor ( Meyer and Boyer, 1981) and minimize the detrimental effect of the drought ( Morgan, 1984 ). Yakushi ji et al. (1996) found that monosaccharides (i.e. glucose and fructose) were largely r esponsible for active osmoregulation in Satsuma mandarin fruit under water stress condition. They also stated that concentration of sucrose, glucose and fructose increased in fruit under moderate water stress within a relatively short period. Furthermore, the total sugar content (and acidity) per fruit of water stressed trees was significantly higher than that in fruit of well-watered trees, suggesting that sugar accumulation in fruit peel and juice sacs was not caused by dehydration under water stress but rather that sugar were accumulated by active osmoregulation in response to water stress ( Yakushiji et al., 1998) that enhanced the rate of photosynthate partitioning to the fruit more than that to leaves and stems ( Asakura et al., 1991). Cohen and Goell ( 1988) found that the

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58 rate of dry matter (mainly carbohydrates) production and its accumulation in grapefruit are hardly affected during very long periods of moderate drought and this may be the result of continued, near normal levels of photosynthesis during stress periods, or a withdrawal of photosynthates from reserves existing in the tree. Additional accumulation of sugars can triggers a decline in photosynthetic gene exp ression (Wingler et al., 2006) and reduce photosynthesis under drought stress ( Vu and Yelenosky, 1989).This may confirm the results of Cohen and Goell (1988) that sugar accumulation in fruit is a result of continued withdrawal of photosynthates from tree reserves. Newer research findings pointed out that the accumulation of sugars in combination with low nitrogen under water stress conditions induced senescence like symptoms (Pourtau et al., 2004; Wingler et al., 2004) and expression of senescenceassociated genes SAG12, late in the senescence process (Wingler et al., 2006) Glycosidases Glycosidases are a group of enzymes that hydrolyze the chemical bonds between the hemicellulose and the pectic -polysaccharides components of the cell wall, causing deterioration of the cell wall, loss of cell turgor pressure, loss of adhesion between cells (Goren, 1993) and then fruit softening (Mitcham and McDonald, 1993) associated with some other physiological processes like abscission due to changes in abscission zone (Burns et al., 1998). H owever, there are other hydrolytic functions of glycosidases not directly associated with cell wall, such as glycolipid, glycoprotein, and reserve oligosaccharide metabolis m (Bolwel l, 1988) Glycosidases activities have been detected in many growing and senescing plant tissues with a role in cell wall metabolism and modification (Bolwell, 1988) Modification of the neutral sugar side chains in the pectin of granulated juice vesicles of sen escing citrus fruit was reported in grapefruit (Hwang et al., 1990) and pummelo (Shomer et al., 1989). This appears to occur at the expense of soluble sugar and acids within the juice vesicle (Burns, 1990b ). With advanced

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59 maturity of citrus fruit (Ritenour et al., 2004) granulated juice vesicles showed decreases in rhamnose, arabinose, mannose, and galactose and increases in xylose and glucose ( Shomer et al., 1989). In the current study, only two of these neutral sugars ; galactose and mannose, and their related enzymes; -mannosidase (EC 3.2.1.24), respectively, in the context of fruit softening were chosen for evaluation. Burns (1990b ) found in Lee tangelo an galactosidase activity was 2 to 3 -mannosidase activity was only detected in granulated tissue. Other research findings confirmed that the hydrolysis of cell -galactosidase increased during the ripening and softening of apples (Bartley, 1974; Berard et al., 1982) tomatoes (Pressey, 1983; Watkins et al., 1988), and apricots (Kovacs and Nemeth -Szerdahelyi, 2002) The -mannosidase was also increased during the ripening of tomatoes (Watkins et al., 1988) mannosidase was found in the cell walls ( Ahmed and Labavitch, 1980) suggesting that this enzyme is involved in the post -transitional processing of glycoproteins in fruit (Watkins et al., 1988) and is a vacuolar enzyme (Boller and Kende, 1979; Faye et al., 1988; Masuda et al., 1990) with a lytic function which can be used as a vacuolar marker. Immunocytochemical evidence -mannosidase is present in the endoplasmic reticulum and the Golgi complex of developing cells, and accumulates in the protein bodies with a role in glycosylation and deglycosylation of proteins and thereby in protein routing and protection against hydrolytic degradation (Faye et al., 1988) Also, it acts on short chain oligosaccharides (Jagadeesh et al., 2004) during its role in glycoprotein processing ( Winchester and Fleet, 1992) galactosidase by comparison has the majority of its activit y bound to the cell wall (Burns, 1990a; Dick et al., 1984; Murry and Bandurski, 1975; Tanimoto, 1985) with a hydrolytic effect on the neutral

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60 glycosidic linkages of pectic polysaccharides and hemicellulose fractions of the cell wall (Ranwala et al., 1992 ). This information is consistent with re ports that citrus pectin has side chains which composed of D -galactose and L arabinose that occur most frequently, while D xylose, D -glucose, D -mannose, D apiose, and L -fucose occur rarely (Darvill et al., 1978). During citrus peel development, flavedo galactosidase remained stable, -galactosidase de creases toward maturity, and then became stable, but -mannosidase increase gradually with maturation. The same trend occurred in albedo tissue, but was more attenuated (Burns and Baldwin, 1994). The activit y of galactosidase is associated with actively growing tissue where high levels of the enzyme were correlated with cell wall loosening mechanisms responsible for growth (Tanimoto, 1985). It has been reported in acid lime ( Selvaraj and Raja, 2000) and Kinnow mandarin (Ram et al., 2002) -galactosidase activity in peel and juice tissue increased from stage 1 to stage 3 of fruit development, then decreased at subsequent stages. This activity was higher in juice tissue than in peel tissue throughout the maturation period, and the enzyme was positively correlated (r2 = 0.505) in peel and negatively correlated (r2 = 0.585) in juice tissue with fruit firmness. Burns (1990a ) found in Valencia oranges jui galactosidase activity declined after eight -week of storage at 10oC (50oF). Gibberellic acid retarded the rate of peel softening by retarding the seasonal change and amounts of cell wall galactosyl, arabinosyl and fucosyl residues (Mitcham and McDonald, 1993) -galactosidase was remarkably enhanced by 2,4 D only in the concentration range which induced growth (107105 M), while higher concentrations (104103 M) inhibited enzyme activity (Tanimoto and Igari, 1976 ). Water stress stimulated the activity of -mannosidase causing de terioration of cell walls (El Tayeb and Ahmed, 2007)

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61 Absc isic Acid (ABA) Plant growth and development are controlled by the coordinated actions of positive and negative regulators, as well as environmental conditions (Davies, 1987). Early experiments led to the identification of a group of growthinhibiting compounds, including a substance known as dormin purified from sycamore leaves during the dormancy period i n autumn (Ohkuma et al., 1963), which was chemically identical ( Addicott et al., 1968) to a substance th at promotes the abscission of cotton fruits, abscisin II, that was renamed abscisic acid to reflect its involvement in the abscission process (Cornforth et a l., 1965). Subsequently, it was noticed that abscisic acid increased considerably when wheat leaves wilt (Wright and Hiron, 1969), and it had a n inhibitory effect that caused stomata to close (Mittelheuser and Van Steveninck, 1969) reflecting its role as an anti-stress hormone that functions as an endogenous regulator of plant transpiration, ameliorating the effects of water stress (Creelman, 1989). Due to its potent function in plant and seed dormancy, in retrospect, dormin would have been a more appropriate name for t his hormone, but the name abscisic acid (ABA) is firmly entrenched in the literature (Taiz and Zeiger, 2002). Abscisic acid (C15H20O4) is one of the five major plant hormones (auxins, gibberellins, cytokinins, ethylene, and abscisic acid), and a member of the sesquiterpenoid group of the terpenoid family of chemicals, which includes a number of essential oils, insect hormones, steroids, gibberellins, carotenoids, and natural rubber. Its biosynthesis takes place in several dif ferent parts of the plant ( Kojima et al., 1994 ; Milborrow and Robinson, 1973), and mainly in chloroplasts then move to every living tissue in plant from the root cap to the apical bud (Taiz and Zeiger, 2002; Xion and Zhu, 2003) This plant hormone is usually produced in roots in response to environmental stress (i.e. drought), and works as a root signal to inhibit stomatal opening and reduce transpiration in leave s (Artsaenko et al., 1995; Davies and Zhang, 1991;

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62 Gomes et al., 2003) by inhibiting K+ inward channels which are required for stomatal opening (Schwartz et al., 1994). ABA also promotes root growth and inhibits shoot growth under water stress conditions (Sharp et al., 2000) The hormone concentrations in leaves under drought stress can increase up to 50 times (from biosynthesis rather than fr om conversion of ABA -conjugate), which is the most dramatic change in the concentrations of any hormone in response to environmental stress (Norman et al., 19 90). It is clearly involved in leaf and fruit senescence and directly (by increasing ACC synthesis; precursor of ethylene) (Gomez -cadenas et al., 2000; Goren, 1993; Goren et al., 1979; Okuda, 1999; Riov et al., 1990; Sagee et al., 1980) or indirectly (by accelerating senescence) (Jackson and Osborne, 1972; Lieberman et al., 1977) increasing ethylene production that increases cellulase and polygalacturonase activity ( Jackson and Osborne, 1972; Kazokas and Burns, 1998; Rasmussen, 1974) and pr omotes leaf and fruit abscission ( Lieberman et al., 1971 ; Rasmussen, 1974; Sagee et al., 1980 ; Taiz and Zeiger, 2002). On the other hand, ethylene can be activated by water stress (Yang and Hoffman, 1984) and may also increase free abscisic acid in th e flavedo of citrus fruit enhancing senescence (Goldschmidt et al., 1973; Lafuente et al., 1997; Lafuente and Sala, 2002) Abscisic acid (ABA) of citrus was first detected in lemon fruit skin and pulp (Milborrow, 1967) then shown to occur in orange ( Goren and Goldschmidt, 1970) and grapefruit ( Ali Dinar, 1980). Among fruit trees, citrus is very high in ABA, and young developing leaves have substantial capacity for synthesis of stress induced ABA (Norman et al., 1990) within 2 3 hours of stress (Milborrow, 2001) to induce stomatal closure (Neales and McLeod, 1991) .The content of ABA (free and conjugated) in citrus fruit is significantly correlated with fruit weight, and it is higher than that of leaves ( Aung et al., 1991). The chloroplast has been po stulated as a major site for ABA biosynthesis (Milborrow, 1974a) except in guard cells where chloroplasts are unable to

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63 convert carotenoids into ABA ( Parry et al., 1988), so that stomata are entirely depend ent on ABA coming through the apoplast, as guard cell lack plasmodesmata ( Sanchez, 1977). Flavedo has been shown to accumulate large amounts of both free and conjugated ABA during senescence ( Goldschmidt et al., 1973), and this indicates that citrus fruit peel (flavedo) may have a high capacity for ABA biosynthesis ( Aung et al., 1991; El Otmani et al., 1995). This varies with cultivar, for example Red Blush grapefruit peel has significantly less ABA than that of Marsh white grapefruit (Hodgson, 1967). Also, Washington navel oranges and Satsuma mandarins differed significantly in ABA content ( Furio et al., 1981). In general, the amount of conjugated ABA (10 -fold that of free ABA (3 depending on citrus species ( Aung et al., 1991). Free ABA is an active form of ABA localized in the cytosol, while conju gated ABA is an inactive form of ABA that covalently conjugated with another molecule, s uch as a monosaccharide (ABA D -glucosyl ester), which accumulates in the vacuoles and serves as a storage form of the hormone (Taiz and Zeiger, 2002). ABA may also be held by some proteinaceous materials, and in the form c alled fettered ABA ( Vittone and Milborrow, 1999). The highest ABA content reportedly was found in the fruit exocarp, and the highest ABA -conjugate was found in the central axis of the fruit ( Harris and Dugger, 1986). During fruit development, the activity of endogenous ABA is greater in the peel than that in the pulp (Josan et al., 1999). The decline of ABA levels was associated with a delay in color change ( Valero et al., 1998), and the increase in ABA of the flavedo was associated with peel senescence and the development of the chloroplasts into chromoplasts (Harris and Dugger, 1986; Martinez -Romero et al., 1999; Nooden, 1988). It was reported that conjugated ABA of navel orange fruit increased 12.6 fold at the time of the color break stage (Harris and Dugger, 1986), and there was a shift in free ABA at the time of chloroplast transformation to chromoplast in

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64 mandarin (Lafuente et al., 1997) It has been reported that increase in ABA level after water stress was associated with increase in sugar content of flavedo tissue of Satsuma mandarin (Kuraoka et al., 19 77; Okuda et al., 2002), and this may be related to the effect of ABA to play a role in the accumulation of assimilates from the phloem into the fruit by strengthen sink capaci ty (Brenner et al., 1989; Kojima et al., 1994; Kojima et al., 1995; Okuda et al., 2002) This increase in ABA level could be related to the beginning of fruit senescence ( Valero et al., 1998 ). Senescence of citrus fruit involves an increase in ABA, which occurs within the first 48 hours before other senescence phenomena become visible. Lafuente et al (1997) found that ABA levels in the flavedo of Fortune mandarins increased concomitantly with the susceptibility to chilling injury and color change from green to orange. The hormonal balance between ABA and other plant hormones affects the intensity of senescence phenomena (Goldschmidt et al., 1970). For example, increases in cyto kinins and gibberellins have inhibitory effect on ABA formation and senescent color change (Brisker et al., 1976; Goldschmidt et al., 1970; Murti, 1988). Increases in fruit ABA along with decreases in gibberellins are closely related to the onset of senescence and the appearance of puffing physiologica l disorder in mandarins (Goldschmidt et al., 1970; Kuraoka et al., 1977). Increases in ABA along with decreases in auxins induce the onset of endogenous ethylene production that influences fruit abscission in water -stressed citrus trees (Gomez cadenas et al., 2000; Guinn and Brummett, 1988; Rasmussen, 1974; Taiz and Zeiger, 2002; Takahashi et al., 1975 ). In this regard, IAA and AB A are antagonistic and have opposing effects (Jackson and Osborne, 1972) delaying or accelerating senescence, respectively (Osborne, 1967). Volatile C omponents The oil glands in the flavedo of the peel contain most of the essential oils. This oil contains chemicals discharged by metabolic processes of the fruit, which contri bute to each type of citrus

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65 to its characteristic aroma and flavor. The prominent chemical classes present in the oil are terpenes, and the hydrocarbon, dlimonene, is the major constituent, but provides little to no direct aroma impact to citrus. The char acter of the flavor is mostly dependent on oxygenated terpene derivatives, aldehydes, ketones, esters, alcohols, and acids ( Braddock, 1999). In orange oils, 111 volatile components have been found, including 5 acids, 26 alcohols, 25 aldehydes, 16 esters, 6 ketones, and 31 hydrocarbons but not all of them have aroma activity Nonvolatile compone nts compose about 1.5%, including waxes, coumarins, flavonoids, carotenoids, tocopherols, fatty acids, and sterols. Grapefruit oil consists of 20 alcohols, 14 aldehydes, 13 esters, 3 ketones, and 14 monoterpenes and sesquiterpenes. The nonvolatile portion composes about 7% of the oil, including coumarins, flavonoids, tocopherols, and waxes ( Kimball, 1999). The quality of citrus oil is dependent on several factors, such as soil, climate, method of extraction, weather during extraction fruit cultivar, and fruit maturity (Wolford et al., 1971). Concentrations of many rind oil constituents vary during maturation and senescence ( Scora and Newman, 1967 ). Quantitative analyses of these constituents are important, p articularly in relation to fruit quality ( Wilson and Shaw, 1980). Volatile aldehydes, such as octanal, decanal, dodecanal, neral and geranial (citral), are among the compounds believed important to grapefruit flavor, and total aldehyde content is often used as a quality index for citrus oils ( Kesterson et al., 1971). Total aldehydes are measured as decanal, since it is often the main aldehyde in grapefruit oil (Wilson and Shaw, 1980). Nootkatone, a ketone, is a major flavor impact compound in grapefruit (MacLeod and Buigues, 1964; Shaw and Wilson, 1981) with a typical grapefruit aroma and low odor threshold ( Berry et al., 1967). It increases with fruit maturity (Del Rio et al., 1992) and was associated with a linear increase in maturity index ( TSS: acid ratio), and it also increased durin g storage (Biolatto et al., 2002 ; Sawamura et al., 1989). H owever, cold storage

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66 and wax application decreased it ( Sun and Petracek, 1999). Oils processed late in the season (May June) contain the highest nootkatone levels (0.75 0.81%) ( MacLeod and Buigues, 1964; Wilson and Shaw, 1980). So, it might be seen as an indicator of maturity ( Drawert et al., 1984) and quality ( Biolatto et al., 2002 ) in grapefruit. Nootk atone is formed from a sesquiterpene called valencene (Drawert et al., 1984; Shaw and Wilson, 1981), and therefore nootkatone is classified as a valencene-class sesquiterpene ketone (Sauer et al., 2003). Valencene can be detected 9 weeks after anthesis and reaches its maximum level 19 weeks after anthesis, then decreases and becomes stable betw een weeks 20 and 53, whereas nootkatone can be detected after 29 weeks of anthesis and then rapid increase takes place between weeks 33 and 53 (Del Rio et al., 1992 ; Del Rio et al., 1991 ). Valencene is present in a minor quantity in orange, but plays an important role in the overall flavor and aroma of orange fruit ( Vora et al., 1983; Weiss, 1997). It is the character impact compound of Valencia orange (Choi, 2003; Tonder et al., 1998), formed in flavedo and increases as the fruit matures (Coggins et al., 1969a ; Del Rio et al., 1992; Elston et al., 2005; Maccarone et al., 1998 ; Sharon -Asa et al., 2003; Shaw and Coleman, 1974 ). Sharon -Asa et al (2003) examined the expression of the Cstps1 gene, a key gene in the production of valencene and found that it is developmentally regulated and encoded to valencene synthase that accumulates only towards fruit maturation, which corresponds with the timing of valencene accumulation. Moreover, they found that valencene accumulation and valencene synthase gene expression responded to ethylene, providing further evidence for the role of ethylene in the final stages of citrus fruit maturation, althou gh citrus are non -climacteric. Most of the nootkatone (97%) and valencene (99.5%) are found in the flavedo, and both of them are found in the pulp, but only nootkatone was found in the albedo (Del Rio et al., 1992; Ting and Attaway, 1971). In addition to valencene, Choi et al (2001) and Sawamura et al (2005) reported some other terpenes

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67 -pinene. O ther minor carbonyl -containing components that may be important to the flavor and aroma of grapefruit oils are esters, such as octyl acetate, citronellyl acetate, neryl acetate, and geranyl acetate. These acetate esters are among the major oxygenated compo nents of grapefruit oil, although not of orange oil (Moshonas, 1971). Oxygenated compounds, mainly alcohols (octanol & linalool) and aldehydes -sinensal) are important for characteristic orange aroma ( Kealey and Kinsella, 1979; Sawamura et al., 2005). Early season oil showed lower linalool, decanal, and dodecanal than late and midseason oils, which were similar in composition ( Shaw and Coleman, 1974 ). Kesterson and Hendrickson (1958) observed an increase in the aldehyde content from 1.50% to 1.85% in the more mature fruit. Linalool was the major alcohol in orange oils (Sawamura et al., 2005) and the most phytotoxic compound in citrus oil glands (Wild, 1992). Its oxidatio n product linalool oxide was reported in mandarins (Njoroge et al., 2005) Generally, alcohols have been identified as being the most important contributor to orange fl avor (Shaw, 1979). Gibberellic acid delays sen escence and increases oil content of the peel ( McDonald et al., 1997; Wilson et al., 1990). H owe ver, it also affects the concentrations of oil constituents, which are related to the degree of peel senescence (Coggins et al., 1969a ). GA3 reduced the rate of increase of nootkatone during maturation ( Wilson et al., 1990). The concentration of many oil components was not affected by GA3, but valencene concentration was concomitant with biochemical and physiological changes associated with senescence, which makes it possible to use it as an indicator of the maturity of orange s (Coggins et al., 1969a ; Lewis et al., 1967). Octanol fluctuated wide ly from one harvest date to another, which makes it unreliable as a senescence index (Coggins et al., 1969a ).

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68 Water stress affects the content of volatile components of the fruit. Since it induces carbon caryophyllene and trans -ocimene, which represent about 82% of the total terpenoid volatil e emission from citrus leaves, and is reduced to 6% under severe drought stress. The effect of drought stress is closely related to the surrounding temperature. Mild drought stress induced decreases in trans -caryophyllene showed no res ponse (Hans en and Seufert, 1999) These results were confirmed in Mediterranean Cypress (Yani et al., 1993), Holm Oak (Bertin and Staudt, 1996), and Aleppo pine (Ormeno et al., 2007) Volatile components tend to be lost during prolonged storage (Ting and Attaway, 1971). Wax application to grapefruit before storage decreases the amount of nonanal and nootk atone in -phellandrene, 3 -carene, ocimene, and octanol increased as temperature increased during storage, but limonene level decreased. As the storage time is -pinene, limonene, linalool, citronell -terpineol, neral, dodecanal, and humulene decreased, but levels of nootkatone, ocimene, 3-phellandrene increased. None of these compounds were affected by the interactive effect of waxing and temperature except nootkatone, which decre ased at low temperature. Nonanal is the only components affected by the interactive effect of waxing and storage duration, decreasing with long storage time, and thus, both components may be considered as candidates for senescence indicators because long s torage time and high temperature promote senescence ( Sun and Petracek, 1999). It is also known that when oranges were kept in cold storage for periods longer than 6 weeks prior to oil extraction, the physiochemical properties of the oil changed, accompanied by a decrease in the aldehyde content ( Wolford et al., 1971), and formation of some components, such as p terpinene due to oxidation of terpenes, indicating that

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69 the oil lost it s freshness ( Smith et al., 2001; Usai and Arras, 1992). Water loss (~5%) from lime fruit caused marginal quantitative changes in composition of terpenes, alcohols and esters (Yada v et al., 2004). Extraction of the volatile constituents from oil samples can be done by a solid phase microextraction (SPME) technique. SPME is a fast, solventless alternative to conventional sample extraction techniques. It is useful in many diverse an alyses, including characterization of flavor components in foods and beverages and fragrance compounds in a wide range of products. SPME requires no solvents or complicated apparatus. It is an adsorption/desorption technique, where analytes establish equil ibria among the sample matrix, the headspace above the sample, and stationary phase coated on a fused silica fiber. They are thermally desorbed from the fiber onto the gas chromatography (GC) column for separation, and then move to the mass spectrometry (M S) for identification, using a GC MS device (Marsili, 1999; Supelco, 2000) Like a good marriage, both GC and MS bring something to their union. GC can separate volatile and semivolatile compounds with great resolution, but it cannot identify them. MS can provide detailed structural information on most compounds such that they can be exactly identified, but it cannot readily separa te them. Therefore, it was not surprising that the combination of the two techniques was suggested shortly after the development of GC in the mid1950s (Hites, 1997)

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70 CHAPTER 2 CHARACTERIZATION OF CITRUS PEEL MATURATI ON AND THE EFFECT OF WATER STRESS, GROWTH REGUL ATORS AND DATE OF HA RVEST ON POSTHARVEST LIFE OF THE FRUIT DURING STORAG E State governments in citrus producing regions have established maturity laws requiring that citrus fruits meet certain quality standards before they can be shipped or sold to prevent the shipment of immature and unpalatable fruit in order to protect the consumer by assuring that only high quality fruit is offered for sale on the fresh market. They also protect the producer by insuring customer confidence in the fruits offered for sale, and by promoting an orderly procession of mature, quality fruit to the market place. These laws result in permitted grades of citrus that have the following common requirements: all fruit must be mature, of similar varietal characteristics and be free from bruises, cuts not healed, decay, growth cracks and insect larvae (Taylor et al., 1994) Fruit maturity can be determined by measuring total soluble solids (TSS), acidity, TSS: acid ratio, and juice volume, but not by peel color, which is related to cold temperature during winter ra ther than fruit maturity ( Young and Erickson, 1961). A citrus fru it whose skin is green may be ready for harvest based on good internal quality. Citrus fruits do not "ripen" in the general sense of the word as it is applied to other fruits; instead, they mature to good eating quality. Peel color is not an indicator of p ulp maturity, as the peel undergoes natural degreening in November and December, and peel color of 'Valencia' oranges often regreens after the spring growth flush occurs (Sauls, 1998) Citrus fruit is nonclimacteric and, as such, do not exhibit a well defined period of rapid conversion of storage starch to sugars or other soluble products ( Soule and Grierson, 1986). I nstead, compositional changes occur gradually, and hence the internal portion of the fruit, the endocarp, becomes edible. This period has been termed maturation. The inedible portion of the fruit, the peel or exocarp, also matures. Although many compos itional changes occur in the peel, maturity in

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71 this tissue often refers to susceptibility of the peel to injur ies and decay (Burns and Baldwin, 1994). The point at which citrus peel passes from an immature t o a mature state, as well as from mature to senescent has been difficult to define, in part, because of the gradual and slower nature of physiological changes in this tissue, however physiological changes (e.g. enzyme activity, polyamines, volatile compone nts etc) that occur during these two transition periods could be used to define peel maturity more accurately (Burns and Baldwin, 1994). A unique aspect of citrus is the potential that maturity and qualit y of peel may be separate from maturity and quality of the internal edible portions. Further, even edible quality is a human perception and may not be tightly linked to internal maturity. External peel characterization may or may not follow internal grade standards of the pulp. Since, it is imperative to make sure that citrus fruits meet the internal maturity standards when harvested, it is also important to characterize peel maturation to determine its susceptibility to be damaged during pre -packing and ha ndling treatments, as well as its susceptibility to decay and physiological disorders during storage and marketing. Florida citrus may be harvested over a very long season compared to other horticultural crops. For example, Florida grapefruit may meet inte rnal maturity standards and be harvested by September or October or not harvested until March or April. Hence, the harvesting window extends beyond 6 months. Early and late season fruit often show higher susceptibility to peel disorders, such as chilling i njury (Ritenour et al., 2003) In the fresh citrus industry, use of growth regulators is a way to control citrus fruit maturation, and hence, harvest date, based on the market demand. GA3 application either singly or c ombined with 2, 4 D has been shown to modify color development by delaying chlorophyll degradation (Coggins and Hield, 1958; Embleton et al., 1973), improve peel strength and reduce

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72 softening rate (Coggins, 1969b ; Coggins, 1981) and to reduces or control several physiological blemishes and certain diseases of the peel (Coggins, 1969b ; Grierson 1981) allowing later harvesting ( Agusti et al., 1981 ). On the other hand, water stress is a way to promote early harvesting since it increase juice sugar and citric acid (Hilgeman, 1977) and improve chlorophyll degradation and color development of the peel ( Morinaga et al., 1985 ), however, water stress is not used commercially to alter maturity, so far. Based on the above information and previous research work, it is apparent that no maturity indices of citrus peel exist, no one has defined exactly the maturity indices of citrus peel; therefore the first aim of this work is to try to define citrus peel maturity and senescence accurately. In order to define citrus peel matu ration more accurately, the first aim of this work is to define citrus peel maturity and senescence accurately by determin ing peel changes that characterize citrus peel maturation and senescence, and also by studying factors or conditions that might affect citrus peel maturity and storability. These factors (i.e. water stress and growth regulators) were used to presumably make differences in maturity (i.e. advance or delay) and see if any physical or chemical measurements show corresponding changes, which might indicate that they could be used to indicate stage of peel development (immature, mature, senescent). Materials and Methods Field Experiment (Season 2004/2005), Effect of Harvest Date This experiment was carried out during the 2004/2005 season using fruit from grapefruit trees ( Citrus paradis i Macf. c v. Marsh) at the University of Floridas Citrus Research and Education Center (CREC) in Lake Alfred, Florida (28O O

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73 Marsh grapefruit fruits were harvested every month starting from Sept ember 2004, whenever the TSS: acid ratio = 6:1 to represent immature, as legal maturity starts at 7 .5 :1 (Soule and Grierson, 1978; Wardowski et al., 1995) until June 2005. Appropriate number of fruits were harvested out of each replicate from all sides of the trees and from three different height levels (high medium and low) of the canopy then fru it were transported to the CRECs packinghouse where fruit were washed over polyethylene brushes with commercial detergent, waxed (Sta Fresh 925, FMC Food Technologies, Houston, TX, USA), dried and visually selected free of external defects. This was one way experimental design with three replications. Field Experiment (Season 2005/2006), Effect of Harvest Date, Water Stress and Growth Regulators This experiment was carried out during the 2005/2006 season using fruit from grapefruit (Citrus paradis i Macf. c v. Marsh) and Valencia orange (Citrus sinensis [L.] Osbeck) trees in two different locations at the University of Floridas CREC in Lake Alfred, Florida (28O 81O Water stress was developed in grapefruit and Valencia orange trees in two different groves by with-holding irrigation for two and half months during the period from November 15th through February 1st. Fruit from these plots were compa red to fruit from irrigated plots. Each irrigated and non irrigated plot had 10 trees with three replications. The plant growth regulators (GA3 and 2, 4 D) were applied to subplots of the irrigated and non irrigated plots by spraying 5 trees per plot wi th GA3 20 mg/L (ProGibb 4% [Gibberellic acid 4% w/w], Valent Corp., Walnut Creek, CA, USA) in combination with 2,4 D, 10 mg \ L (2,4 D selective weed killer [2,4 -Dichlorophenoxyacetic acid 11.84%] American brand, Nufarm Americas Inc., Burr Ridge, IL, USA). The spray was applied December 5th, at the stage of fully enlarged green fruit according to Tadeo et al (1988), Medeiros et al (2000) and Ram et al (2002).

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74 This was a factorial experimental design with three replications. Within each irrigation treatment plot, five trees had growth regulators (GA3 and 2, 4 D) and five trees did not. Another experime nt on Valencia orange trees in a different grove at CREC was carried out to compare non -irrigated plots, covered with Tyvek (DuPont Tyvek, Wilmington, DE, USA) for four months during the period from November 17th through March 21st, to irrigated trees. E ach irrigated and non irrigated plot had ten trees with three replications. Marsh grapefruit fruits were harvested every two months starting from September 2005, when the TSS: acid ratio = 6:1 to represent immature, until July 2006. Valencia orange fruits were harvested every two months starting from January 2006 until July 2006. Appropriate numbers of fruit (100) were harvested out of each one of the 3 replicate s from all sides of the trees (north, east, sout and west) and again from three differe nt heights in the canopy (low, medium and high) the n fruit were transported to the CRECs packinghouse where fruit were washed over polyethylene brushes with commercial detergent, waxed (Sta -Fresh 925, FMC (now JBT) Food Technologies, Houston, TX, USA), d ried and visually selected free of external defects. Field Experiment (Season 2006/2007), Effect of Harvest Date, Water Stress and Growth Regulators This experiment was carried out the same as the second season with some minor changes, as follow: Water st ress by not irrigating was carried out during the period from November 14th through February 5th For the Valencia orange experiment with soil covered by Tyvek, the period was for three and half months from December 5th through March 23rd, and this was done in a different grove at CREC. No harvest was collected in July for grapefruit.

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75 Storage Experiment (Season 2004/2005 and Season 2005/2006) Fruit from each replicate at each harvest date were stored in two cartons; one carton (30 fruits) at 40OF (4.4OC ) and 85 90% RH and another carton (30 fruits) at 70OF (21.1oC) and 8590% RH for 12 weeks (3 months). This 3 months of storage is mainly to carry fruit post normal harvest dates weather every month (first season) or every two months (second season) to see fruit behaviour by the new harvest date, and the possibility to store fruit for long periods to extend the maketing window. Fruits were analyzed at harvest and after 2, 4, 6, 8, 10 and 12 weeks. Factors evaluated at harvest were TSS, acid, TSS: acid ratio, fruit weight, fruit color, peel peel turgidity and FDF. From each replicate, 10 fruit were harvested with their stems to measure FDF at harvest. Factors evaluated during and after storage were fruit weight loss, decay and chilling injury (CI). Factors eva luated at the end of storage period were fruit color, peel peel turgidity, TSS, acid and TSS/acid ratio. TSS, acidity, and TSS: acid ratio Fruit juice analyses were done in the juice processing laboratory at CRECs packinghouse, using FMC equipment for jui ce extraction and analysis, during the first and second seasons. During the third season, juice analysis was done using a benchtop Refractometer (Abbe 3L, Bausch & Lomb, Rochester, New York, USA) on the translucent part of the juice after decantation to me asure TSS. Titratable acidity was done according to the AOAC method (using 25 ml juice, 3 drops of phenolphthalein, NaOH, 0.1N and end pH 8.3) using a regular pipette with acidity calculated and a stirrer plate ( AOAC, 1997). All analyses were done by the same person at room temperature and results were expressed as oBrix and % citric acid. Peel color Fruit color was measu red using a Konica Minolta chromometer (measuring head model CR 300 and data processor model DP 301, Konica Minolta Sensing America Inc., Ramsey, NJ,

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76 USA). Fruit color was analyzed using the Hunter L (chroma) a (hue) and b (density) scale (Hunter associ ates Laboratory, Inc. Reston, Virginia, USA), and the color index was calculated using the following formula; (1000 a) / (L b) (JimenezCuesta et al., 1981). Peel color was measured on three dif ferent spots on the equatorial part of the fruit and averaged. Color index changes from minus values in green fruit to positive values in orange fruit. Firmness / peel turgidity / turgor pressure Peel peel turgidity was measured at room temperature using a Wagner fruit tester (3mm probe diameter) (FT Series, Wagner Instruments, CT, USA) on three different spots on the equatorial part of the fruit and averaged. Readings were recorded in Kg when disruption of oil glands occurred. Fruit detachment force (FDF) Detachment force was measured with a Wagner Force One device (Wagner Instruments, CT, USA). Fruit stem (peduncle) was clipped to 5cm in length, inserted into the gauge, and the stem pulled parallel to the fruit axis. Readings were taken in Kg when detachm ent of the fruit from the stem occurred. Weight loss Measurement of fruit weight loss was done for 10 fruit per replicate by weighing the fruit at harvest date and every two weeks during storage until the end of the storage period. The difference as a per centage from the original weight (at harvest) was calculated. Decay & c hilling injury (CI) Fruit decay and CI were measured visually every two weeks until the end of storage period. The number of infected/injured fruit were counted and presented as the p ercentage from the original number of fruit/box (30). Fruits that had a minimum of three different chilling-

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77 injured spots were considered chilling -injured and removed from the box. Fruits that had any kind of decay were removed from the box. Statistical An alysis Data were analyzed by SAS 8.2 (Statistical Analysis System) using ANOVA (Analysis of Variance) and means were compared using DMRT (Duncan Multiple Range Test). Standard error of the means were also calculated by SAS ( SAS Institute Inc., 1999). Results and Discussion Effect of Harvest Date and Storage Conditions TSS, acidity, and TSS: acid ratio During three seasons of experiments, grapefruit were harvested in September when the TSS: acid ratio = 6:1 to represent immature fruit, before the legal maturity was reached with a ratio of 7 .5 :1 (Soule and Grierson, 1978; Wardowski et al., 1995) Evaluation of this ratio was done using some fruit samples from different positions in the field, as well as from different leve ls of the canopy (low, medium and high) using a hand held refractometer to measure TSS and a pipette to measure acidity H owever, during the second season this ratio was closer to the legal maturity, and it was below 6:1 in the third season, as shown in Ta ble 2 1 and Figure 2 1. An increase in TSS and a decrease in acid content were obtained with delayed harvest date, and this was reflected in the increasing TSS: acid ratio as harvest date and maturity progressed. These findings are in agreement with previous reports and mainly relate to the consumption of acid in respiration rather than increasing soluble solids (Rana and Sing, 1992; Samson, 1986; Ting and Attaway, 1971 ). This acid consumption resulted in highly significant TSS: acid ratio by the end of the harvest season, compared with the earlier harvest dat es during all three seasons. As seen in Figure 2 1, the maturity index ( TSS: acid ratio) increased gradually during the season

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78 (Daito and Sato, 1985) and remain constant between January and March, then increase after March. T he month of March was the change point in TSS: acid ratio of grapefruit b ecause of the reduction in acids due to respiration This is also reflect that may be January is the beginning of fruit maturity according to Harding and Fisher (1945) who stated that a fter maturation, there is a tendency for the TSS to remain more or less constant. The changes in TSS: acid ratio took place more slowly in grapefruit than in Valencia orange (Chace and Church, 1924 ), as shown in Table 2 2 and Figure 2 2. Valencia oranges were harvested in January; one month early before the legal maturity ( TSS: acid ratio = 12:1) starts in February ( Ladaniya, 2008 ). Maturity index w as 10.5:1 and 11.5:1 in 2005/06 and 2006/07 seasons, respectively, and it increased consistently from January to May, Table 2 2, and became significantly different after May of the 2005/06 season only, which can be considered the change point in TSS: acid ratio of Valencia. The higher TSS: acid ratio in July of 2005/06 season compared to that in July of 2006/07 season is possibly related to acid loss at higher temperature; 78.4oF (25.8oC) compared to 77.2oF (25.1oC), respectively, Figure B 1, appendix. P eel color The color index of grapefruit changed significantly between September and November during the three seasons as shown in Table 2 3 and Figure 2 3, and then increased insignificantly with the progress of the season (may be with increase in temperat ure with the progress of the season) By the end of the sampling period there was an insignificant reduction in color index, perhaps due to natural regreening of the fruit. This is in agreement with previous report stated that peel undergoes natural degree ning in November and December, and peel color of 'Valencia' oranges often regreens after the spring growth flush occurs (Sauls, 1998) A similar trend was also observed in Valencia orange, with a change in color i ndex by March, Table 2 4 and Figure 2 4. The significant change in color index from January to March of 2005/06 season, Table 24,

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79 was coincided with increase in temperature from 63.7oF (17.6oC) in January to 68.4oF (20.2oC) in March, Figure B 2, appendix. Peel color is not an indicator of pulp maturity (Sauls, 1998) Natural regreening of citrus fruit can occur at warm temperature during late spring and summer season, and is considered a serious problem in Valencia orange in Southern California and some years in Florida (Hsu et al., 1989; Thomson et al., 1967) Firmness / peel turgidity / turgor pressure In grapefruit, tissue turgidity fluctuated from one harvest date to another over the three seasons, Table 2 5, but the general pattern of fruit firmness was decreasing with fruit maturation (Grierson, 2006a ), and the correlation between harvest date and fruit firmness showed small R2 values, as shown in Figure 2 5. The values was higher in Valencia orange wit h a strong correlation in the third season ( R2 = 0.99) ( Figure 2 6 ). Also, Valencia orange showed a gradual decrease in fruit firmness over the 2005/06 and 2006/07 seasons (Figure 26), compared to grapefruit, which was almost constant in the first two s easons (Figure 2 5). Moreover, Valencia orange showed a higher range of peel turgidity (4.71 9.71 Kg), Table 2 6, compared to grapefruit (4.67 7.07 Kg), Table 2 5. This is may be due to the thinner peel and the lower peel water content of Valencia orange compared to Marsh grapefruit (Oberbacher, 1965). Fruit detachmen t force (FDF) As the growing season progressed and the internal fruit maturity increased, FDF decreased gradually, with a tendency to increase again toward the end of the season. This was clearer in the second and third season for Marsh grapefruit and th e second season for Valencia orange, Tables 2 7 and 2 8, and Figures 2 7 and 2 8. The FDF of grapefruit during the first season was nearly stable until the last sampling, Table 2 7. These results are in agreement with previous reports (Juste et al., 1988; Ladaniya, 2008 ). The increase of FDF at the end of the growing season may be due to the later time of harvest (March to May in grapefruit and May to July in

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80 Valencia) coinciding with new flushes, young fruit development for the following seasons crop, and more root grow. These young growing tissues are rich sources in endogenous plant hormones (Goldschmidt, 1976; Hofman, 1990; Plummer et al., 1991). Therefore, it has been speculated that endogenous hormones from these young tissues reduce the abscission of mature fruit. T his is mainly related to the high ratio of IAA to ABA at the abscission zone in the calyx (Rasmussen, 1973; Yuan et al., 2001) Weight loss Fruit stored at 40oF did not show any significant change in water loss duri ng storage (Fig. 2 9 and 2 10). F ruit harvested in September and December had the lowest % weight loss during storage at 70oF, compared to other harvest dates, Figure 2 11. At 40oF, the percent of weight loss of December -harvested fruit after 12 weeks was 3.39%, Figure 2 10, at which grapefruit showed slight shrinkage, compared to full deformation of December -harvested fruit stored at 70oF, which lost 8.45% of their initial weight Figure 2 11. In general, as shown in Table 2 9 and Figure 2 9, during the fi rst season, fruit collected in December lost 2.16% and 5.17% at 40oF and 70oF, respectively, but the lowest percent of weight loss during the second season was 2.56% for January harvested fruit stored at 40oF and 2.83% for March fruit stored at 70oF. At th ese two harvest dates, fruit showed the lowest percent of weight loss every two weeks during storage, as shown in Figure 2 12 and 2 13. Valencia orange numerically showed the lowest percent of weight loss at 40 and 70 degrees (3.64% and 4.44%) for fruit harvested in March, Table 2 10 and Figures 2 14, 2 15 and 2 16. Although some of these values were not significantly differ from other harvest dates, they still showed the best fruit condition, fruit were still marketable, except for the first seasons gra pefruit stored at 70oF, which lost more than 5% of water. According to Grierson and Wardowski (1978), oranges start shrinkage at weight loss of 2.5%, and become unsalable at 5% of the original weight. In general, these data are in agreement wit h previous

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81 reports in terms of the affect of higher storage temperature resulting in higher % weight loss, because of higher transpiration rate of the fruit under higher evaporative demand (Grierson and Wardowski, 1975; McCorna ck, 1975) Also, from Tables 2 9 and 2 10 and Figures 2 9 and 214, it is obvious that at 40oF % weight loss in Valencia orange is higher than that of Marsh grapefruit at all harvest dates, and this is mainly related to fruit size, because transpirat ion rate is greater in smaller fruits, apparently due to large surface area : volume ratio in orange than grapefruit (Haas, 1927). Peel shriveling due to water loss is immediately visible, causing fruit dull appearance, softening, and peel senescence (Ben Yehoshua, 1969; El Otmani, 2006). It can be concluded that the period from December to March is the best time to harvest Marsh grapefruit, which has long harvest season (September May), and the month of March is the best time to harve st Valencia orange, which has short harvest season (February June) ( USDA, 2004). This is mainly to harvest fruit with less senescent peel which means less susceptibility to postharvest problems such as decay and physiological disorders compared to mature peel (Burns and Baldwin, 1994). Decay Data displayed in Table 2 11 and Figures 2 17, 218 and 219 showed that during the first season, stored grapefruit at both 40oF and 70oF had numerically the lowest % decay when harvested in November and the highest for the March harvest. During the second season, September and November showed no decay for fruit stored at 40oF, and fruit for these dates showed the lowest decay compared to other harvest dates at 70oF, whereas the highest decay was in July at both temperatures, as sho wn in Figure 2 20 and 2 21. Regarding Valencia orange, the highest % decay was in July at both temperatures, but the difference was only significant at 40oF, and the lowest % decay, numerically, was in January at 40oF and March at 70oF, Table 2 12 and Fi gures 2 22, 2 23 and 2 24. The percent of decayed fruits increased as the fruit maturity

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82 progress, and the effect was higher at high temperature, Tables 2 11 and 212. These results are in agreement with previous reports; as fruits become older, susceptibi lity to postharvest diseases increases, because peel becomes less firm and hence less force is required to puncture the peel (Coggins et al., 1969a ). Storage at low temperature and high humidity is beneficial to the maintenance of the natural resistance of both the peel and the button (fruit calyx) of the fruit to infection (Eckert and Eaks, 1989), as well as to retard the growth of latent pathogens in infected fruit (Smilanick et al., 2006). From the data in Figures 2 18 and 2 20, the grapefruit storage period at 40oF can be extended to 12 weeks for all harvest dates. However, this is only based on decay results up to this point of discussion with no consideration to chilling injury results that will discuss in the next section. The maximum decay was 27.78 % in April 2005 compared to 13.33 % in May 2006, almost a 50% reduction in infected f ruit between the two seasons. On the other hand, at all harvest dates except March, grapefruit stored at 70oF during the first season had 50% decay after 12 weeks and 50% decay after 10 weeks for fruit harvested in March, Figure 2 19. During second season at 70oF, grapefruit harvested in November and May were the only two groups that reached 12 weeks of storage without 50% decayed fruit, Figure 2 21. Also, fruit harvested in September, January, March and July only reached 11, 7, 8 and 4 weeks, respectively with less than 50% infected fruits. Valencia orange showed less % decay compared to Marsh grapefruit after 12 weeks of storage at 40oF, Figures 2 20 and 2 23 whereas fruit harvested in March, May, and July only reached 10, 6, and 4 weeks of storage a t 70oF, respectively, before having 50 % decay, Figures 2 21 and 2 24. Average w eight loss % data of both seasons and two storage temperatures suggest that the period from January to March is the beginning of significant infection of Marsh grapefruit dur ing storage, whereas for Valencia orange this

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83 onset of significant decay is on May. This period of time when fruit peel starts to be less resistant to pathogens may be related to the onset of peel senescence. Chilling injury (CI) During the first two se asons of experiments, grapefruit collected in November and stored at 40oF had a significantly higher percentage of CI compared to other harvest dates, as shown in Table 2 13 and Figure 2 25. For November harvest, 73.3% of the fruit showed injured by the en d of storage period, whereas all the fruit were injured by the 10th week of storage during the second season, Figures 2 26 and 227, respectively. Fruit collected early and late the first seasons were less injured during storage compared to other harvest dates, whereas during the second season, mid -season fruit were less injured, Table 2 13. During both seasons, November harvested fruit showed higher percent CI compared to earlier harvests, and this is possible because chilling injury is a time by temperatu re problem (Skog, 1998) and may depend upon climate (Young, 1961), winter field temperature ( Kawada et al., 1978 ), picking date ( Pantastico et al., 1968), and temperature before refrigeration (Purvis and Yelenosky, 1993). Figure B 1, appendix shows that field temperature in November of both seasons was about 70oF, compared to 77oF and 82oF in October and September, respectively. Almost one third of fruit harvested in September (34.4%) and May (33.3%) were injured by the end of storage period during the first season, Figure 2 26, but more than half (53.3%) to two -third (65.5%) of fruit harveste d in March and January, respectively, were injured during the second season, Figure 2 27. As shown in Table 2 13 and Figure 2 28, Valencia orange harvested in July had the highest CI during storage and the difference was significant compared to other har vest dates. However, the percent of Valencia fruit with CI for all harvest dates during storage was less than that of Marsh grapefruit. The maximum percent of injured Valencia fruit was 31.1% after 12 weeks of storage for the July harvested fruit, Fi gure 2 28. This result is in agreement with previous reports stated that

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84 grapefruit is more susceptible than oranges to CI caused by storage temperature (Pantastico et al., 1968; Ritenour et al., 2003) Lafuente et al (1997) found that the susceptibility of Fortune mandarins to CI increased concomitantly with color change from green to orange. They suggested that chlorophyll may play an important role in CI resistance. This f inding is in agreement with Valencia orange results, but not with Marsh grapefruit during the second season of this study where the less colored grapefruit was more susceptible to CI. These data indicate that although chilling injury incidence started in September, but the month of November is the beginning of noticeable CI of Marsh grapefruit during storage, and this may be the onset of peel changing from an immature to a mature stage, whereas this period appeared to occur in July, a period of near s enescence, for Valencia orange. These results (from November toward the end of the season) are in agreement with Ritenour et al. (2003) who stated that in Floridas climate, fruits are most susceptible to CI early and late in the season, however, early harvested fruit in September and October of the first season Figure 2 25, disagree Ritenours results. Such injuries of CI may be the result of a loss of cellular integrity caused by damage to cell membr anes ( McCollum and McDonald, 1991) due to fairly high percentage of water loss (2.96%) in November, Table 2 9, and later these injuries are often accompanied by an increase in susceptibility to decay ( Cohen et al., 1990). This was confirmed above as the period from January to March was the beginning of noticeable decay of Marsh grapefruit during storage, Table 2 11. Effect of Water Stress and Growth Regulators TSS, acidity, and TSS: acid ra tio To determine the effect of water stress and growth regulators on citrus peel maturation and senescence, TSS: acid ratio of the fruit juice was also measured trying to correlate between internal and external fruit quality, so fruit can be picked in a su itable time that merge between

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85 juice quality and less peel injuries. Table A 1, appendix and Figure 2 29 show that grapefruit juice quality was not affected by any treatment, and there were no significant difference between all treatment s and the control, except during the second season where the treatment of WS*GR was significantly different from the control during the months of January and March. The c ontrol showed the highest value of TSS: acid ratio, followed by the GR treatment, then WS treatment, and WS*GR. These results are in agreement with previous report, in that, acid content in juice, soluble sugars content in juice and peel, peel thickness, and peel to -pulp ratio increase in drought -stressed trees (Boman e t al., 1999; Hockema and Etxeberria, 2001; Huang et al., 2000; Mostert and Van Zyl, 2000) Acidity increases more rapidly than TSS under dry condition. Consequently, TSS: acid ratio decreases ( Wittwer, 1995). Also, t he role of GA3 in delaying senescence of the peel is reflected in preservation of more functional mitochondrial and plasma mem branes resulting in a higher rate of O2 uptake, a higher rate of respiration, a lag in sugar accumulation, reduced solute efflux, and the survival of protoplasts (Nolte et al., 1990 ). Generally, GA3 does not affect juice quality when it is used to delay peel senescence and extend harvest season (Coggins, 1981). It has been reported that 16ppm 2,4 D and/or 10ppm G A3 when applied preharvest to Clementine mandarin, Marsh grapefruit, and Valencia orange caused a delay in peel aging and prolonged the harvest season, but had no effect on fruit weight loss in storage, acidity, TSS, or TSS:Acid ratio (El -Otmani and Coggins, 1991; Gilfillan et al., 1973 ). Any reduction in TSS: aci d ratio of WS and WS*GR treatments is mainly related to lower sugar production due to reduction in photosynthesis under water stress conditions (Vu and Yelenosky, 1989). By comparing fruit from the GR and WS treatment, there is one more reason why fruit treated with growth regulators have hi gher TSS: acid ratio than water stressed -fruit because 2,4 -

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86 D increases sink capacity of endocarp and thus of fruit resulting in greater mobilization activity of water, carbohydrates and minerals to the fruit leading to good juice quality ( Agusti et al., 1992) represented with high oBrix at harvest (El Zeftawi, 1980b ). Regarding Valencia orange, Table A 2, appendix and Figure 2 30 showed that there were no significant difference among all treatment during both season of experiments. Unlike grapefruit, Valencia control could not be distinguished from WS, GR, or WS*GR treatments, and this may be due to irrigation usually reducing TSS and acidity, and it has greater effect on acidity in grapefruit than in orange (Barbera and Carimi, 1 988; Boman et al., 1999) The only noticeable and significant difference appeared when severe water stress treatment was applied by covering the soil using Tyvek, as shown in Table A 3, appendix and Figure 231, and this was only happened during 2005/2006 season. This difference may be due to what Yakushiji et al. (1998) found that photosynthetic rate (Vu and Yelenosky, 1989) and stomatal conductance (Syvertsen, 1982) of severely drought stressed Satsuma mandarin trees were significantly lower than those of well -watered trees. So, the amount of metabolites that been used to build soluble solids and acids were low, and then TSS: acid ratio was low in severely stressed fruit. Peel color To evaluate the influence of WS and GR on peel senescence and coloratio n, results of Marsh grapefruit were normal and in agreement with previous reports; the control treatment had the highest color index followed by WS, then WS*GR, and GR, Table A 1, appendix and Figure 2 32. The difference was significant between GR and al l treatments in March 2005/06, as well as between GR and WS and control in May 2005/06. The reduction in color index in May, Figure 2 32, is hard to be justified or re ferred to weather information that was in normal trend at that time, Figures B 1 and B 2, appendix, or colorimeter calibration error, because colorimeter shows normal result trend for Valencia orange from March to May of the same season, Figure

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87 2 33. Results of the second season 2006/07 were not available except in May, where the difference between GR and all treatments was significant. Gibberellic acid (GA3) is used primarily as a preharvest treatment to delay certain aspects of rind senescence of Navel orange and seedless grapefruit (Coggins, 1981). It was used to mo dify color development by delaying chlorophyll degradation (Coggins and Henning, 1988; Coggins and Hield, 1 968; El-Zeftawi, 1980a ; Garcia Luis et al., 1992; Goldschmidt et al., 1970; Porat et al., 2001 ) via inhibition of chlorophyllase synthesis (Trebitsh et al., 1993) and inhibition of carotenoid biosynthesis (Jun et al., 2002). These results are also in agreement with previous records in terms of time of GR application. Autumn application of GA3 to citrus fruit approaching maturity (just prior to color break) delay color change and delay peel senescence (Chitzanidis et al., 1988; Coggins, 1981; Greenberg et al., 1992; McDonald et al., 1997; Pozo et al., 2000; Stover, 2000) allowing a late harvest ( Agusti et al., 1981). Two, four D also has the same effect like GA3 effect. It was reported that preharvest application of oranges and lemon with 2,4 D delay color development (Stewart, 1954). A combination of GA3 and 2,4 D d elay color development and extend the harvest season of grapefruit and oranges (Chitzanidis et al., 1988 ; El Otmani et al., 1990; Ferguson et al., 1982; Kinay et al., 2005; Medeiros et al. 2000; Sen et al., 2001), however, apart from the delay in rind coloration, GA3 and 2,4 D do not affect legal maturity (Ro use and Zekri, 2006). The second treatment after GR, in terms of better peel coloration was WS*GR and this is in agreement with previous findings which stated that insufficient irrigation decrease the effectiveness of GA3 in South Africa (Gilfillan et al., 1973 ), whereas it has little effect on GA3 efficacy for mature Marsh grapefruit trees in Florida ( Ferguson et al., 1983). Water stress treatment was the third one in this order, and this is normal result because water stress induces the process of cell sen escence through specific changes in cell ultrastructure (e.g. chromatin

PAGE 88

88 condensation, thylakoid swelling, plastoglobuli accumulation, and chlorophyll degradation) (Munne Bosch and Alegre, 2004 ). In Valencia orange, Table A 2, appendix and Figure 2 33, results showed that GR and WS*GR treatment s have more green color, and the difference was significant compared to the control during Marsh and May of the 2005/06 season. The WS treatment during the same season gave higher peel coloration compared to the control at all harvest dates, and this may b e due to peel color break being delayed when irrigation is increased, because water availability improves nitrogen nutrition, which delays color break and enhances regreening (Boman et al., 1999) Results of the second season 2006/07 for May and July indicate the GR treatment showed less peel coloration, as well. The trend for color was green c olor increase from May to July for all treatments This may be due to the natural regreening of the fruit (Hsu et al., 1989; Thomson et al., 1967) Data of the Tyvek experiment, Table A 3, appendix and Figure 2 -34, showed higher color index for the t reated fruit, compared to the control, and the difference was significant only at March 2005 / 200 6 This is in agreement with findings found in humid Japan, where color break was accelerated in Satsuma mandarin when soil was covered by plastic sheet to indu ce drought stress (Boman et al., 1999) The low values of Valencia color index during th e 2006/07 season compared to the 2005/06 season may be due to the difference in monthly precipitation between two seasons that shows increase in the amount in June 2007 and July 2007 that may dilute the color during this season, Figure B 2, appendix, or it may be related to change in the colorimeter calibration due to maintenance early in the 2006/07 season, and this is the reason of unavailable data in January and March. From the abovementioned data, we can conclude that the G R treatment either alone, or i n combination with WS treatment was the best treatment to delay color break but it did not reduce

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89 percent decay or chilling injury in Marsh grapefruit and Valencia orange. Thus, it can be said that based on the difination or peel maturity, GR did not r etard pell maturity. Fruit harvested in March showed the best results in this regard for both varieties. The results for Valencia orange were good for March in 2005/06 as was good juice quality ( TSS: acid ratio). So, the month of March was the best time to harvest Valencia orange with good juice quality and less senescent peel, which means less susceptibility to postharvest problems such as, decay and physiological disorders (Burns and Baldwin, 1994). Thi s harvest date can be extent until May, since there were no significant difference in juice quality between GR and control, and fruit still in good condition with less CI compared to the control Table A 2, appendix. For Marsh grapefruit, good results we re also obtained with GR in January harvested fruit, such as peel peel turgidity and low % decay during storage at 40oF, as well as in May, such as fruit juice quality, peel peel turgidity % weight loss and % decay at 40oF, Table A 1, appendix. This indic ates that Marsh grapefruit can be early harvested in January or late harvested in May. Firmness / peel turgidity / turgor pressure In citrus, there is a negative correlation between peel turgidity and water content of the peel and pulp. For this reas on, peel turgidity has never been used as an index of fruit firmness, instead, it has been used as an index of tissue turgidity (Oberbacher, 1965). The word firmness is therefore more associated with fruit turgidity and peel thickness ( Grierson, 2006b ). In general, fruit becomes less firm with maturation, and senescing pe el has an albedo layer with small cells of low cytoplasmic content, low metabolic activity, larger intercellular spaces and weakened cell wall, which break easily ( Coggins, 1969b ). Data of Marsh grapefruit, Table A 1, appendix and Figure 2 35, indicate that application of GR had fruit of higher values of peel turgidity either with GR only or wh en GR was combined with WS treatment in both seasons, compared to the control and WS treatment. This is possible

PAGE 90

90 to be related to a reduction in peel maturation rate with GR H owever, it may not be related to change in maturation rate, especially with WS t reatment that may shrink albedo cells and cause more peel turgidity as in January of 2005/06 season, although the difference is not significant compared to GR or WS*GR, Table A 1 and Figure 2 35. The difference between GR and control was only significant in May of the 2005/06 seasons and both GR and WS*GR were significantly differed from the control in March of the 2006/07 season. In Valencia orange the results were different from grapefruit, in terms of the GR and WS*GR treatments. During the 2005/06 sea son, Table A 2, appendix and Figure 236, higher values of peel turgidity occurred for the GR and WS*GR treated fruit, compared to the control, early in the season in March and January, respectively but as the season progress, the control had the highest values in July In March of both seasons, the difference was significant between GR and the control. Peel turgidity values of severely drought stressed Valencia orange fruit, Table A 3, appendix, were closer to the values of the control in both seasons, and the difference was only significant in January and March of the 2005/06 season, and January, March and July of the 2006/07 season, Figure 2 37. The high values of severely stressed fruit in 2006/07 season may be related to that severe drought did shrink the albedo cells and cause higher value of peel turgidity This also can be confirmed and referred to the amount of precipitation during both seasons of study, Figure B 2, appendix, that shows the higher the amount of rainfall, the lower the values of pe el turgidity and this is may also be the reason of higher peel turgidity in the middle of 2005/06 season, and relatively constant values in 2006/07 season for both grapefruit, Figure 2 35, and orange, Figures 2 36 and 2 37. In general, there was a reduc tion in fruit turgor pressure in water stressed fruit (Huang et al., 2000). Also, water stress stimulates the activity of glycosidases causing deterioration of cell

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91 walls, resulted in fruit softening (Konno et al., 1986) Application of GA3 to citrus fruit approaching maturity (just prior to color bre ak) enhances peel firmness and delays peel senescence (Chitzanidis et al., 1988; Coggins, 1981; McDonald et al., 1997; Pozo et al., 2000; Stover, 2000) Preharvest application of 2,4 D to oranges and lemons has been reported to delay rind softening (Erner et al., 1993) on the tree and in cold room storage. A combination of GA3 and 2 4 D enhanced peel firmness and exten t the harvest season of grapefruit and oranges (Chitzanidis et al., 1988 ; El Otmani et al., 1990; Ferguson et al., 1982; Kinay et al., 2005; Medeiros et al., 2000; Sen et al., 2001). In gen eral, data showed that peel turgidity values were high in March for both Marsh grapefruit and Valencia orange, a ssociated with other previously mentioned parameters of TSS: acid ratio and peel color, supporting the conclusion that March is the best tim e to harvest these fruits H owever, for Marsh grapefruit harvested in January of 2005/06 season, the significant difference between GR and control, as well as between the WS*GR and control, Table A 1, appendix and Figure 2 35, reflect that grapefruit can be still early harvested in January, especially GR treated fruit and control had the same TSS: acid ratio, and both GR and WS*GR showed less % decay during storage at 40oF compared to the control. Also, the significant difference in peel turgidity between GR and control in May reflect the possibility of extending the harvest season until May with same TSS: acid ratio as the control, and small nonsignificant % weight loss and % decay than the control, Table A 1, appendix and Figure 2 35. For Valencia orange, Table A 2, appendix and Figure 2 36, the difference between GR and control was not significant in January or May, but Figure 2 36 reflect that fruit may be harvested one month before and one month after March Fruit detachment force (FDF) Detachment force (FDF) is the force required to separate the fruit from the peduncle, and may be an indicator of fruit maturity and senescence (Coppock, 1961). All treatments and the

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92 control of Marsh grapefruit during the 2005/06 season, Table A 1, appendix and Figure 2 38 showed a decreasing pattern from January to March, but increasing after March until July. These results do not ag ree with the previous work reported by Coppock (1961) where detachment force decreased during the season. Also, GR treatment either alone or in combination with WS showed lower FDF compared to the control and WS treatments, but supposedly GA delays maturat ion and reduces fruit drop expressed by high FDF. This variation in results may be due to small sample size (n=10 fruits), especially since the 10 fruit were collected from different positions in the canopy. Kender and Hartmond (1999) stated that a t maturity, citrus fruit remain firmly attach ed to the stem, and FDF is extremely variable within the tree and generally higher in the top and exterior parts of the canopy, where fruits are more exposed to the sun and develop stronger stems. The most interesting results for March of both season s was that FDF was decreasing at high tissue turgidity (control vs. WS), Table A 1, appendix and Figure 2 38. T his is logical: at high tissue turgidity, fruit detachment force is low because less pressure is required to break cells This is in agreement with Co ppocks (1961) statement that less force is required to remove oranges following rains and early in the mo rning when the fruit is in a turgid condition (Coppock, 1961). During the 2006/07 season, there were a reduction in FDF fr om January to March, and GR and WS*GR treatments had significant higher FDF compared to the control Figure 2 38. Fruit detachment force is positively correlated with the ratio of endogenous IAA to ABA or endogenous IAA, but negatively to endogenous ABA i n the fruit abscission zone (Yuan et al., 2001). Acting like the natur al auxins in plant, 2,4 D is used primarily as a preharvest treatment to delay and reduce abscission of mature citrus fruit (Coggins, 1981) and delay harvesting ( Monselise, 1977) by inhibiting the ethylene effect that promotes the activity of the hydrolytic enzyme s; endo -beta -glucan and polygalacturonase at the abscission zone (Burns and

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93 Lewandowski, 2000; Goren, 1993) thus reducing fruit drop by maintaining high fruit detachment force (Chitzanidis et al., 1988; Goren et al., 2000). Results show higher FDF with GR and WS*GR treatments than the control in January and March of both season, Table A 1 and Figure 2 38. The increase in FDF from March to May is may be associated with the natural regreening of fruit. Rasmussen (1981) stated that in Valencia orange, high level of GA3 in the flaved o of the fruit with the regreening of the peel may help to prevent ABA from inducing ethylene and promoting abscission. GA3 and 2,4D are excellent companion growth regulators for delaying rind senescence and reducing drop of mature fruit, respectively (Coggins, 1981). In Valencia orange, Table A 2, appendix and Figure 2 39, the same trend of variable data was noticed as for Marsh grapefruit and again may have been due to small sample size. The higher values of FDF of the GR and WS*GR treatments compared to the control in the 2005/06 season were insignificant, Table A 1, appendix and Figure 238. Severe water stressed Valencia orange, covered with Tyvek, Table A 3, appendix and Figure 2 40, had low FDF compared to the controls in both seasons, except for the Ma y harvest in 2006/07, however the differences were only significant in January and May of 2005/06 and July of 2006/07. The trend of high values of FDF in the 2006/07 season compared to 2005/06 season is opposite to that of peel turgidity but it is hard to relate this difference in FDF between seasons to weather condition, as related for peel turgidity The reduction in FDF may be related to an increase in ABA at the abscission zone of the fruit (Taiz and Zeiger, 2002), which is clearly involved in leaf and fruit senescence directly by increasing ACC synthesis; precursor of ethylene (Gomez cadenas et al., 2000; Goren, 1993; Goren et al., 1979; Okuda, 1999; Riov et al., 1990; Sagee et al., 1980) or indirectly by accelerating senescence (Jackson and Osborne, 1972; Lieberman et al., 1977) that increase ethylene production, which increase cellulase and polygalacturonase activity

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94 (Jackson and Osborne, 1972; Kazokas and Burns, 1998; Rasm ussen, 1974) and promote leaf and fruit abscission ( Lieberman et al., 1971; Sagee et al., 1980; Taiz and Zeiger, 2002). On the other hand, ethylene can be activated by water stress (Yang and Hoffman, 1984) and may also increase free abscisic acid in the flavedo of citrus fruit enhancing senescence (Goldschmidt et al., 1973; Lafuente et al., 1997; Lafuente and Sala, 2002) Weight loss Water loss is a serious factor that adversely affects the quality of citrus fruit (Ben Yehoshua et al., 1979). Water loss of stored Marsh grapefruit increased with a prolonged storage period, and water loss was higher at 70oF than at 40oF, Table A 1, appendix and Figures 2 41 and 2 42. This is in agreement with previous reports by Grierson (1974) and Grierson and Miller (2006c ). Fruit from WS and WS*GR treatments and harvested in March had the lowest % wei ght loss compared to other treatments and the differences was significant compared to the control every two weeks during storage at 40oF, whereas, at 70oF, fruit treated with GR and the control fruit when harvested in March had the lowest % weight loss com pared to all other treatments at each measurement, every two weeks of storage H owever the difference between GR and the control was not significant. F or Valencia oranges harvested in March and stored at 70oF, control had the lowest % weight loss during the first 8 weeks of storage then GR was the lowest during the last 4 weeks, whereas at 4 0oF WS*GR treatment had the lowest % weight loss, Table A 2, appendix and Figure s 2 43 and 2 44. According to Grierson and Wardowski (1978), fruit show sh rinkage at 2.5 % weight loss, and become unsalable after 5% loss of the original weight. WS treated Marsh grapefruit had about 2.5% loss after 8 weeks at 40oF, and GR -treated fruit had about 2.5% loss after only 6 weeks at 70oF H owever, fruit lost less than 5% weight and were still salable at the end of storage period, Figures 2 41 and 2 42. All treatments and control of Valencia oranges harvested in January and March had lost about 2.5% weight

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95 after 4 weeks at 40oF and fruit were still salable until 10 weeks of storage, Figure 2 44. GR treated fruit stored at 70oF lost less than 2.5 % weight after the second week of storage and were only salable after the 6th week, but the control fruit were salable until the 8th week of storage, Figure 2 43. W ater s tress is known to induce fruit senescence ( Munne Bosch and Alegre, 2004), and growth regulators (gebberellic acid and auxins) delay fruit senescence ( Coggins, 1981). This finding is applicable for grape fruit stored at 70oF, but the situation was opposite at low temperature storage (40oF), and this may be due to an interaction between temperature and WS that may reduce the effect of WS during storage. By the end of storage period at 70oF, the GR treatment had the lowest water loss for Marsh grapefruit, Table A 1, appendix and Figure 2 41, and Valencia orange, Table A 2, appendix and Figure 2 43. On the other hand, Valencia orange stored at 40oF showed the best results with WS*GR treatments either in January or in March, and the difference was significant compared to the control for each month, Table A 2, appendix and Figure 2 44. Valencia trees treated with Tyvek for severe water stress treatment, Table A 3, appendix, had fruit with reduced weight loss than that of the control at 70oF, Figure 2 45, but not at 40oF, Fig ure 2 46. The lowest % weight loss at 70oF was in March and May. On the other hand, at 40oF, the Tyvek treatment showed the lowest numerical percentage weight loss with March harvested fruits during the first 6 weeks of storage, Figure 2 46. Fucik (1981) found that GA3 reduced water loss from grapefruit during postharvest storage. GA3treated fruits have peel with a structure and wax characteristics similar to that of young frui t (Coggins, 1969b ), less wax accumulation and retarded wax ultrastructure changes ( El Otmani and Coggins, 1985a ), but this reduction in wax quantity had no significant effect on fruit weight loss in their study (El Otmani et al., 1986 ). This is an indication that not only wax quantity but also morphology and

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9 6 composition play a role in restricting water vapor and gas diffusi on ( El Otmani and Coggins, 1985a ; El Otmani and Coggins, 1985b ). From the abovementioned results of weight loss and other parameters such as, juice quality and percentage decay, it appears that fruit harvested in March showed the best results for both Marsh grapefruit and Valencia orange in terms of small percentage water loss H owever this time can be adjusted and fruit can be harvested early in January for Marsh grapefruit only. WS and WS*GR treatment s are effective in this regard for fruit stored at 40oF. On the other hand, GR treatment showed good results for fruit stored at 70oF, Table A 1, reflected that harvest date can be extended to May. Valencia orange harvest date can b e extended to May, with some little effect for WS and WS*GR treatments. Valencia orange life in storage is shorter than that of Marsh grapefruit, and this suggest that May harvest should be the stop point of Valencia orange, as well as for Marsh grapefruit, because fruit after May start to be senescent and their storage life is shortened. Decay As fruits become older, susceptibility to postharvest diseases increases, because the peel becomes less firm and hence less force is required to puncture the peel (Coggins et al., 1969a ). This happened with Marsh grapefruit and Valencia orange from the WS and GR treatments stored at 40oF and 70oF, Tables from 2 14 to 216, and Figures from 2 47 to 252. For grapefruit the maximum % decay at the end of storage period at 40oF considering all treatments was 13.33%, whereas this was the lowest % decay we have from the control after four weeks of storage at 70oF. Storage at low temperature and high humidity is beneficial to the maintenance of the natural resistance to infection of both the peel and the button (fruit calyx) of the fruit ( Eckert and Eaks, 1989), but the most obvious effect of low temperature is to retard the growth of pathogens in the fruit (Smilanick et al., 2006).

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97 Generally, at 40oF, Figure 2 47, grapefruit controls showed the highest % decay at all harvest dates except January where WS had significantly higher % decay. The same result in the January harvest happened at 70oF, Figure 2 48. This may have been due to the age of fruit, because when the y are still young, their susceptibility to decay is less However, but treating the m with WS reduced this resistance and increased their susceptibility to pathogen invasion. Water stress enhances fruit maturation and senescence stimulating the activity of glycosidases causing deterioration of cell walls, resulted in fruit softening. At 7 0oF storage, Figure 2 48, for the March, May, and July harvests the situation was completely different from 40oF, Figure 2 47. At 7 0oF the control showed low % decay compared to some other treatments. This may be due to some interaction between the treatm ent and the storage temperature. The difference was highly significant between the control and the other treatments at only the May harvest throughout storage. The GR treatment had less % decay compared to the control in January at 40oF and 70oF, but WS ha d less % decay in March and May at 40oF Figures 2 47 and 2 48. Valencia oranges had less % decay compared to the control at 40oF with WS and WS*GR in March, WS*GR in May, and GR in July. The highest % decay (6.76%) was for GR treated fruit in July, Ta ble A 2, appendix and Figure 2 49. At 70oC, Figure 2 50, fruit treated with WS*GR and harvested in January and May had the lowest, significantly different, % decay compared to the control until 10 weeks of storage, but in March, the control was the best co mpared to other treatments, and the difference was significan t until the end of the storage period. These results were highly variable and hard to interpret, especially at 70oF. However, at 40oF storage with harvest in March and May, any WS treatment alone or combined with GR reduced decay. This contradicts the effect of water stress enhancing fruit senescence ( Munne Bosch and Alegre, 2004; Sharon -Asa et al., 2003), but this treatment could cause less fruit turgor

PAGE 98

98 pressure ( Huang et al., 2000) and a reduction in the incidence of green mold (Ritenour et al., 2002) due to a reduction in mechanical injuries during harvest and packaging. For the March harvest, especially the WS treatment had better results than WS*GR, Figure 2 49. WS can reduce the effectiveness of GR in delaying peel senescence as insufficient irrigation decreased the effectiveness of GA3 in South Africa ( Fergu son et al., 1983). However the WS*GR treatment was more effective than WS in May, and in a previous report WS has little effect on GA3 efficacy for mature Marsh grapefruit trees in Florida ( Ferguson et al., 1983). So, because G R was not that effective in March and became effective in May, we can state that may be the period from March to May is the transition between less mature stage (less susceptibility as shown in control) and more mature stage (more susceptibility as shown i n control) of the peel, Figure 2 49, especially GR showed the best and significant results compared to the control and other treatments in July when the peel start to be senescent in all treatment, but GR. Also, it might be related to some other factors, s uch as difference in handling between harvests and treatments. The growth regulators used in this experiment, 2,4 -dichlorophenoxyacetic acid (2,4 D) and gibberellic acid (GA3), reportedly are used to control fruit disease (Coggins, 1981; Wilson, 1983). GA3 maintains a compact structure in the albedo (Monselise, 1979) and reduces several physiological blemishes and diseases of the peel (Coggins, 1969b ). It has also been reported that spraying orange with GA3 at color break reduced preharvest fruit drop (Cameiro de Me deiros et al., 2000) and postharvest decay (El -Otmani and Coggins, 1991). Autumn application of GA3 to citrus fruit approaching maturity (just prior to color break) enhances peel firmness, delays color change, and delays peel senescence and the subse quent peel disorders and decay (Chitzanidis et al., 1988; Coggins, 1981; McDonald et al., 1997; Pozo et al., 2000; Stover, 2000) allowing a late harvest ( Agu sti et al., 1981). Two, Four D is also recommended to control stem -end rots of

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99 oranges and grapefruit during long-distance shipment and prolonged storage (Schiffman Nadel et al., 1972). Soil coverage with Tyvek, Table A 3, appendix, and Figures 2 51 and 2 52, gave better results compared to the control, and the difference was significant in March and May at 8 weeks and 10 weeks of sto rage, respectively, for fruit stored at 40oF (% decay = 0), but fruit stored at 70oF still had good and significant results compared to the control by the end of storage period in January and March. These results confirm that severe WS may also be a reason for less turgor pressure, less water content (Huang et al., 2000) and a reduction in the incidence of green mold (Ritenour et al., 2002) due to less mechanical injuries to the peel inflicted during harvesting and handling which are the principal sites of infection by the wound -invading pathogens Penicillium and Geotrichum (El Otmani, 2006). Chilling injury (CI) Ritenour et al. (2003) stated that in Floridas climate, citrus fruit are most susceptible to CI early and late in the season. This is very noticeable from the data in Table A 1, appendix and Figure 2 53 for Marsh grapefruit stored at 40oF, where March -harvested fruit had less CI compared to other harvest dates. Within March, fruit treated with WS either alone or in combination with GR had less CI compared to the control and GR treatment. This difference from the control was only significant until the 10th week of storage for the WS treatment (CI = 34%) and until the end of storage for the WS*GR treatment (CI = 42%). These results are in agreement with previous reports that showed that GR delayed peel senescence (Chitzanidis et al., 1988; Coggins, 1981; McDonald et al., 1997; Pozo et al., 2000; Stover, 2000) whereas water stress enhance peel senes cence ( Munne Bosch and Alegre, 2004; Sharon -Asa et al., 2003). This may be the reason why GR treatment showed more CI than WS in March, because fruit is less mature more like January harvested fruits, and WS treatment advanced peel maturity and the

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100 effect was very n oticeable in January with high % CI, but in March showed less % CI and this may be because peel already passed the immaturity stage however, the interaction between WS and GR showed the best results compared with WS or GR, and this is possibly related to that WS can reduce the effectiveness of GR in delaying peel senescence ( Ferguson et al., 1983) and keep the fruit in medium stage between immaturity and maturity. These results are consistent with the results for weight loss and decay, in that less CI, water loss and decay occurred in the WS*GR in March compared to other treatments and control that showed higher % CI. CI may be the result of a loss of cellular integrity caused by damage to cell membranes ( McCollum and McDonald, 1991) and partially due to fairly high water loss, as from the control and GR treated fruit, Figure 2 42. These CI injuries are often accompanied by an increase in susceptibility to decay ( Cohe n et al., 1990), as shown in March harvested control and GR treated fruit, Figure 2 47. For Marchharvested grapefruit, CI symptoms started to be noticeable from the 4th week of storage, which is in agreement with previous reports stating that CI sympt oms generally require 3 to 6 weeks to develop at low storage temperature (Ritenour et al., 2003) Oranges are susceptible to CI when stored at temperatures below 35oF ( Skog, 1998). Grapefruit are more susceptible than oranges to CI with development at storage temperatures below 50oF for mid and late season fruit and 60oF for early season fruit (Pantastico et al., 1968; Ritenour et al., 2003) This is seen in Table A 2, appendix and Figure 2 54. Moreover, the results of Va lencia orange are completely opposite to those of Marsh grapefruit. As shown in Figure 2 54, CI of March harvested oranges is higher than that of January and May for all treatments. The symptoms did not start to appear until after the 10th week of stor age for January harvested fruit. The high % CI in March followed by low % CI in May, Figure 2 54, as well as the previous mentioned data of decay at 40oF, Figure 2 49, that shows low % decay in March

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101 followed by high % decay in May, can be related to the p hysiological age of the peel and the change in fruit susceptibility to CI and decay as the fruit transit from less mature stage in March to more mature stage in May. In March and May, both of WS and GR treatments had less symptoms of CI compared to the con trol and WS*GR. Previous reports indicate that GR delays peel senescence and subsequent peel disorders and decay In Florida (Chitzanidis et al., 1988; Coggins, 1981; McDonald et al., 1997; Pozo et al., 2000; Stover, 2000) It has also been reported that spraying orange with GA3 at color break reduced chilling injury of fruit stored at 1 5oC (Arpaia and Eaks, 1990). Regarding water stress effect on reducing CI, this can be referred to previous reports stating that water stress increases the level of endogenous ABA (Lafuente and Sala, 2 002) IAA (Koshita and Takahara, 2004) GA3 ( Sardo and Germ ana, 1988), and ethylene formation (Ben Yehosh ua and Aloni, 1974). ABA increases fruit resistance to chilling injury (Kawada et al., 1979; Serrano et al., 1997) Similar findings were observed for fruit from the Tyvek -treated trees; their fruit had less symptoms of CI during storage, Table A 3, appendix and Figure 2 55. Low % CI in May suggests that the month of May is may be the transition period between immaturity and senescence of Valencia orange, and fruit should not be harvested after May. Conclusion The above data indicates that the months of March and May are the time of increases in ma turity index of Marsh grapefruit and Valencia orange, respectively based on storage data suh as percentage decay that showed increase by mid-season The WS or GR treatments either alone or combined did not affect juice quality represented by TSS: aci d ratio in both species. The only exception of this was the significant reduction in TSS: acid ratio of Marsh grapefruit with WS*GR treatment compared to the control in January and March of the 2006/07 season as well

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102 as the significant reduction with Ty vek treatment during 2005/06 season, which confirm the role of severe water stress reducing juice quality Although color is temperature dependent, GR treatment both alone or in combination with WS treatment delayed color break, and may give an indicatio n about retarding peel senescence in Marsh grapefruit and Valencia orange. Fruit harvested in March showed the best results in this regard. The effect of GR on color index extended until the month of May for grapefruit only. Tyvek treatment improved co lor index significantly compared to the control only in March and May of the 2005/06 season. Fruit firmness ( peel turgidity ) of both species decreased with fruit maturation, but this trend was more noticeable for Valencia orange than Marsh grapefruit WS treatment advanced the peel maturation of Valencia orange and reduced peel turgidity compared to the control in January of both seasons. GR in March of the 2005/06 season only showed the best (significant) results for Valencia orange, and Tyvek tre atment increased peel peel turgidity significantly in March of both seasons. GR and WS*GR showed the best (significant) results for the May harvest of the 2005/06 season and March harvest of the 2006/07 season for Marsh grapefruit which may reflect diff erence in peel maturation rate of grapefruit between seasons. The general trend of f ruit detachment force w as decreas ing with fruit maturation. WS *GR treatment showed significant increase compared to the control in detachment force in March of both season s for Marsh grapefruit, but only the GR treatment for the March harvest of the first season for Valencia orange. Tyvek treatment reduced FDF compared to the control in January of both seasons confirming the role of WS advancing peel maturation rate Water loss was significantly low for WS and WS*GR treatment fruit stored at 40oF for March -harvested grapefruit, but they both increased % weight loss by the end of storage period

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103 significantly compared to the control and the GR treatments at 70oF For fru it harvested in March and May, and then stored at 70oF WS increased % weight loss by the end of storage period significantly compared to the control and the GR treatments, which may reflect the effect of WS advancing maturation rate versus the effect of G R retarding it. WS*GR reduced weight loss of Valencia orange harvested in January and March and stored at 40oF. At 40oF, grapefruit had lower % decay from WS and WS*GR treatment in March, and from WS treatment in May. Valencia had less decay a t 40oF f rom WS in March WS*GR in May and GR in July. At 70oF, grapefruit showed less decay for the GR treatment harvested in January and orange showed this with WS *GR and Tyvek in January. March -harvested grapefruit showed less % CI compared to other harvest dat es, and within March, fruit treated with WS GR showed the lowest % CI compared to the control. These results are also consistent with the results of w eight loss and decay that showed less percent of CI, w eight loss and decay with WS*GR in March compared to other dates that showed higher % CI. For Valencia orange, the % CI of March -harvested fruit was higher than that of January and May harvests for all treatments. WS and GR reduced CI insignificantly compared to the control in May, and this probably because CI is not a common thing in Valencia orange. These results showed the reduction in fruit susceptibility to CI between Marsh grapefruit in March and Valencia orange in May which may be related to the difference in the time of peel maturity in both species. Weight loss, decay and chilling injury are three factors that limit the postharvest life of fruit. Results showed that at 40oF, grapefruit control fruit harvestd in November showed high % weigh loss during the first 6 weeks, and highest % chillin g injury between 6 and 8 weeks of storage, whereas fruit harvested in May showed high % weight loss during the last 6 weeks of

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104 storage. This is something interesting that can be considered in future studies about the difference in storage period between ea rly and late harvested fruit. Collectively, based on the best time of harvest and the best treatment for both Marsh grapefruit and Valencia orange it looks like the period from January to March is the best time to harvest Marsh grapefruit during the long harvest season extending from September to May, and the period from March to May is the best time to harvest Val e ncia orange during the short harvest season extending from February to July. However, combining the effects of harvest date, WS and GR on physical characteristics of the peel with their effects on chemical characteristics from the next chapters may give a broader window of harvest date based on the effect of WS and GR on the levels of peel sugars, glycosidases, ABA and volatile component s of the peel. These chemical constituents may reflect the stage of peel maturation, and indicate how WS and GR can/cant be used as tools to adjust the internal level of the chemical constituents of the peel, associated with changes in physical characteri stics, and then to determine the safe harvest window and/or the cut off points of harvest period to avoid periods of immature peel or senescent peel to reduce peel postharvest problems, such as physiological disorders and decay, while still ha ving acceptab le internal quality.

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105 Table 2 1. Changes in Marsh grapefruits TSS: acid ratio with harvest date over three seasons. Data are average of 3 replicates from control trees. Date 2004/2005 2005/2006 2006/2007 September 6.40 e 6.93 e 5.35 d October 7.03 d e November 7.64 dc 7.62 de 6.77 c December 7.81 c January 7.89 c 8.56 dc 8.58 b February 8.05 c March 8.03 c 9.07 c 8.68 b April 8.30 c May 10.56 b 10.15 b 9.89 a June 11.85 a July 13.31 a Means with the same letters in each column ar e not signi Table 2 2. Changes in Valencia oranges TSS: acid ratio with harvest date over t wo seasons. Data are average of 3 replicates from control trees. Date 2005/2006 2006/2007 January 10.49 c 11.50 c March 15.77 bc 17.20 bc May 25.89 b 25.53 ab July 49.28 a 32.93 a Table 2 3. Changes in Marsh grapefruits color index with harvest date over three seasons. Data are average of 3 replicates from control trees. Date 2004/2005 2005/2006 2006/2007 September 9.47 c 10.85 c 10.79 c October 5.92 b November 2.40 a 3.33 b 2.66 b December 0.59 a January 0.94 a 0.86 a NA February 0.90 a March 0.65 a 0.66 a NA April 1.14 a May 1.09 a 1.03 a 0.59 a June 1.53 a July 0.94 a

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106 Table 2 4. Changes in Valencia oranges color index with harvest date over t wo seasons. Data are average of 3 replicates from control trees. Date 2005/2006 2006/2007 January 1.43 b NA March 3.22 a NA May 4.01 a 3.23 a July 3.67 a 2.04 b Table 2 5. Changes in Marsh grapefruits tissue turgidity (Kg) with harvest date over t hree seasons. Data are average of 3 replicates from control trees. Date 2004/2005 2005/2006 2006/2007 September 4.45 c 4.83 c 7.07 a October 5.28 a November 4.01 d 6.34 a 4.85 d December 4.76 b January 4.44 c 4.67 c 6.14 b February 4.92 b Ma rch 4.73 b 5.84 ab 5.36 c April 4.73 b May 4.38 c 5.34 bc 5.64 c June 4.37 c July 4.89 c Means with the same letters in each column are not significantly different ( P .. Table 2 6. Changes in Valencia oranges tissue turgidity (Kg) with harvest date over t wo seasons. Data are average of 3 replicates from control trees. Date 2005/2006 2006/2007 January 9.04 a 6.56 a March 9.71 a 5.92 b May 7.85 b 5.47 b July 7.63 b 4.71 c Means with the same letters in each column are not significantly different ( P

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107 Table 2 7. Changes in Marsh grapefruits detachment force (Kg) with harvest date over three seasons. Data are average of 3 replicates from co ntrol trees. Date 2004/2005 2005/2006 2006/2007 September 9.08 abc 10.59 a 12.65 a October 9.60 ab November 9.63 ab 7.73 b 9.59 b December 10.26 a January 9.89 a 7.71 b 9.29 b February 9.87 a March 9.48 abc 3.89 c 6.92 c April 8.18 c May 8.19 c 9.49 ab 8.56 bc June 8.45 bc July 8.86 ab Means with the same letters in each column are not significantly different ( P Table 2 8. Change s in Valencia oranges detachment force (Kg) with harvest date over t wo seasons. Data are average of 3 replicates from control trees. Date 2005/2006 2006/2007 January 11.50 a 16.29 a March 8.42 b 14.20 ab May 10.56 a 12. 33 b July 10.01 ab 15.14 a Means with the same letters in each column are not significantly different ( P =0.05) Table 2 9. P ercentage w eight loss of control Marsh grapefruit stored for 12 weeks at two temperatures over two seasons (average percent age / 12 weeks storage / harvest date ). 2004/2005 2005/2006 Date 40 o F 70 o F 40 o F 70 o F September 2.37 a 4.88 d 3.89 ab 6.08 a October 3.07 a 7.56 ab November 2.96 a 7.35 abc 3.84 ab 6.43 a December 2.16 a 5.17 cd January 2.64 a 8.12 ab 2.56 b 5.53 a February 2.95 a 9.77 a March 2.54 a 8.21 ab 2.96 ab 2.83 b April 2.90 a 9.27 a May 2.52 a 6.53 bcd 3.61 ab 3.42 b June July 4.48 a 5.16 a Means with the same letters in each column are not significantly different ( P .

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108 Table 2 10. Percentage w eight loss of control Valencia orange stored for 12 weeks at two temperatures over one season (average percentage / 12 weeks storage / harvest date ). 2005/2006 Date 40 o F 70 o F January 3.65 b 8.06 a March 3.64 b 4.44 c May 3.77 ab 5.35 bc July 5.03 a 7.08 ab Means with the same letters in each column are not significantly different ( P Table 2 11. Percentage decay of control Marsh grapefruit stored for 12 weeks at two temperatures ove r two seasons (average percentage / 12 weeks storage / harvest date ). 2004/2005 2005/2006 Date 40 o F 70 o F 40 o F 70 o F September 2.96 bc 11.48 c 0.00 b 16.29 c October 4.07 bc 13.89 bc November 0.93 c 10.74 c 0.00 b 16.67 c December 3.70 bc 19.07 bc January 3.89 bc 15.00 bc 0.56 b 47.22 ab February 14.26 a 20.00 bc March 14.44 a 35.74 a 3.89 a 39.82 b April 14.26 a 23.89 b May 9.44 ab 22.78 b 3.52 a 22.78 c June July 5.56 a 59.07 a Means with the same letters in each column are not significantly different ( P Table 2 12. Percentage decay of control Valencia orange stored for 12 weeks at two temperatures over one season (average percentage / 12 weeks storage / harvest date ). 2005/2006 Date 40 o F 70 o F January 0.74 b 26.11 b March 3.15 b 24.44 b May 5.18 b 46.29 a July 9.81 a 52.22 a Means with the same letters in each column are not significantly different ( P .

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109 Table 2 13. Percentage chilling injury of control Marsh grapefruit (two seasons) and Valencia orange (one season) stored for 12 weeks at 40oF (average percentage storage / 12 weeks / harvest date ) Marsh grapefruit Valencia orange Date 2004/2005 2005/2006 2005/2006 September 12. 41 d 43.33 b October 26.48 bc November 41.85 a 65.78 a December 24.26 c January 25.56 bc 29.44 c 6.11 b February 21.85 c March 29.44 b 26.67 c 9.07 b April 21.85 c May 9.44 d 46.67 b 5.37 b June July 52.41 b 17.59 a Means with the same letters in each column are not significantly different ( P 4 5 6 7 8 9 10 11 Sept Nov Jan Mar May Harvest date TSS: acid ratio 2004/05 2005/06 2006/07 Figure 2 1. Change s in Marsh grapefruits TSS: acid ratio with harvest date over three seasons Data are average of 3 replicates from control trees.

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110 0 10 20 30 40 50 60 Jan Mar May Jul Harvest date TSS: acid ratio 2005/06 2006/07 Figure 2 2. Change s i n Valencia orange s TSS: acid ratio with harvest date over t wo seasons. Data are average of 3 replicates from control trees. -14 -12 -10 -8 -6 -4 -2 0 2 Sept Nov Jan Mar May Harvest date Color index 2004/05 2005/06 2006/07 Figure 2 3. Change s in Marsh grapefruits color index with harvest date over three seasons. Data are average of 3 replic ates from control trees.

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111 0 1 2 3 4 5 Jan Mar May Jul Harvest date Color index 2005/06 2006/07 Figure 2 4. Change s in Valencia orange s color index with harvest date over t wo seasons. Data are average of 3 replicates from control trees. y = -0.0181x + 4.7058 R2 = 0.0243 y = -0.0217x + 5.4487 R2 = 0.0153 y = -0.1182x + 6.4017 R2 = 0.1963 3 4 5 6 7 8 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date Puncture Resistance (Kg) 2004/05 2005/06 2006/07 Linear (2004/05) Linear (2005/06) Linear (2006/07) Figure 2 5. Change s in Marsh grapefruits tissue turgidity (Kg) with ha rvest date over three seasons. Data are average of 3 replicates from control trees.

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112 y = -0.0101x + 10.11 R2 = 0.6397 y = -0.0099x + 6.72 R2 = 0.9911 0 2 4 6 8 10 12 14 Nov Jan Mar May Jul Month Puncture Resistance (Kg) 2005/06 2006/07 Linear (2005/06) Linear (2006/07) Figure 2 6. Change s in Valencia orange s tissue turgidity (Kg) with harvest date over t wo seasons. Data are average of 3 replicates from control trees. 0 2 4 6 8 10 12 14 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date FDF (Kg) 2004/05 2005/06 2006/07 Figure 2 7 Change s in Marsh grapefruits fruit detachment force (FDF) (Kg) with harvest date over three seasons. Data are average of 3 replicates from control trees.

PAGE 113

113 0 2 4 6 8 10 12 14 16 18 Jan Mar May Jul Harvest date FDF (Kg) 2005/06 2006/07 Figure 2 8. Change s in Valencia orange s fruit detachment force (FDF) (Kg) with harvest date over t wo seasons. Data are average of 3 replicates from control trees. 0 2 4 6 8 10 12 Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date (month) Weight loss (%) 04/05 @ 40F 04/05 @ 70F 05/06 @ 40F 05/06 @ 70F Figure 2 9. Percentage w eight loss of control Marsh grapefruit stored for 12 weeks at two temperatures over two seasons ( data are average percentage / 12 weeks storage / harvest d ate) .

PAGE 114

114 0 1 2 3 4 5 0 2 4 6 8 10 12 Storage period (week) Weight loss (%) Sept Oct Nov Dec Jan Feb Mar Apr May Figure 2 10. Percentage w eight loss of Marsh grapefruit during storage at 40oF for 12 weeks in 2004/2005 season ( data are average percentage / 3 replicates / 2 weeks storage)

PAGE 115

115 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 Storage period (week) Weight loss (%) Sept Oct Nov Dec Jan Feb Mar Apr May Figure 2 11. Percentage w eight loss of Marsh grapefruit duri ng storage at 7 0oF for 12 weeks in 2004/2005 season (data are average percentage / 3 replicates / 2 weeks storage)

PAGE 116

116 0 1 2 3 4 5 6 7 8 9 0 2 4 6 8 10 12 Storage period (week) Weight loss (%) Sept Nov Jan Mar May July Figure 2 12. Percentage w eight loss of Marsh grapefruit during storage at 40oF for 12 weeks in 2005 /2006 season (data are average perc entage / 3 replicates / 2 weeks storage)

PAGE 117

117 0 2 4 6 8 10 0 2 4 6 8 10 12 Storage period (week) Weight loss (%) Sept Nov Jan Mar May July Figure 2 13. Percentage w eight loss of Marsh grapefruit during storage at 7 0oF for 12 weeks in 2005 /2006 season (data are average percentage / 3 replicates / 2 weeks storage)

PAGE 118

118 0 1 2 3 4 5 6 7 8 9 Jan Mar May Jul Harvest date (month) Weight loss (%) 40F 70F Figure 2 14. Percentage w eight loss of Valencia orange during storage at 40oF and 7 0oF for 12 weeks in 2005 /200 6 season (data are average percentage / 3 replicates / 2 weeks storage) 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 12 Storage period (week) Weight loss (%) Jan Mar May July Figure 2 15. Percentage w eight loss of Valencia orange during storage at 40oF for 12 weeks i n 2005 /200 6 season (data are average percentage / 3 replicates / 2 weeks storage)

PAGE 119

119 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Storage period (week) Weight loss (%) Jan Mar May July Figure 2 16. Percentage w eight loss of Valencia orange during storage at 7 0oF for 12 weeks in 2005 /200 6 season (data are average percentage / 3 replicates / 2 weeks stor age) 0 10 20 30 40 50 60 70 Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date Decay (%) 2004/05 @ 40F 2004/05 @ 70F 2005/06 @ 40 2005/06 @ 70F Figure 2 17. Percentage decay of control Marsh grapefruit stored for 12 weeks at two temperatures over two seasons (data are average percentage / 12 weeks storage / harvest date) .

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120 0 5 10 15 20 25 30 35 0 2 4 6 8 10 12 Storage period (week) Decay (%) Sept Oct Nov Dec Jan Feb Mar Apr May Figure 2 18. Percentage decay of Marsh grapefruit during storage at 40oF for 12 weeks in 2004/2005 season (data are average percentage / 3 replicates / 2 weeks storage)

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121 0 10 20 30 40 50 60 70 0 2 4 6 8 10 12 Storage period (week) Decay (%) Sept Oct Nov Dec Jan Feb Mar Apr May Figure 2 19. Percentage decay of Marsh grapefruit during storage at 7 0oF for 12 weeks in 2004/2005 season (data are average percentage / 3 repl icates / 2 weeks storage)

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122 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 Storage period (week) Decay (%) Sept Nov Jan Mar May Jul Figure 2 20. Percentage decay of Marsh grapefruit during storage at 40oF for 12 weeks in 2005 /200 6 season (data are average percentage / 3 replicates / 2 weeks storage)

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123 0 10 20 30 40 50 60 70 80 90 0 2 4 6 8 10 12 Storage period (week) Decay (%) Sept Nov Jan Mar May Jul Figure 2 21. Percentage decay of Marsh grapefru it during storage at 7 0oF for 12 weeks in 2005 /200 6 season (data are average percentage / 3 replicates / 2 weeks storage) .

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124 0 10 20 30 40 50 60 Jan Mar May Jul Harvest date (month) Decay (%) 40F 70F Figure 2 22. Percentage decay of Valencia orange during storage at 40oF and 7 0oF for 12 weeks in 2005 /200 6 season (data are ave rage percentage / 3 replicates / 2 weeks storage) 0 5 10 15 20 25 0 2 4 6 8 10 12 Storage period (week) Decay (%) Jan Mar May Jul Figure 2 23. Percentage decay of Valencia orange during storage at 40oF for 12 weeks in 2005 /200 6 season (data are average percentage / 3 replicates / 2 weeks storage)

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125 0 10 20 30 40 50 60 70 80 90 0 2 4 6 8 10 12 Storage period (week) Decay (%) Jan Mar May Jul Figure 2 24. Percentage de cay of Valencia orange during storage at 7 0oF for 12 weeks in 2005 /200 6 season (data are average percentage / 3 replicates / 2 weeks storage) 0 10 20 30 40 50 60 70 80 Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date (month) Chilling injury (%) Grapefruit 2004/05 Grapefruit 2005/06 Valencia 2005/06 Figure 2 25. Percentage chilling injury of control Marsh grapefruit (two seasons) and Valencia orange ( one season) stored for 12 weeks at 40oF (data are average percentage / 12 weeks storage / harvest date)

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126 0 10 20 30 40 50 6070 80 0 2 4 6 8 10 12 Storage period (week) Chilling injury (%) Sept Oct Nov Dec Jan Feb Mar Apr May Figure 2 26. Percentage chilling injury of Marsh grapefruit during storage at 40oF for 12 weeks in 2004/2005 season (data are average percentage / 3 replicates / 2 weeks storage)

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127 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 Storage period (week) Chilling injury (%) Sept Nov Jan Mar May Jul Figure 2 27. Percentage chilling injury of Marsh grapefruit during storage at 40oF for 12 weeks in 2005 /2006 season (data are average percentage / 3 replicates / 2 weeks storage)

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128 0 5 10 15 20 25 30 35 40 0 2 4 6 8 10 12 Storage period (week) Chilling injury (%) Jan Mar May Jul Figure 2 28. Percentage chilling injury of Valencia orange during storage at 40oF for 12 weeks in 2005 /2006 season (data are average percentage / 3 replicates / 2 weeks storage)

PAGE 129

129 6 7 8 9 10 11 12 13 14 15 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Harvest date (month) TSS:acid ratio CONT WS GR WS & GR Figure 2 29. Effect of field water stress and growth regulators treatments on TSS: acid ratio of Marsh grapefruit at harvest during the 2005/2006 and 2006/2007 season.

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130 0 10 20 30 40 50 60 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date (month) TSS: acid ratio CONT WS GR WS & GR Figure 2 30. Effect of field water stress and growth regulators treatments on TSS: acid ratio of Valencia orange at harvest during the 2005/2006 and 2006/2007 season.

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131 0 10 20 30 40 50 60 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date (month) TSS: acid ratio CONT Tyvek Figure 2 3 1. Effect of soil coverage with Tyvek on TSS: acid ratio of Valencia orange at harvest during the 2005/2006 and 2006/2007 season.

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132 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Harvest date (month) Color index CONT WS GR WS & GR Figure 2 32. Effect of field water stress and growth regulators treatments on color index of Marsh grapefruit at harvest during the 2005/2006 and 2006/2007 seasons

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133 0 12 3 4 5 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date (month) Color index CONT WS GR WS & GR Figure 2 33. Effect of field water stress and growth regulators treatments on color index of Valencia orange at harvest during the 2005/2006 and 2006/2007 seasons

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134 0 1 2 3 4 5 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date (month) Color index CONT Tyvek Figure 2 34. Effect of soil cove rage with Tyvek on color index of Valencia orange during at harvest during the 2005/2006 and 2006/2007 seasons

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135 4 5 6 7 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Harvest date (month) Tissue turgidity (Kg) CONT WS GR WS & GR Figure 2 35. Effect of field water stress and growth regulators treatments on Marsh grapefruit s tissue turgidity at harvest during t he 2005/2006 and 2006/2007 seasons.

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136 4 5 6 7 8 9 10 11 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date (month) Puncture resistance (Kg) CONT WS GR WS & GR Figure 2 36. Effect of field water stress and growth regulators treatments on Valencia oranges tissue turgidity at harvest during the 2005/2006 and 2006/2007 seasons.

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137 4 5 6 7 8 9 10 11 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date (month) Puncture resistance (Kg) CONT Tyvek Figure 2 37. Effect of soil coverage wit h Tyvek on Valencia oranges tissue turgidity at harvest during the 2005/2006 and 2006/2007 seasons.

PAGE 138

138 0 2 4 6 8 10 12 14 2005/06 Sept Nov Jan Mar May Jul 2006/07 Sept Nov Jan Mar May Harvest date (month) Detachment force (Kg) Control WS GR WS & GR Figure 2 38. Effect of field water stress and growth regulators treatments on fruit detachment force of Marsh grapefruit at harvest during the 2005/2006 and 2006/2007 seasons.

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139 0 2 4 6 8 10 12 14 16 18 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date(month) Detachment force (Kg) Control WS GR WS & GR Figure 2 39. Effect of field water stress and growth regulators treatments on fruit detachment force of Valencia orange at harvest during the 2005/2006 and 2006/2007 seasons.

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140 6 8 10 12 14 16 18 2005/06 Jan Mar May Jul 2006/07 Jan Mar May Jul Harvest date (month) Detachment force (Kg) CONT Tyvek Figure 2 40. Effect of soil coverage wi th Tyvek on fruit detachment force of Valencia orange at harvest during the 2005/2006 and 2006/2007 seasons.

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141 0 2 4 6 8 10 12 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Weight loss (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 41. Changes in cumulative percentage weight loss of water stress and growth regulators treated Marsh grapefruit during storage at 70oF during the 2005/2006 season.

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142 0 1 2 3 4 5 6 7 8 9 10 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Weight loss (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 42. Changes in cumulative percentage weight loss of water stress and growth regulators treated Marsh grapefruit during storage at 4 0oF during the 2005/2006 season.

PAGE 143

143 0 2 4 6 8 10 12 14 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Weight loss (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 43. Changes in cumulative perc entage weight loss of water stress and growth regulators treated Valencia orange during storage at 70oF during the 2005/2006 season.

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144 0 2 4 6 8 10 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Weight loss (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 44. Changes in cumulative percentage weight loss of water stress and growth regulators treated Valencia oran ge during storage at 40oF during the 2005/2006 season.

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145 0 2 4 6 8 10 12 14 Jan CONTROL Jan Tyvek Mar CONTROL Mar Tyvek May CONTROL May Tyvek July CONTROL July Tyvek Harvest date (month) & Treatment Weight loss (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 45. Effect of soil coverage with Tyvek on cumulative percentage weight loss of Valencia orange during storage at 70oF during the 2005/2006 season.

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146 0 1 2 3 4 5 6 7 8 9 10 Jan CONTROL Jan Tyvek Mar CONTROL Mar Tyvek May CONTROL May Tyvek July CONTROL July Tyvek Harvest date (month) & Treatment Weight loss (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 46. Effect of soil coverage w ith Tyvek on cumulative percentage weight loss of Valencia orange during storage at 40oF during the 2005/2006 season.

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147 0 2 4 6 8 10 12 14 16 18 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Decay (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 47. Changes in cumulative percentage decay of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season.

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148 0 20 4060 80 100 120 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Decay (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 48. Changes in cumulative percentage decay of water stress and growth regulators treated Marsh grapefruit during storage at 70oF during the 2005/2006 season.

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149 0 5 10 15 20 25 30 35 40 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Decay (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 49. Changes in cumulative percent age decay of water stress and growth regulators treated Valencia orange during storage at 40oF during the 2005/2006 season.

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150 0 10 20 30 40 50 60 70 80 90 100 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Decay (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 50. Changes in cumulative percentage decay of water stress and growth regulators treated Valencia orange during stor age at 70oF during the 2005/2006 season.

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151 0 5 10 15 20 25 30 Jan CONTROL Jan Tyvek Mar CONTROL Mar Tyvek May CONTROL May Tyvek July CONTROL July Tyvek Harvest date (month) & Treatment Decay (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 51. Effect of soil coverage with Tyvek on cumulative percentage decay of Valencia orange during storage at 40oF during the 2005/2006 season.

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152 0 10 20 30 40 50 60 70 80 90 100 Jan CONTROL Jan Tyvek Mar CONTROL Mar Tyvek May CONTROL May Tyvek July CONTROL July Tyvek Harvest date (month) & Treatment Decay (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 52. Effect of soil coverage with Tyvek on cumu lative percentage decay of Valencia orange during storage at 70oF during the 2005/2006 season.

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153 0 10 20 30 40 50 60 70 80 90 100 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) &Treatment Chilling injury (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 53. Changes in cumulative percentage chilling injury of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season.

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154 0 10 20 30 40 50 60 Jan CONTROL Jan WS Jan GR Jan WS & GR Mar CONTROL Mar WS Mar GR Mar WS & GR May CONTROL May WS May GR May WS & GR July CONTROL July WS July GR July WS & GR Harvest date (month) & Treatment Chilling injury (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 54. Changes in cumulative percentage chilling injury of water stress and growth regulators treated Valencia orange during storage at 40oF during the 2005/2006 season.

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155 0 5 10 15 20 25 30 35 40 Jan CONTROL Jan Tyvek Mar CONTROL Mar Tyvek May CONTROL May Tyvek July CONTROL July Tyvek Harvest date (month) & Treatment Chilling injury (%) 2 Weeks 4 Weeks 6 Weeks 8 Weeks 10 Weeks 12 Weeks Figure 2 55. Effect of soil coverage with Tyvek on cumulative percentage chilling injury of Valencia orange during storage at 40oF during the 2005/2006 season.

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156 CHAPTER 3 CHARACTERIZATION OF CITRUS PEEL MATURATI ON AND THE EFFECT OF WATER STRESS, GROWTH REGUL ATORS AND DATE OF HA RVEST ON SUGAR CONTE NT In citrus fruit, sugar content increases rapidly during development (Lowell et al., 1989 ), resulting from an increase in photoassimilate supply and a decrease in sugar consumption as respiratory rates decline (Goldschmidt and Koch, 1996). Sucrose is the major translocated carbohydrate in most plants ( Avigad, 1982), including citrus plants ( Kriedemann, 1969; Sawamura et al., 1975). Sugars increase gradually during maturation, until reach their peak then start declining gradually while the fru it remain on the tree (Grierson, 2006a ). Biochemical changes after harvest remain more or less constant, and any increase in sugar: acid ratio during postharvest storage is mainly because acids tend to decrease faster than sugars due to respiration ( Ting and Attawa y, 1971). Sugars are potential effectors of juice quality (Ting, 1969), peel color (Huff, 1984), and have a role in peel resistance to chilling injury (Purvis and Grierson, 1982; Purvis et al., 1979) and plant response to water stresses (Price et al., 2004) Soluble sugars could be contributing effectors of the GA3-mediated delay in chloroplast -chromoplast conversions by flavedo (Fidelibus et al., 2008). Water stress triggers the physiological function of osmoregulation, which involves solute, such as sugars accumulation in cells, sufficient to lower osmotic potential of cells and allow them to absorb water to maintain cell turgor (Meyer and Boyer, 1981) and minimize the detrimental effect of the drought (Morgan, 1984). In this study, our targeted sugars are sucrose and the main reducing sugars; glucose and fructose. Changes in flavedo and albedo sugars are monitored over the harvest season and combined to changes in physical measuremnts at harvest and weight loss, decay and chilling injury after storage. T he hypothesis is ; a s harvest dates become later, peel sugars will change

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157 from immature to mature to senescent, and peel physical changes will reflect level of peel maturity. Materials and Methods Field Experiments All three field experiments over three seasons 2004/2005, 2005/2006 and 2006/2007 were described before in chapter 2, pages 737 5 Storage Exp eriment (Season 2005/2006) Fruit from each replicate at each harvest date were stored in two cartons; one carton (30 fruits) at 40OF (4.4OC) and 85 90% RH and another carton (30 fruits) at 70OF (21.1oC) and 8590%RH, for 12 weeks. Tissue P reparation for C h emical A nalyses At harvest, as well as after 12 weeks storage, starting from the January harvest date of both Marsh grapefruit and Valencia orange, ten fruits out of each replicate were chosen for chemical analyses, by peeling the flavedo layer off, us ing an apple peeler (Back to Basics Products, Inc., Draper, UT, USA), and then the albedo layer was removed with a regular stainless steel knife. Flavedo and albedo were immediately frozen in liquid nitrogen ( Valero et al., 1998) and stored at 80oC (Forma 86oC ULT Freezer, Forma Scientific Inc., Marietta, OH, USA) until sugars analyses were performed. Soluble Sugars Extraction and Determination For each sample, 100mg of flavedo was mixed with 2ml of 80% ethanol in a centrifuge tube, and left at room temperature for 30 minutes. The same process was done for the albedo tissue. After 30 minutes, the mixture was centrifuged again for 5 minutes, then 50 L of the supernatant were taken for sucrose analysis and 20 L of the supernatant were taken for reducing sugars analysis.

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158 Sucrose was estimated by the method of Handel (1968) well and boiled at 100oC, then cooled down to room temperature for 10 minutes. Three ml anthrone solution (150mg anthrone / 100 ml sulfuric acid 70%) was added to the mixture, and then the mixture was placed in a water bath at 40oC for 20 minutes. The solution was then left at room temperature for 15 minutes to develop color before reading by spectrophotometer at 620nm. Samples were compared to a standard range including 0, 25, 50, 75 and 100 L of sucrose. Reducing sugars were estimated by the method of Nelson (1944) and Somogy (1952) by and mixed well. The solution was boiled for 10 minutes at 100oC and cooled down to room temperature. Reagent B mixed well. Finally, 1.4 ml of distilled water was added to the solution, and the solution was left at room temperature for 40 minutes to 3 hours (depending on the color development) before reading by spectrophotometer at 520nm. Statistical Analysis Data were analyzed by SAS 8.2 (Statistical Analysis System) using ANOVA (Analysis of Variance) and means were compared using DMRT (Duncan Multi ple Range Test). Standard error of the means were also calculated by SAS ( SAS Institute Inc., 1999). Results and Discussio n Effect of Harvest Date The seasonal patterns of sugar accumulation in flavedo and albedo tissue for the three harvesting seasons are shown in Table 3 1. As reported previously by Purvis et al. (1979) and

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159 Purvis and Grierson (1982), results showed that sucrose increased throughout the fall and winter, and reach the maximum in April and February for both flavedo and a lbedo, respectively during the first season, as in Figure 3 1, then start to decrease again toward the end of the season. On the other hand, as the growing season progressed, reducing sugars levels increased gradually in flavedo, but this increase in reduc ing sugar started in Nov ember in albedo tissue when the mean minimum temperatures started to drop below 70oF Figure B 1, appendix Reducing sugars reached their maximum level in March and February for flavedo and albedo, respectively, then start to declin e again when the temperature start to increase over 65oF Figure B 1 appendix. These results are in agreement with Grierson (2006a ), and this change in sugars level with temperature is mainly due to the effect of temperature on the activity of invertase ( Purvis and Rice, 1983). The level of sucrose and reducing sugars of flavedo and albedo during three seasons, Figures 3 1, 3 2 and 3 3 was interesting because the data show a low sucrose level compared to high reducing sugars in the flavedo in November T his is the period that showed a jump in color index values from September ( 9.47, 10.85, and 10.79) to November ( 2.40, 3.33, and 2.66) in all three seasons, respectively, as in Table 2 3 and Figure 23 in the previous chapter, because color change of flavedo tissue should be preceded by increasing in reducing sugars levels (Fidelibus et al., 2008; Pourtau et al., 2006; Price et al., 2004; Rolland et al., 2006) although sugar levels were not the same in all s easons. Color break is stimulated by low winter temperature (Erickson, 1968) and high level of peel reducing sugars (Goldschmidt and Koc h, 1996; Huff, 1984; Iglesias et al., 2001) This can be noticed from F igures 3 -1, 3 2 and 33, where the level of flavedo reducing sugars is higher than flavedo sucrose, and meanwhile the level of flavedo reducing sugars is increasing from September to N ovember, whereas the level of albedo reducing sugars is decreasing during all three seasons because of ascending reducing sugars

PAGE 160

160 gradient from albedo to flavedo. The time of color break can be advanced if sugar level in the peel is increased by stem inject ions of sucrose, which stimulate the conversion of chloroplast to chromoplast (Iglesias et al., 2001) The inverse correlation between the extent of green color retention and hexoses (e.g. glucose and fructose), but not sucrose, in flavedo is well known. Hexoses availability ca used downregulation of genes encoding enzymes for chlorophyll synthesis and photosynthesis (Fidelibus et al., 2008; Pourtau et al., 2006; Price et al., 2004; Rolland et al., 2006) and upregulation of chloroplast -ch romoplast interconversion ( Koussevitzky et al., 2007). The reduction in reducing sugars of flavedo and albedo tissues toward the end of the season during all three seasons (Figures 31, 3 2 and 3 3) maybe due to sugar movement from albedo to juice sacs, because albedo tissue works as a transit sink or reservoir for sucrose enroute by symplastic pathway via plasmodesmata (Garcia Luis et al., 1991) to juice sacs during late -season fruit development (Koch, 1984; Koch and Avigne, 1990; Yen and Koch, 1990) and this is also maybe have minor role f or the high TSS: acid ratio toward the end of the season, as in Table 2 1 and Figure 2 1 in the previous chapter, which is mainly related to reduction in acids due to respiration (Samson, 1986). Results of Valencia orange in the second and third season are presented in Table 3 2, and Figures 3 4 and 3 5, respectively. The general trend of sucrose and reducing sugars in 2005/06 and 2006/07 seasons, was to incre ase and to decrease, respectively, toward the end of the season, and this is the reason why fruit showed some late -season regreening and reduction of color index which was significant in 2006/07 (2.04 in July vs. 3.23 in May), as shown in Table 2 4 in the previous chapter, because soluble sugars in the peel corresponded with color break in Valencia orange. Huff (1984) noticed that regreening of Valencia orange in spring is preceded by a decrease in peel soluble sugars. The reduction of reducing sugars of albedo toward

PAGE 161

161 the end of the season is due to the movement of sugars to juice sacs that for some minor extent can play a minor role increasing TSS: acid ratio in July, as in Table 2 2 in the previous chapter, which is mainly related to acid consumption toward the end of season (Samson, 1986). From the above mentioned results of both Mar s h grapefruit and Valencia orange, it can be noticed that most of the prominent changes of sugars accompanied by physiological changes in peel color and TSS: acid rati o of juice were taking place from November to April in 2004/05 season and from November to March in 2005/06 and 2006/07 seasons in grapefruit and from March to May in both seasons of Valencia These changes can be related to some other physiological diso rders, such as reduction of mid-season chilling injury, Table 2 13, as happen in March for grapefruit, Figure 2 22, and May for Valencia orange, Figure 2 -23, which was in agreement with reports of Purvis et al. (1979) and Purvis and Grierson (1982) stated that high levels of reducing sugars in grapefruit peel are considered to indicate greater resistance to chilling injury, and hence these result can be related for some extent to the less percent decay appeared during this period, as in Table 2 11 and Table 2 12 for both grapefruit and Valencia, respectively. So, from all of these information, it can be concluded th at fruit can be harvested within this mid -season period ; January March for Marsh grapefruit and March May for Valencia orange, with good juice quality and less senescent peel, which means less susceptibility to postharvest physiological disorders a nd decay ( Burns and Baldwin, 1994). Effect of Water Stress and Growth Regulators To investigate the effect of WS and GR on Marsh grapefruit in terms of sugar level and its relation to other physiological processes related to fruit quality, experiment was run for two seasons, and results are presented in Table 3 3. The results are interesting where they show reduction in fruit growth rate and less mature fruit with the use of GR expressed as low soluble sug ars compared to improved growth rate and more mature fruits with the use of WS expressed

PAGE 162

162 as more soluble sugars. The only significant difference was in March for flavedos sugars and in March and May for albedos sugars during 2005/06 season. In March, the level of reducing sugars of flavedo was less with GR (140.04 g/mg DW) compared to WS (232.61 g/mg DW) and control (195.06 g/mg DW) which means less fruit coloration, as well as less soluble sugars of albedo of GR treated fruit (164.46 g/mg DW) compar ed to the control (259.93 g/mg DW) Kuraoka et al. (1977) reported that one and two applications of GA3 at about the time of color break (which is the same application time of this experiment) could suppress total solubl e sugars accumulation in the flavedo of Satsuma mandarin fruit. The level of sucrose and reducing sugars is also shown in Table 3 3, and can be compared to the color index results of grapefruit in 2005/06 season, Table A 1, appendix, that shows low color index of GR -treated fruit ( 1.18) compared to the WS treated fruit ( 0.68) and control fruit ( 0.66) in March 2005/06 season These results also clear in Figure 3 6 that shows a peak in reducing sugars of flavedo and albedo in May of GR treated fruits, c ompared to that in March of WS -, WS*GR and control treated fruit, which confirm the role of growth regulators delaying maturation rate compared to water stress advancing it. Gibberellic acid (GA3) is used primarily as a preharvest treatment to delay cert ain aspects of rind senescence of Navel orange and seedless grapefruit ( Coggins, 1981), and to modify color development by delaying chlorophyll degradation (Coggins and Henning, 1988; Coggins and Hield, 1968; El Zeftawi, 1980a ; Garcia Luis et al., 1992; Goldschmidt et al., 1970; Porat et al., 2001 ) via inhibition of chlorophyllase synthesis (Trebitsh et al., 1993) and inhibition of carotenoid biosynthesis (Jun et al., 2002). Soluble sugars could be contributing effectors of the GA3-mediated delay in chloroplast -chromoplast conversions by flavedo ( Fidelibus et al., 2008). The role of GA3 in retarding color break may be through the involvement of ethylene and sugars. There is evidence in Sat suma mandarin that sucrose stimulates chloroplast to chromoplast

PAGE 163

163 conversion in flavedo, and this conversion requires initial nitrogen depletion and subsequent sucrose accumulation. The sucrose stimulation operates via ethylene, whereas GA3 acts as represso r of the sucrose ethylene stimulation (Iglesias et al., 2001) Also, GA3 can alter sucrose metabolism in plants (Cheikh et al., 1992), and alter expression of photosynthetic genes in plants (Fujii et al., 2008 ). The reduction of glucose and fructose levels in flavedo after application of GA3 was previously confirmed in Shamouti orange (Goldschmidt et al., 1977; Monselise and Goren, 1965) and Satsuma mandarin ( Kuraoka et al., 1977). Fidelibus et al. (2008) found in Hamlin orange that GA3 treatment maintained a descending sucrose gradient from the albedo to the flavedo typical of young, photosynthetically active fruit, as shown in Table 3 3, reducing flavedo glucose and fructose levels (140.04 g/mg D.W.) below those of nontreated fruit (195.06 g/mg D.W.). In general, acid content in juice, and soluble sugars content in juice and peel increased in drought -stressed trees (Boman et al., 1999; Hockema and Etxeberria, 2001; Huang et al., 2000; Mostert and Van Zyl, 2000) Acidity increased more rapidly than TSS under dry condition. Consequently, TSS: acid ratio decreased ( Wittwer, 1995), and this is the reason for low TSS: acid rat io of WS -fruit in March 2005/06, as shown in Table A -1, appendix. Water stress triggers the physiological function of osmoregulation, which involve solute (i.e. sugars) accumulation in cells sufficient to lower osmotic potential of cells and allow them to absorb water to maintain cell turgor (Meyer and Boyer, 1981) and minimize the detrimental effect of the drought ( Morgan, 1984). Yakushiji et al. (1996) found that monosaccharides (i.e. glucose a nd fructose) were largely responsible for active osmoregulation in Satsuma mandarin fruit under water stress condition, and the concentration of sucrose, glucose and fructose increased in fruit sap under moderate water stress within a relatively short peri od. Furthermore, the total sugar content per fruit of water stressed trees was numerica ly higher than that in fruit of well -watered

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164 trees, as in Table 3 3 where flavedo reducing sugars in March of WS fruit and WS*GR fruit were numerically higher (232.61 an fruit (195.06 g/mg D.W.), suggesting that sugar accumulation in fruit peel and juice sacs was not caused by dehydration under water stress but rather that sugar were accumulated (from carbon ass imilates and/or tree reserve) by active osmoregulation in response to water stress (Yakushiji et al., 1998) that enhanced the rate of photosynthates por tioning in the fruit more than that of leaf and stem ( Asakura et al., 1991). It can also be noticed from Table A 1, appendix that in March, the percent chilling injury of water stressed -fruit stored at 40oF was smaller compared to the control and GR treatment. It w as 18.52% and 16.30% for WS and WS*GR treatments, respectively, compared to 26.67% for the control and 23.15% for the GR, and this meets the highest amount of reducing sugars of flavedo and albedo in WS and WS*GR compared to GR and control, as shown in Table 3 3 This was also followed by lower % decay of WS (0.74%) and WS*GR (0.55%) compared to GR (2.22%) and control (3.88%) at the same temperature in March These results confirmed the results of Purvis et al. (1979) and Purvis and Grierson (1982) stated that high levels of reducing sugars in grapefruit peel are considered to indicate greater resistance to chilling injury and hence these result can be related for some extent to the less percent decay In the third season 2006/07, Table 3 3 and Figure 3 7, reducing sugar levels of flavedo increased significantly with WS treatment in May compared to the control and GR tr eatments, with higher color index ( 0.66) compared to GR ( 1.19) but not the control ( 0.59) which showed better coloration, as in Table A 1, appendix. Also, it can be noticed that peel turgidity and FDF of water stressed fruit (5.48 Kg and 8.20 Kg ) w ere less than that of the control (5.64 Kg and 8.56 Kg ), GR (5.96 Kg and 8.32 Kg ) and WS*GR (5.83 Kg and 8.86 Kg ), respectively, which indicates that water stressed fruit are more mature. By comparing the amount

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165 of reducing sugars in flavedo during 2005/06 an d 2006/07 seasons, it can be noticed from Table 3 3 that despite no significant differences from the control, the difference was significant between GR and WS treatments in March of the 2005/06 season and in May of the 2006/07 season, which indicates that there may be some cumulative effect of GRs on the growth rate of the peel, expressed as color delay, as Table A 1, appendix shows almost the same color index value of GR treated fruits in March 2005/06 season ( 1.18) compared to May 2006/07 season ( 1.19), and both values significantly differed from the control ( 0.66 and 0.59, respectively) In other word, this seems to confirm the role of GR in delaying the rate of peel senescence, and hence, the time of fruit harvesting It also shows the difference in peel maturity between seasons. Application of all treatments to Valencia orange in the second season showed little difference among treatments, and the general trend was almost the same, Table 3 4 and Figure 38. Reducing sugars of flavedo and albedo we re high for all treatments compared to the sucrose of both tissues, and the GR treatments showed high amount of albedo reducing sugars in May compared to the control and other treatments, but the difference was only significant with the control Generally the amount of reducing sugars of flavedo and albedo started to decrease significantly by March, toward the end of the season. This is normal due to two things; movement of reducing sugars from peel to juice sacs by the end of the season (Koch, 1984; Koch and Avigne, 1990; Yen and Koch, 1990) and the natural regreening of the peel in the late season that must be preceded by a decline in the amount of reducing sugar of the peel ( Huff, 1984). This amount of albedo reducing sugar in March remained almost the same until May in the GR treatment, which delayed the rate of physiol ogical processes and peel senescence ( Coggins, 1981). A similar trend of sucrose and reducing sugars during the season was noticed in the 2006/07 season, Figure 3 9, with higher amounts of sugars in both flavedo and albedo for all

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166 t reatments compared to the 2005/06 season. This may have been due to the difference in the amount of monthly precipitation between the two seasons Figure B 2 appendix. Rainfall was higher in the first season than in the second season, allowing more concentrating of sugars in fruit peel and pulp in the second season. It can not be related to the cumulative effect of WS and GR because the control also had the same trend of sugar increase between the two seasons. There were no prominent differences between tr eatments in both seasons on sugar levels of flavedo and albedo, and this can be noticed in Table A 2, appendix, with no significant differences among treatments for color index or TSS: acid ratio. This also was noticed from the Tyvek experiment Table 3 5 and Figure s 3 10 and 311. During the period of soil coverage with Tyvek, the significant increase in the amount of albedo sucrose in January of the 2005/06 season and albedo reducing sugars in March of the 2006/07 season can be related to what Yakushiji e t al. (1996) found; that reducing sugars (i.e. glucose and fructose) were largely responsible for active osmoregulation under water stress conditions. Furthermore, the total sugar content (and acidity) per fruit of water stressed trees was significantly higher than that in fruit of well -watered trees, suggesting that sugar accumulation in f ruit peel was not caused by dehydration under water stress but rather that sugars were accumulated by active osmoregulation in response to water stress ( Y akushiji et al., 1998) that enhanced the rate of photosynthates partitioning to the fruit (Asakura et al., 1991) and influence gene expression for biosynthesis and perception of ABA (Rognoni et al., 2007; Rolland et al., 2006) and its interaction with ethylene initiating fruit color in response to stress (Price et al., 2004; Rognoni et al., 2007) The reduction in the amount of flavedo sucrose and albedo reducing sugars in July of 2005/06 and 2006/07 seasons maybe related to additional accumulation of sugars that can triggers a decline in photosynthetic gene expression (Wingler et al., 2006) and reduce photosynthesis under drought stress ( Vu and

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167 Yelenosky, 1989). All of these physiological processes enhance the rate of pe el maturation and senescence. Previous research findings pointed out that the accumulation of sugars in combination with low nitrogen under water stress conditions can induce senescence like symptoms ( Pourtau et al., 2004; Wingler et al., 2004), and expression of senescence associated genes, like SAG12, were expressed late during senescence processes (Wingler et al., 2006) Data of juice quality and peel color of Valencia orange were not significant enough and are hard to relate to WS, GR, WS*GR and Tyvek treatments. This may be due to color break being stimulated by low winter temperature ( Erickson, 1968) and level of peel sugar (Goldschmidt and Koch, 1996; Huff, 1984; Iglesias et al., 2001) Low temperature (5oC) can increase invertase activity and level of reducing sugars in flavedo of Marsh grapefruit on the tree and during storage (Purvis and Rice, 1983). In addition to this, as air and soil temperatures fall below 13oC (55.5oF), chlorophyll degradation takes place, revealing the underlying carotenoids and giving fruit a bright yellow or orange color (Young and Erickson, 1961). Unfortunately, Florida weather has warm winter nights with not enough low temperatures for that purpose, as shown in Figure B1, appendix. Areas with hot dry summer and cool humid winter, like California (Mediterranean cli mate) generally produce fruit with better color, but thicker and coarser peel than areas having more humid growing season and warmer winter nights, like Florida (subtropical climate) ( Young and Erickson, 1961). Effect of Storage Condition s Biochemical changes after harvest remain more or less constant (Ting and Attaway, 1971 ). Sugar content of flavedo of Marsh grapefruit stored at both 40oF and 70oF did not show any specific pattern during storage compared to the amount at each harvest date. The only noticeable change is the increase of flavedo reducing sugar of GR treat ed fruit at the end of storage period at 40oF, compared to the control, with all harvest dates except for July, and their reduction at

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168 70oF with all harvest dates except January as in Table 3 6 and Figure B 3, appendix. Regarding albedo sugars, the only p attern of change was the increase of sucrose of GR treated fruit stored at 70oF, and this was corresponding to the increase of sucrose at harvest date after March, as in Table 3 6 and Figure B 3, appendix. By comparing these data with results of Table A 1 appendix, weight loss and decay at 40oF decreased with the increase in reducing sugars. Fucik (1981) found that GA3 reduced water loss from grapefruit during postharvest stora ge because GA3-treated fruits have peel with a structure similar to that of young fruit (Coggins 1969b ). The reduction in decay may be related to GA3 maintaining a compact structure in the albedo (Monselise, 1979), and hence the whole peel structure, expressed as high peel turgidity of GA3treated fruit compared to the control, as shown in Table A 1, appendix, that reduced the possibility of patho gens invasion. Data of Valencia orange did not show any specific pattern of sugar changes for fruit stored at both 40oF and 70oF compared to their level at harvest The only noticeable change was the increase of albedo reducing sugars of fruit stored at 40oF treated with GR in comparison to the control, Table 3 7 and Figure B 4, appendix H owever the most prominent effect was with soil coverage with Tyvek that showed significant reduction in flavedo reducing sugars at 40oF in March, however the significa nt reduction of chilling injury at 40oF in March and decay at both 40oF and 70oF in May Table A 3, appendix, can be related to the increase in the amount of reducing sugars at harvest not during storage, as in Table 3 8 and Figure B -5, appendix. On the o ther hand, the reduction in sugar amount of infected fruits can be related to what Sinclair and Crandall (1949) reported about the physiological breakdown of the fruit while on the tree and in storage, and the deleterious effects caused by the invasion of fungi, result in chemical changes in the carbohydrate constituents of grapefruit peel. Generally, Yakushiji et al. ( 1998) found that

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169 photos ynthetic rate ( Vu and Yelenosky, 1989) and stomatal conductance ( Syvertsen, 1982) of severely drought stressed Satsuma mandarin trees were significantly lower than those of well watered trees, and this is the reason of reduction of sugar content of the peel in Tyvek treated fruit To summarize the above mentioned results of both March grapefruit and Valencia orange, most of the prominent changes of sugars accompanied by physiological changes in peel color and TSS: acid ratio of juice were taking place from November to April in 2004/05 season and from January to March in 2005/06 and 2006/07 seasons in grapefruit and from March to May in both seasons of Valencia, which can be related to some other physiological disorders, such as reduction of mid -season chilling injury; March for grapefruit, and May for Valencia orange, as well as less percent decay appeared during this period for both grapefruit and Valencia. So, it can be concluded that fruit can be harvested within this mid-season period, with good juice quality and less senescent peel, which means less susceptibility to postharvest physiological disorders and moderate percentage of decay. Also, this change i n sugar level by t he beginning of this harvest window is possibly the transition from immaturity stage to maturity stage of the peel. Adjusting sugar level using external application of GR, and hence changing harvest time can also be done. Results showed that GR delay the peak of albedo reducing sugar to May, compared to WS, WS*GR and control that with no differences for both Marsh grapefruit and Valencia orange only in the 2005/06 season, but no changes took place in the 2006/07 season. Gibberellic acid (GA3) is used primarily as a preharvest treatment to delay certain aspects of rind senescence of Navel orange and seedless grapefruit ( Coggins, 1981). Furthermore, the total sugar c ontent (and acidity) per fruit of water stressed trees was significantly higher than that in fruit of well -watered trees. Sugars, in combination with low

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170 nitrogen supply due to drought, can induce senescence -like symptoms ( Pourtau et al., 2004; Wingler et al., 2004), such as increasing color index compared to GR treatment H owever, combining WS treatment with storage at low temperature (40oF ) showed less senescent like symptoms, such as reduction of percent chilling injury and percent decay during mid -season period compared to GR treatment and control. So, it seems that GR treatment reduce the senescence like symptoms, but when this treatment combined with cold storage, the results becomes worse than those of WS treat ment that presumably showed more senescent peel than GR treatment and control. This means that low temperature delay the appearance of senescencelike symptoms of water stressed -fruit. On the other hand, GR delay the rate of peel growth keeping the peel in less mature state and in a structure similar to that of young fruit ( Coggins, 1969b ). These results also showed that t he effect of GR on peel senescence is cumulative from one season to another, and this is appeared from the same rate of color index in March 2005/06 and May 2006/07 for Valencia orange. So, this is proving the role of GR in dela ying peel senescence to harvest fruit with higher TSS: acid ratio. Conclusion Sugar level in the peel of Marsh grapefruit and Valencia orange increas ed as fruit maturation progressed, reaching a peak in mid -season, and then decreased toward the end of the season. Fruit can be harvested within this mid-season period, with good juice quality and less senescent peel, which means less susceptibility to postharvest physiological disorders and decay (Burns and B aldwin, 1994). The role of growth regulators in delaying senescence was noticed through a lag in sugar accumulation, as well as no improvement (or no change) in juice quality as reported before by Coggins (1981). Water stress induced the process of cell senescence and chlorophyll degradation

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171 (Munne Bosch and Alegre, 2004 ), expressed as better fruit coloration than the control and GR treated fruit Treatments with growth regulators had more effect in adjusting the time of harvest than that of water stres s. Interactions between WS and GR with the storage temperature played a role on the appearance of senescence like symptoms, with no effect on sugar content of the peel, which remains almost constant during storage (Ting and Attaway, 1971) and mai nly is determi ned by the harvest date So, the cut off point, at which harvest should be stopped during the season to prevent more senescent peel and still provide good juice quality is may be sometime between March and April for Marsh grapefruit, because the amount of soluble sugars of the peel changes from one year to another (for instance; February to April in the 2004/05 season compared to January to March in the 2005/06 and 2006/07 seasons in grapefruit) probably based on environmental factors such as cold tempera ture in winter and rainfall during the growing season, especially in Florida. Moreover, the effect of growth regulators seems to be cumulative and affect the suitable time of harvest. For Valencia orange s maybe May is the cut off point of this window be cause of the reduction in sugar level after May, which may be related to the start of peel senescence, and then no more sugars can be accumulated in the peel Combining data of soluble sugars with the next three chapters; glycosidases, ABA, and volatile co mponents, in addition to future research may help to define peel senescence more accurately.

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172 Table 3 1. Effect of harvest date on the amount of sucrose and reducing sugars ( g / mg D.W.) of Marsh grapefruit peel during three seasons 2004/2005 Flavedo Albedo Harvest date Sucrose Reducing Sugar Sucrose Reducing Sugar September 0.00 e 17.01 e 112.76 c 129.59 a October 16.94 e 36.94 de 128.09 bc 100.99 a November 37. 24 d 45.33 dc 145.06 bc 96.23 a December 43.53 dc 60.44 bcd 148.76 bc 134.61 a January 55.94 bc 71.31 abc 136.14 bc 111.50 a February 61.81bac 93.32 a 196.41 a 176.42 a March 68.28 ab 98.45 a 171.44 ab 123.77 a April 79.29 a 82.64 ab 172.96 ab 165.42 a May 49.02 dc 86.11 ab 141.86 bc 172.45 a June 75.15 a 61.28 bcd 161.94 ab 98.71 a 2005/2006 Flavedo Albedo Harvest date Sucrose Reducing Sugar Sucrose Reducing Sugar September 6.55 c 32.79 d 81.36 b 143.46 c October November 26.80 b 39.3 3 d 115.56 a 106.87 d December January 39.82 a 89.19 dc 124.65 a 126.60 dc February March 10.97 c 195.06 a 68.79 b 259.93 a April May 39.38 a 166.81 ab 67.51 b 225.37 b June July 39.44 a 109.23 bc 84.33 b 223.24 b 2006/2007 F lavedo Albedo Harvest date Sucrose Reducing Sugar Sucrose Reducing Sugar September 17.33 b 53.48 c 79.72 c 165.99 ab October November 58.52 a 83.97 bc 145.18 a 110.20 b December January 42.38 ab 144.14 ab 93.60 bc 241.53 a February Marc h 47.52 a 190.11 a 121.71 ab 195.88 ab April May 37.36 ab 121.75 abc 118.13 ab 149.39 ab June July Mean separation within columns by DMRT (P

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173 Table 3 2. Effect of harvest date on the amount of sucrose and reducing sugars ( g / mg D. W.) of Valencia orange peel during two seasons 2005/2006 Flavedo Albedo Harvest date Sucrose Reducing Sugar Sucrose Reducing Sugar January 48.87 a 123.64 a 29.04 b 139.63 ab March 38.11 a 106.06 b 34.93 b 157.42 a May 59.71 a 57.48 c 90.67 a 1 03.60 bc July 70.34 a 26.68 d 83.82 a 85.33 c 2006/2007 Flavedo Albedo Harvest date Sucrose Reducing Sugar Sucrose Reducing Sugar January 56.63 ab 134.50 a 47.55 a 126.78 b March 48.27 b 144.15 a 47.10 a 192.19 a May 85.93 ab 83.83 ab 97.15 a 108.30 b July 115.76 a 24.55 b 94.81 a 78.20 b

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174 Table 3 3. Effect of water stress and growth regulators on sucrose and reducing sugars ( g / mg D.W.) of Marsh grapefruit peel du ring two seasons. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. 2005/2006 Flavedo Albedo Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar January CONT 39.82 a 89.19 a 124.65 a 126.60 a WS 45.71 a 82.26 a 116.24 a 122.39 a GR 39.42 a 64.73 a 98.54 a 136.49 a WS*GR 45.36 a 88.25 a 107.35 a 168.01 a March CONT 10.97 b 195.06 ab 68.79 b 259.93 a WS 26.25 ab 232.61 a 120.42 ab 235.80 ab GR 36.96 a 140.04 b 136.58 a 164.46 b WS*GR 27.07 ab 236.78 a 157.42 a 243.83 a May CONT 39.38 a 166.81 a 67.52 b 225.37 ab WS 23.13 a 162.99 a 101.15 ab 243.09 a GR 30.06 a 192.55 a 117.97 a 223.43 ab WS*GR 33.17 a 182.79 a 110.82 a 220.69 b July CONT 39.44 a 109.23 a 84.34 a 223.24 a WS 70.40 a 91.82 a 114.82 a 187.87 a GR 71.39 a 175.90 a 131.99 a 197.50 a WS*GR 66.26 a 130.80 a 117.55 a 234.00 a 2006/2007 Flavedo Albedo Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar January CONT 42.38 a 144.14 a 93.60 b 241.53 a WS 47.86 a 148.60 a 159.48 a 189.85 ab GR 32.71 a 188.40 a 155.62 a 158.45 ab WS*GR 33.15 a 128.03 a 153.45 a 135.70 b March CONT 47.52 a 190.11 a 121.71 a 195.88 a WS 51.10 a 140.17 a 103 .73 a 196.44 a GR 49.24 a 144.95 a 119.48 a 158.77 a WS*GR 35.70 a 97.39 a 117.37 a 191.86 a May CONT 37.36 a 121.75 b 118.13 a 149.39 a WS 34.46 a 192.16 a 89.91 a 161.94 a GR 46.03 a 135.86 b 85.73 a 199.93 a WS*GR 53.4 5 a 151.31 b 102.35 a 182.05 a July CONT WS GR WS*GR

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175 Table 3 4. Effect of water stress and growth regulators on sucrose and reducing sugars ( g / mg D.W.) of Valencia orange peel during two seasons CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. 2005/2006 Flavedo Albedo Month Treat. Sucrose Reducing sugar Sucrose Reducing suga r January CONT 48.87 a 123.64 a 29.05 b 139.63 a WS 38.82 a 103.72 a 23.08 b 128.88 a GR 51.30 a 121.72 a 44.59 a 154.65 a WS*GR 48.72 a 94.85 a 45.15 a 133.65 a March CONT 38.11 a 106.06 a 34.93 a 157.42 a WS 36.23 a 133.8 3 a 42.28 a 157.34 a GR 35.66 a 132.61 a 37.43 a 158.23 a WS*GR 40.51 a 119.68 a 55.50 a 153.19 a May CONT 59.71 a 57.48 a 90.67 a 103.60 b WS 61.08 a 65.63 a 75.61 a 138.33 ab GR 61.73 a 77.94 a 82.83 a 157.77 a WS*GR 61.47 a 46.56 a 79.72 a 133.75 ab July CONT 70.34 a 26.68 a 83.82 a 85.33 ab WS 42.77 ab 45.90 a 85.50 a 108.34 a GR 57.36 ab 22.24 a 86.20 a 75.43 ab WS*GR 27.74 b 31.98 a 68.30 a 33.85 b 2006/2007 Flavedo Albedo Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar January CONT 56.63 a 134.50 a 47.55 b 126.78 a WS 54.26 a 162.49 a 54.60 ab 136.90 a GR 56.74 a 85.80 a 59.62 ab 93.18 a WS*GR 47.40 a 111.39 a 76.17 a 139.63 a March CONT 48.27 a 144 .15 a 47.10 a 192.19 a WS 33.66 ab 171.44 a 46.42 a 200.77 a GR 37.34 ab 179.43 a 39.85 a 201.59 a WS*GR 26.94 b 170.32 a 41.10 a 178.75 a May CONT 85.93 a 83.83 a 97.15 a 108.30 a WS 105.46 a 97.35 a 89.48 a 156.98 a GR 63.20 a 95.41 a 68.70 a 152.81 a WS*GR 60.12 a 89.63 a 87.58 a 138.74 a July CONT 115.76 a 24.55 a 94.81 b 78.20 ab WS 105.39 a 50.70 a 119.95 ab 109.86 a GR 107.85 a 43.15 a 135.11 a 71.54 ab WS*GR 97.35 a 49.51 a 129.31 a 54.31 b

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176 Table 3 5. Effect of soil coverage with Tyvek on the amount of sucrose and reducing sugars ( g / mg D.W.) of Valencia orange peel during two seasons CON T; control, Tyvek; soil covered with tyvek. 2005/2006 2006/2007 Flavedo Albedo Flavedo Albedo Month Treat. Sucrose Reducing Sugar Sucrose Reducing Sugar Sucrose Reducing Sugar Sucrose Reducing Sugar January CONT 48.87 a 123.64 a 29.04 b 139.63 a 33.68 a 228.28 a 40.04 a 168.92 a Tyvek 45.45 a 107.93 a 34.31 a 136.25 a 36.41 a 145.33 a 42.77 a 164.01 a March CONT 38.11 a 106.06 a 34.93 a 157.42 a 45.68 a 104.42 a 36.49 a 164.70 b Tyvek 48.18 a 116.20 a 37.03 a 151.77 a 46.07 a 150.35 a 34.47 a 181.95 a May CONT 59.71 a 57.48 a 90.67 a 103.60 a 54.25 a 85.34 a 72.58 a 126.14 a Tyvek 66.76 a 45.68 a 72.38 a 108.42 a 58.17 a 58.75 a 76.65 a 99.17 a July CONT 70.34 a 26.68 a 83.82 a 85.33 a 6 1.29 a 39.52 a 105.69 a 161.51 a Tyvek 39.24 b 19.03 a 92.06 a 59.41 a 72.25 a 24.60 a 112.82 a 69.17 b Mean separation of treatments under each harvest date

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177 Table 3 6. Effect of water stress, growth regulators and storage temperature on the amount of sucrose and reducing sugars ( g / mg D.W.) of Marsh grapefruit peel during storage for 12 weeks at 40oF and 70oF in 2005/2006 season CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are means S E Flavedo Harvest 12 weeks @ 40 o F 12 weeks @ 70 o F Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar Sucrose Red ucing sugar Jan. Cont. 39.823.15 89.1913.82 33.2814.33 180.3226.04 13.741.51 81.861.78 WS 45.716.42 82.264.87 19.770.99 158.0517.67 27.5920.82 59.916.73 GR 39.424.40 64.736.42 23.312.64 192.1418.23 21.006.60 102.4511.36 WS*GR 4 5.364.51 88.256.13 12.002.18 122.5610.57 10.140.82 91.7622.10 Mar. Cont. 10.970.50 195.0633.56 11.001.75 146.079.54 36.325.37 264.0119.84 WS 26.253.96 232.6121.03 8.500.89 165.115.13 46.7616.44 217.6424.36 GR 36.9610.46 140.04 13.68 13.640.67 199.6157.46 33.328.88 204.6332.83 WS*GR 27.074.89 236.789.93 10.733.35 186.3226.92 20.945.86 240.4422.01 May Cont. 39.387.15 166.8114.00 25.475.75 241.9657.11 30.756.87 239.8541.01 WS 23.134.79 162.9915.02 35.2 613.72 105.1713.66 21.777.04 170.5844.73 GR 30.068.30 192.5515.35 31.604.20 249.6952.25 36.930.75 148.859.59 WS*GR 33.172.12 182.7914.92 14.144.81 127.1511.13 24.933.54 127.7610.38 Jul. Cont. 39.442.88 109.2323.38 18.836.13 22 9.0537.57 100.0215.0 166.8941.80 WS 70.4013.65 91.825.14 12.920.63 191.7710.19 55.383.39 171.3851.61 GR 71.3913.86 175.9053.79 10.713.07 213.2810.46 82.646.73 81.4213.95 WS*GR 66.264.97 130.8016.60 11.842.28 198.394.56 80.32 75.97 104.2153.70 Albedo Harvest 12 weeks @ 40 o F 12 weeks @ 70 o F Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar Sucrose Reducing sugar Jan. Cont. 124.656.45 126.6016.06 71.186.03 198.6424.52 44.211.34 152.5823.19 WS 116.2416.67 122.3914.50 55.219.68 186.375.46 31.595.86 142.0248.65 GR 98.5419.78 136.4919.93 71.121.60 195.1630.09 45.173.21 210.289.65 WS*GR 107.3515.53 168.0117.21 49.996.82 196.0913.84 40.131.67 205.077.63 Mar. Cont. 68.7914.03 259.9313. 19 54.695.95 226.7918.42 71.898.20 344.9713.26 WS 120.4210.99 235.801.50 59.062.92 245.2519.37 93.0014.58 296.4721.60 GR 136.5827.58 164.4643.48 63.873.32 267.537.38 117.795.30 274.2753.99 WS*GR 157.424.73 243.835.39 66.584 .56 274.862.27 73.686.04 331.4416.84 May Cont. 67.523.44 225.374.42 61.8817.84 219.6545.44 54.002.48 144.4831.97 WS 101.156.97 243.096.51 53.113.89 204.2913.56 55.762.63 153.6629.91 GR 117.9713.80 223.435.98 41.5010.55 217.495 0.71 81.732.88 250.9716.55 WS*GR 110.8214.21 220.695.77 54.689.55 224.5440.92 54.676.33 209.3630.61 Jul. Cont. 84.3412.65 223.244.19 59.4411.27 313.4712.67 139.9813.8 189.4732.02 WS 114.8222.97 187.8721.41 68.115.70 321.569.24 125.082.38 257.6266.82 GR 131.9923.73 197.5022.78 73.992.26 311.7918.65 190.9119.5 139.3117.06 WS*GR 117.5516.59 234.0019.07 39.9212.63 265.1030.04 87.6258.91 210.6873.87

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178 Table 3 7. Effect of water stress, growth regulators and stor age temperature on the amount of sucrose and reducing sugars ( g / mg D.W.) of Valencia orange peel during storage for 12 weeks at 40oF and 70oF in 2005/2006 season CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are means S E Flavedo Harvest 12 weeks @ 40 o F 12 weeks @ 70 o F Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar Sucrose Reducing sugar Jan. Cont. 48.873.38 123.64 3.94 16.571.59 120.7329.30 4.781.43 69.3418.65 WS 38.823.66 103.729.79 21.151.99 111.9320.91 4.540.98 56.4810.27 GR 51.303.74 121.726.39 19.962.07 114.8813.65 6.370.94 56.847.32 WS*GR 48.726.59 94.8515.16 20.211.69 83.537.32 4.790.18 69.952.59 Mar. Cont. 38.117.99 106.06 6.75 9.960.51 104.639.72 30.974.79 56.897.64 WS 36.233.44 133.8316.32 7.181.24 103.098.54 45.4220.16 67.5522.98 GR 35.668.46 132.6112.55 7.270.76 100.400.59 24.235.10 52.8310.17 WS*GR 40.513.09 119.68 6.81 8.540.74 118.572. 75 28.940.65 52.1211.56 May Cont. 59.7120.01 57.484.83 20.730.83 104.7213.53 32.841.37 31.159.07 WS 61.088.88 65.638.84 28.768.73 79.5615.16 26.184.99 53.1130.89 GR 61.7314.19 77.9426.49 19.423.46 112.1112.08 24.444.63 36.704 .73 WS*GR 61.4711.53 46.564.10 25.001.65 60.4335.32 27.502.76 20.825.42 Jul. Cont. 70.3413.87 26.683.46 20.934.70 103.4321.12 40.1312.29 67.2916.28 WS 42.773.37 45.9018.29 12.061.11 110.515.19 15.835.35 51.101.75 GR 57.369.8 4 22.244.99 17.343.06 113.892.40 24.986.28 53.849.98 WS*GR 27.744.14 31.981.50 13.542.72 123.3114.72 45.6011.65 54.9721.65 Albedo Harvest 12 weeks @ 40 o F 12 weeks @ 70 o F Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar Sucros e Reducing sugar Jan. Cont. 29.051.62 139.633.31 30.940.65 71.282.40 9.002.60 141.7027.2 WS 23.087.59 128.8824.64 34.694.47 138.8318.82 8.521.41 100.0812.8 GR 44.593.93 154.651.02 43.711.78 154.0622.59 10.213.71 92.7728.11 WS* GR 45.151.45 133.6511.27 37.554.82 111.6711.58 7.632.87 106.9917.9 Mar. Cont. 34.936.88 157.424.06 33.836.10 122.2723.15 48.442.96 152.4521.4 WS 42.2810.30 157.345.87 37.643.60 113.819.10 53.823.58 185.2914.5 GR 37.435.66 158 .236.86 43.564.51 140.8315.11 35.937.99 136.0041.4 WS*GR 55.503.09 153.1914.76 39.920.65 119.818.39 49.786.53 141.1223.6 May Cont. 90.6716.62 103.606.81 55.176.67 91.3612.39 62.9311.27 103.087.38 WS 75.6112.93 138.3313.87 5 0.9910.93 77.149.03 46.655.78 81.1816.22 GR 82.8312.64 157.774.07 53.362.91 98.7943.21 67.279.78 133.1017.1 WS*GR 79.7216.33 133.7518.00 52.797.41 88.3111.31 58.3114.13 72.862.74 Jul. Cont. 83.828.64 85.3323.27 51.178.67 110.64 10.63 90.9825.50 90.0122.67 WS 85.5014.12 108.3417.21 45.496.32 124.859.06 32.354.53 145.7221.4 GR 86.208.63 75.4315.12 49.6512.55 128.5818.17 62.6125.72 118.715.47 WS*GR 68.3011.80 33.857.21 40.039.93 160.0726.54 108.7834. 1 119.0514.2

PAGE 179

179 Table 3 8. Effect of soil coverage with Tyvek and storage temperature on the amount of sucrose and reducing sugars ( g / mg D.W.) of Valencia orange peel during storage for 12 weeks at 40oF and 70oF in 2005/2006 season CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Flavedo Harvest 12 weeks @ 40 o F 12 weeks @ 70 o F Month Treat. Sucrose Reducing sugar Sucrose Reducing sugar Sucrose Reducing sugar Jan. Cont. 48.873.38 123.64 3.94 16.571.59 120.7329.30 4.781.43 69.3418.65 Tyvek 45.453.34 107.934.32 17.483.90 91.4327.40 3.660.52 67.8512.84 Mar. Cont. 38.117.99 106.066.75 9.960.51 104.639.72 30.974.79 56.897.64 Tyvek 48.184.30 116.206.93 16.183.65 83.345.97 24.277.19 42.963.79 May Cont. 59.7120.01 57.484.83 20.730.83 104.7213.53 32.841.37 31.159.07 Tyvek 66.7621.16 45.683.39 15.854.90 74.6617.05 33.941.26 25.561.20 Jul. Cont. 70.3413.87 26.683.46 20.934.70 103.4321.12 40.1312.29 67.2916.28 Tyvek 39.24 19.03 10.33 92.79 52.81 10.86 Albedo Harvest 12 weeks @ 40 o F 12 weeks @ 70 o F Month Treat. Sucrose Reducing sug ar Sucrose Reducing sugar Sucrose Reducing sugar Jan. Cont. 29.051.62 139.633.31 30.940.65 71.282.40 9.002.60 141.7027.2 Tyvek 34.311.02 136.256.39 31.302.90 95.6326.60 11.645.32 105.3620.2 Mar. Cont. 34.936.88 157.424.06 33.836.10 1 22.2723.15 48.442.96 152.4521.4 Tyvek 37.032.45 151.773.48 37.044.40 135.5618.93 38.992.09 140.1640.9 May Cont. 90.6716.62 103.606.81 55.176.67 91.3612.39 62.9311.27 103.087.38 Tyvek 72.382.08 108.429.44 47.172.21 150.7823.46 75.7 71.64 185.1113.1 Jul. Cont. 83.828.64 85.3323.27 51.178.67 110.6410.63 90.9825.50 90.0122.67 Tyvek 92.06 59.41 35.27 107.882.76 124.37 98.32 Values are means S E

PAGE 180

180 0 50 100 150 200 250 Sept Oct Nov Dec Jan Feb Mar Apr May Jun Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 1. Effect of harvest date of Marsh grapefruit on sucrose and reducing sugars content during 2004/2005 season. Values are means S E

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181 0 50 100 150 200 250 300 Sept Nov Jan Mar May Jul Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 2. Effect of harvest date of Marsh grapefruit on su crose and reducing sugars content during 2005/2006 season. Values are means S E.

PAGE 182

182 0 50 100 150 200 250 300 Sept Nov Jan Mar May Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 3. Effect of harvest dat e of Marsh grapefruit on su crose and reducing sugars content during 2006/2007 season. Values are means S E.

PAGE 183

183 0 20 40 60 80 100 120 140 160 180 Jan Mar May Jul Harvest date Sugar conten (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 4. Effect of harvest date of Valencia orange on su crose and reducing sugars content during 2005/2006 season. Values are means S E

PAGE 184

184 0 50 100 150 200 250 Jan Mar May Jul Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 5. Effect of harvest date of Valencia orange on su crose and reducing sugars content during 2006/2007 season. Values are means S E.

PAGE 185

185 Figure 3 6. Effect of field water stress and growth regulators treatments o n su crose and reducing sugars content of Marsh grapefruit peel during the 2005/2006 season. Values are means S E. 0 50 100 150 200 250 300 Control Jan Mar May Jul WS Jan Mar May Jul GR Jan Mar May Jul WS & GR Jan Mar May Jul Treatment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug.

PAGE 186

186 Figure 3 7. Effect of field water stress and growth regulators treatments on su crose and reducing sugars con tent of Marsh grapefruit peel during the 2006/2007 season. Values are means S E. 0 50 100 150 200 250 300 Control Jan Mar May WS Jan Mar May GR Jan Mar May WS & GR Jan Mar May Treatment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug.

PAGE 187

187 0 40 80 120 160 200 Control Jan Mar May July WS Jan Mar May July GR Jan Mar May July WS & GR Jan Mar May July Traetment & harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 8. Effect of field water stress and growth regulators treatments on su crose and reducing sugars content of Valencia orange peel during the 2005/2006 season. V alues are means S E.

PAGE 188

188 0 50 100 150 200 250 Control Jan Mar May July WS Jan Mar May July GR Jan Mar May July WS & GR Jan Mar May July Treatment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 9. Effect of field water stress and growth regulators treatments on su crose and reducing sugars content of Valencia orange peel during the 2006/2007 season. Values are means S E.

PAGE 189

189 0 40 80 120 160 200 Control Jan Mar May July Tyvek Jan Mar May July Treatment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 10. Effect of soil coverage with Tyvek on su crose and reducing sugars content of Valencia orange peel during the 2005/2006 season. Values are means S E.

PAGE 190

190 0 50 100 150 200 250 Control Jan Mar May July Tyvek Jan Mar May July Traetment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure 3 11. Effect of soil coverage with Tyvek on sucrose and reducing sugars content of Valencia orange peel during the 2006/2007 season. Values are means S E.

PAGE 191

191 CHAPTER 4 CHARACTERIZATION OF CITRUS PEEL MATURATI ON AND THE EFFECT OF WATER STRESS, GROWTH REGUL ATORS AND DATE OF HA RVEST ON INTERNAL LE VEL OF GLYCOSIDASES IN GRAP EFRUIT RIND The objective of this study was t o focus on glycosidases changes that affect cell wall noncellulosic neutral sugar composition (i.e. glycosides, such as, galactose, mannose, arabinose, xylose etc) in citrus peel during maturation to provide information that could be used to further defin e physiological maturity and senescence. In a previous study, the percentage of these neutral sugars in Marsh grapefruit flavedo decreased from 38% to 27% during fruit development (Mitcham and McD onald, 1993) Glycosidases reflect the changes in cell wall galactosyl and arabinosyl residuals indicating changes in pectin polymers of cell walls, which in turn are associated with the loss of peel firmness over the season (Mitcham and McDonald, 1993) A survey of many types of fruits revealed that 15 out of 17 different fruits experienced a net loss of noncellulosic neutral sugar with ripening and 14 out of 15 experienced net losses of cell wall galactosyl residues and/or arabinosyl residues (Gross and Sams, 1984) Gibberellic acid retards rate of peel softening by retarding the seasonal change and reduction i n the amounts of cell wall galactosyl, arabinosyl and fucosyl residues. While there was a tendency towards higher xylosyl, mannosyl and glucosyl content in gibberellic acid-treated fruit, it seems that gibberellic acid has a greater effect on neutral sugar s associated with pectin polymers than on neutral sugars associated with hemicellulosic polymers (Mitcham and McDonald, 1993) In this study, changes in peel maturation was study in the context of fruit softening. The activity of associated enzymes; -mannosidase (EC 3.2.1.24) were measured as indicators of maturity A -galactosidase was remarkably enhanced by 2,4 D in the concentration range which induced growth (107105 M)

PAGE 192

192 (Tanimoto and Igari, 1976 ), and the same results were rep orted using IAA, as well (Johnson et al., 1974; Tanimoto, 1985 ). In contrast, some other research findings showed that IAA had no significant effect on the activity of -mannosidase (Evans, 1974; Katsumi and Yamamoto, 1979). Water stress stimulates the activity of both enzymes causing cell wall deterioration (El Tayeb and Ahmed, 2007; Konno et al., 1986) -galactosidase is involved in breakdown of pectic polymers of galactose during cell growth (Konno et al., 1986) and in breakdown of the bonds between cell wall polysaccharides during cell wall loosening (Murry and Bandurski, 1975) On 1,3 linked oligosaccharides (Jagadeesh et al., 2004; Li, 1967) during its role in glycoprotein processing (Winchester and Fleet, 1992). Materials and Methods Field Experiments All three field experiments over three seasons 2004/2005, 2005/2006 and 2006/2007 were described before in chapter 2, pages 737 5 Tissue preparation for chemical analyses At every harvest date, tissue was prepared and stored, as described before in chapter 3, page 1 39. Glycosidases Extraction Glycosidases extraction was made accordi ng to the method of Burns and Baldwin (1994). Frozen tissue (5 g) was homogenized in 15 ml cold acetone (3:1, acetone: tissue, v/w) by shaking the mixture for about 2 hours at 4oC. Using vacu um filtration, the insoluble materials were collected on filter paper, then washed with 10 ml cold acetone (two volumes of the original

PAGE 193

193 weight), and then washed again with 10 ml cold acetone. The residue was dried overnight under desiccant. Glycosidases we re extracted from acetone powders at 4oC. Powder was agitated using a platform shaker (Innova 2100, New Brunswick Scientific Inc., Edison, NJ, USA) for 1 hour in cold water (1 g acetone powder of flavedo: 10 ml cold water & 1 g acetone powder of albedo: 25 ml cold water, w/v). The mixture was centrifuged at 10000 g for 30 minutes at temperature 4oC using a Sorvall Evolution RC Centrifuge with SS 34 Rotor (Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was collected and p ellets were agitated a gain in cold water (1 g acetone powder of flavedo: 10 ml cold water and 1g acetone powder of albedo: 25 ml cold water, w/v) for 30 minutes, and then centrifuged again at 10000 g for 30 minutes at temperature 4oC. The supernatant was collected again and com bined with the first supernatant. The final pellets were discarded and the supernatant was kept at 80oC (Forma 86oC ULT freezer, Forma Scientific Inc., Marietta, OH, USA) until assaying for glycosidase activity. Protein Analysis Protein was estimated by the method of Bradford (1976) using bovine serum albumin (BSA) at a concentration of 2.0 mg/ml in a 0.9% aqueous (as standard) with concentrations of 0, 5, 10, 15 and 20 respectively, with water to equal 10 00 L. Samples (supernatant) were brought from the freezer and melted in a water bath at 25oC until liquid, and then 200 l of the sample were added to 800 l of water. Coomassie (1 ml) plus protein assay reagent (Thermo Scientific, Rockford, IL, USA) we re added to all tubes of standards and samples and mixed well, then incubated in a water bath (Isotemp water bath, Fisher Scientific, Pittsburgh, PA, USA) at 25oC for 15 minutes to be read by UV -visible

PAGE 194

194 recording spectrophotometer (Shimadzu UV 160, Shimadz u Scientific Instruments, Inc., Kyoto, Japan) at wave length 595 nm. -Galactosidase Activity This was done according to the methodology of Sigma -Aldrich (St. Louis, MO, USA) according to Distler and Jourdian (1973) with a minor change of incubation time from 10 minutes to 15 minutes. Enzyme substrate (4 D galactopyranoside) and all chemicals used in this experiment were Sigma -Aldrich products. Two tubes from each sample (reaction R & control C ) were prepared. Enzyme substrate (0.5 ml) was added to 0.4 ml sodium acetate buffer (SAB) in all tubes (R & C), and solution was mixed well, then samples (0.02 ml) were only added to the R tubes and all tubes were incubated at 25oC for 15 minutes. After 15 minutes, 3 ml of borate buffer (BB), stop reagent, were added to all tubes to stop the reaction, then solution was mixed well. Samples (0.02 ml) were added again to the C tubes then shaken well. Samples appearance w as measured using a UV -visible recording spectrophotometer (Shimadzu UV 160, Shimadzu Scientific Instruments, Inc., Kyoto, Japan) at 400 nm. Calculations wer e made according to the protocol using the following equation; units enzyme / mg protein = (A400nm sample A400nm blank Total volume of assay) (incubation time 18 protein concentration in reaction mix), where A400nm; spectrophotometer reading at 400 nm, and 18; millimolar extinction coefficient of p Nitrophenol at 400 nm. -Mannosidase Activity This also was done according to the methodology described by Sigma -Aldrich (St. Louis, MO, USA) according to Li (1967) and Lee (1972) with a minor change of th e incubation time from 5 minutes to 10 minutes. Enzyme substrate (4D -mannopyranoside) and all chemicals used in this experiment were Sigma -Aldrich products. Two tubes from each sample (reaction R & control C ) were prepared. At the beginni ng, only 0.1 ml water was added to the

PAGE 195

195 C tubes. Enzyme substrate (0.5 ml) was added to 0.5 ml citrate buffer (CB) in all tubes (R & C), and solution was mixed well, then samples (0.1 ml) were only added to the R tubes and all tubes were incubated at 25oC for 10 minutes. After 10 minutes, 2 ml of borate buffer (BB), stop reagent, were added to all tubes to stop the reaction, then solutions were mixed well. Samples appearance w as measured using a UV -visible recording spectrophotometer (Shimadzu UV 160, Sh imadzu Scientific Instruments, Inc., Kyoto, Japan) at wave length 405 nm. Calculations were made according to the protocol using the following equation; units enzyme / mg protein = (A405nm sample A405nm blank Total volume of assay) (incubation time 18 protein concentration in reaction mix), where A405nm; spectrophotometer reading at 405 nm, and 18; millimolar extinction coefficient of p Nitrophenol at 405 nm. Statistical Analysis Data were analyzed by SAS 8.2 (Statistical Analysis System) using AN OVA (Analysis of Variance) and means were compared using DMRT (Duncan Multiple Range Test). Standard error of the means were also calculated by SAS ( SAS Institute Inc., 1999). Results and Discussion Effect of Harvest Date As known, in citrus fruit, the growth pattern of peel is different from that of pulp, and the first stage of cell division can continue in the peel until cl ose to pulp maturation ( Scot t and Baker, 1947). Contrarily, Coggins in (1969b ) stated that peel senescen ce sometimes begins before fruit has attained internal maturity. Also, it has been found that respiration rate of the peel of grapefruit is considerably higher than that of the pulp, but tends to decrease after harvest while that of the rest of the fruit r emains constant ( Vakis et al., 1970) -galactosidase has been associated with actively growing tissue (Labrador and Nicolas, 1984), where high levels of activity have been correlated with cell wall loosening mechanisms responsible for growth. This

PAGE 196

196 was noticed in all three seasons, Table 4 1 and Figures 41, 42 and 4 3, in both flavedo and albedo tissues, where the high -galactosidase activity was consistent with the first sampling date, September, that probably represent the beginning of stage 3 of fruit growth, that characterized by cession of cell division in the flavedo epidermis, as the fruit approaches to maturity (Bain, 1958; Monselise, 1986) and the involvement of the enzyme in the modification of the neutral sugar cell wall fraction, causing reduction in cell wall turgidity and the whole fruit firmness, Table 2 5, chapter 2. It can also be noticed that the level of flavedo and galactosidase decreased after September then become more or less constant with the progress of harvest dates with a -galactosidase in March o f the first and second seasons and March May of the third season, and galactosidase in April May March May and January March of the first, second and third season, respectively, and this followed by little increase again at the end of the season, after May This is also corresponding with little increase in peel turgidity values during the period from March to April in the first and second seasons, Table 2 5. This difference in the time of enzyme activities among all three season may reflect difference in peel maturity. These results a re consistence with previous reports in acid lime ( Selvaraj and Raja, 2000 ) and Kinnow mandarin (Ram et al., 2002) sho -galactosidase activity in peel tissue increased from stage 1 to stage 3 of fruit development, then decreased at subsequent stages, and the enzyme had low correlation (r2 = 0.505) in peel with fruit firmness. Also, Mitcham and McDonald (1993) reported that the percentage of the neutral sugars in Marsh grapefruit flavedo decreased from 38% to 27% during fruit development In addition to that, the increase in the enzyme act ivity after May, especially in albedo tissue may be related to the over senescent stage of the peel, and this can be referred to Burns (1990b ) who found in Lee tangelo and Dancy tangerine that -

PAGE 197

197 galactosidase activity was 2 to 3 -fold higher in extracts of granulated (senescent) than normal juice vesicles. Other research findings confirmed that the hydrolysis of cell wall and the activity -galactosidase increased during the ripening and soft ening of apples (Bartley, 1974; Berard et al., 1982) tomatoes ( Pressey, 1983; Watkins et al., 1988), muskmelon (Ranwala et al., 1992) and apricots (Kovacs and Nemeth -Szerdahelyi, 2002) -mannosidase of flave do and albedo had no fixed trend with harvest dates galactosidase in both tissues, as shown in Table 4 1, and Figures 4 1, 4 2 and 4 mannosidase showed high level in September and November of the first season, Figure 4 1 and in November of the second season Figure 4 2, and third season, Figure 4 3, that probably represents the beginning of stage 3 of fruit growth. The big difference between flavedos -mannosidase may be related to difference in extractability of the enzyme between the two tissues related to difference in cellular compartmentation and role of the enzyme in maturity and senescence of the flavedo and albedo as suggested by Dick et al. (1984) The level of fl -mannosidase fluctuated over the season during all three seasons, with a trend of little increase toward the end of the season. The highest activity was noticed in January and May of 2004/05, March 2005/06, and in the third season the activity was a lmost constant from March to the end of the season. These results toward the end of the first and second seasons are in agreement with previous reports of Burns and Baldwin (1994) stated that specific activity -mannosidase increased as maturation and senescence progressed. It can also be -mannosidase was in consistence with the bloom period of citrus, and at this time fruit were approximately 12 months of age, which means in senescence stage of the peel. This is confirmed in this study; for instance, the enzyme peak was in February

PAGE 198

198 2005, Table 4 1 and the bloom date was in March 3rd to March 4th 2004 (Albrigo, 2004), whereas the enzyme peak was in January 2006, Table 4 2, and the bloom date wa s from February 25th to March 5th 2005 ( Albrigo 2005). In another research findings, Burns (1990b ) -mannosidase activity was only detected in granulated (senescent) tissue, and this was noticed in the third season; the peak in -mannosidase was in May 2 007, Table 4 3 and the bloom date was little late in March 1st7th 2006 (Albrigo, 2006). These results are also in agreement with other research findings, on climacteric fruits, stating that t mannosidase was increased during the ripening of tomatoes ( Watkins et al., 1988) and pear (Ahmed and Labavitch, 1980). A survey of many types of fruits revealed that 15 out of 17 different fruits experienced a net loss of noncellulosic neutral sugar with ripening and 14 out of 15 experienced net losses of cell wall galactosyl residues and/or arabinosyl residues (Gross and Sams, 1984) From these data, it can be noticed that the period from February to March is a good time to harvest Marsh grapefruit with acceptable fruit firmness associated Table 2 5, with good TSS: acid ratio that start to be 8:1 by this period during all three seasons, Table 2 -1, however, the average of three seasons data in Table 2 1 and 25 refer that fruit still can be harvested earlier in January or little late in April with acceptable taste and firmness, respectively. Effect of Water Stress and Growth R egulators To investigate the effect of WS and GR on the activity of both enzymes during the growing season, this experiment were carried out in two seasons, as shown in Table 4 2 and Figures 4 4 and 45, and results showed that there were no significant difference among all treatments and the control at all harvest dates except at May 2005/06 where GR treatments inc -mannosidase significantly compared to the control and other two treatments, which means the delay of activity until later in the season was accompanied by a

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199 delay in certain aspects of rind senescence (Coggins, 1981); high significant peel turgidity (5.96 Kg) of GR treatment compared to the control (5.34 Kg) Table A 1, apendix. Growth regulators delay changes in albedo associated with maturity and senescence. Senescing peel has an albedo layer with s mall cells of low cytoplasmic amount and low metabolic activity, and larger intercellular spaces and weakened cell wall, which break easily ( Coggins, 1969b ). Overall these changes are associated with postharvest loss of water ( Spiegel -Roy and Goldschmidt, 1996), causing fruit deformation, which increase with fruit maturation and senescence ( Kawada and Albrigo, 1979), and hence, this deformation affect flaved o peel turgidity Also, in January -galactosidase significantly compared with GR treatment, and insignificantly compared with the control and WS treatment, which means that water stress minimize the effect of growth regulators ( Ferguson et al., 1983; Gilfillan et al., 1973 ) and advanced the peak of enzyme activity to January, accompanied with numerically low fruit peel turgidity (5.96 Kg) compared to the control (6.14 Kg). Contrarily, the WS*GR treatment reduced the activity of -mannosidase significantly compared to the control and other two treatments in March of 2005/06 season, but the peel turgidity was almost the same (5.91 Kg for WS*GR vs. 5.84 Kg for the control). The GR ga lactosidase (0.000 units/g DW) in January 2006/07 season, and this is in agreement with previous reports stated that gibberellic acid retards the rate of peel softening by retarding the seasonal change and in the amounts of cell wall galactosyl, arabinosyl and fucosyl residues (Mitcham and McDonald, 1993) -galactosidase was remarkably enhanced by 2,4 D in the concentration range which induced growth (107105 M), but higher conce ntrations (104103 M) inhibited the enzyme activity ( Tanimoto and Igari, 1976). Also, by comparing the effect of GR on the galactosidase (0.000

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200 -mannosidase (1.235 units/g DW) in May 2005/06, Table 4 2 and Figure 4 4, it agrees with data of Mitcham and McDonald (1993) who found that gibberellic acid retarded the rate of peel softening by retarding the seasonal change and reduction in the amounts of cell wall galactosyl, arabinosyl and fucosyl residues, while there was a tendency towards higher xylosyl, mannosyl and glucosyl content in gibberellic acid treated fruit. It appears that gibberellic acid has a greater effect on neutral sugars associated with pectin polymers than on neutral sugars associated with hemicellulosic polym ers, however in this study, as shown in Table 4 -galactosidase in flavedo with fruit maturation in both seasons (0.090 in January 2005/06 > 0.037 in March > 0.012 in May, and 0.090 in January 2006/07 > 0.051 in March > 0.031 in May ). S o in comparison with the control, GR could probably still improve the fruit peel turgidity by the time significantly as in March and May of the 2005/06 season and insignificantly as in March and May of the 2006/07 season Table A 1, appendix, and can be used as a tool to delay the suggest ed harvest time (February March) for some more time, may be before May, because the level of -galactosidase -mannosidase increased in May 2006/07 season in GR treated fruit, Figure 4 5. Little previous information was available about the effect of water stress on glycosidases activity. Data do not show any noticeable ch anges in enzyme activity, except for the numerically -galactosidase of WS*GR (0.096 units/g DW) compared to the control (0.022 units/g DW) in January 2006/07, Table 4 2 and Figure 4 galactosidase of WS (0.434 units/g DW) compared to the control (0.042 units/g DW) in May 2005/06, Table 4 2 and Figure 44. Contrarily WS*GR significantly reduced the activity of -mannosidase (0.548 units/g DW) compared to the control (1.134 units/g DW) in March of 2005/06 season Table 4 2. The WS*GR in January 2006/07 was corresponding to peel

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201 turgidity = 5.71 Kg compared to 6.14 Kg of the control Table A 1, appendix, and this can be referred to what Konno et al. (1986) and El Tayeb and Ahmed (2007) stated about w ater stress that stimulates the activity of glycosidases causing deterioration of cell wall, and fruit softening expre ssed as reduction of fruit peel turgidity H owever results of March and May 2005/06 season can not be referred to the previous reports because the WS*GR treatment in March 2005/06 was corresponding to peel turgidity = 5.91 Kg that numerically compared to 5 .84 Kg of the control, and the WS treatment in May 2005/06 was corresponding to peel turgidity = 5.57 Kg that significantly compared to 5.34 Kg of the control, Table A 1, appendix. So, from all of these results, it can be concluded that water stress either alone or in combination with growth regulators increase the activity of the glycosidases, and this is contradictory to the reports of Konno et al. (1986) and El Tayeb and Ahmed (2007) because our peel turgidity results did not cope with so called cell wall deterioration by the effect of glycosidases, and this may be due to some effect of growth regulators when c ombined with water stress, because growth regulators retard the rate of peel softening by retarding the seasonal change and reduction in the amounts of -mannosidase is a vacuolar enzyme (Boller and Kende, 1979; Faye et al., 1988; Masuda et al., 1990) with a lytic function ( Boller and Kende, 1979) -galactosidase where the majority of its activity is bound to the cell wall (Burns, 1990a; Dick et al., 1984; Murry and Bandurski, 1975; Tanimoto, 1985) with a hydrolytic effect on the neutral linkage s of pectic polysaccharides of the cell wall (Ranwala et al., 1992 ). -man nosidase is present in the endoplasmic reticulum and the Golgi complex of developing cells, and accumulates in the protein bodies with a role in glycosylation and deglycosylation of proteins and thereby in protein routing and protection against hydrolytic degradation (Faye et al., 1988) So, it can be concluded that the activity of glycosidases under the

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202 e ffect of water stress needs more investigation to provide information that could be used to further define physiological maturity and senescence. Conclusion Glycosidases cause increases in cell wall galactosyl and arabinosyl residuals indicating degradati on of pectin polymers of cell wall s which in turn are associated with the loss of peel firmness over the season (Mitcham and McDonald, 1993) -galactosidase increased gradually during the first stages of fruit development then decreased with the progress of fruit maturation toward senescent condition with little increase again late season in senescent peel -mannosida se is fluctuating early season with a general trend to increase in senescent peel The increase in both enzymes activities was accompanied by a significant decrease in fruit firmness and significant increase in juice quality from March to May as in chapter 2 (Table 2 1 and 2 5) suggesting the February-March period as a good time to harvest Marsh grapefruit. Application of water stress and growth regulators to affect glycosidases activity and adjust the time of harvest needs more investigation, but at lea st, from the few significant results, it can be not ed that growth regulators increased the activity of glycosidases later in the season, by May, accompanying peel senescence. Also, water stress in combination with growth regulators treatment increased glycosidases activity earlier in January, followed by a decrease in enzyme activity in March, which is the suggested date of harvest. This also supports that water stress minimizes the effect of growth regulators to some extent (Ferguson et al., 1983; Gilfillan et al., 1973). These results also show t hat GR was more effective than WS in terms of altering glycosidases level s and the relationship with peel maturation rate. Suggested harvest time is February March period, which can still be extended with GR treatment to April only, to avoid high level o f enzymes, associated with senescent peel later in season.

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203 Table 4 1. Effect of harvest date on the activity of glycosidases (units / g D.W.) of Marsh grapefruit peel during three seasons 2004/2005 Flavedo Albedo Harvest date galact osidase mann osidase galact osidase mann osidase September 0.213 a 0.949 a 0.279 a 0.652 ab October 0.175 ab 0.797 a 0.209 ab 0.603 ab November 0.108 dc 0.926 a 0.114 b 0.276 b December 0.062 de 0.896 a 0.121 b 0.304 ab January 0.064 de 1.0 17 a 0.164 b 0.462 ab February 0.100 cde 0.718 a 0.136 b 0.936 ab March 0.042 e 0.704 a 0.135 b 0.499 ab April 0.142 bc 0.847 a 0.118 b 0.533 ab May 0.078 de 0.908 a 0.119 b 0.660 ab June 0.059 de 0.736 a 0.180 b 1.474 a July 2005/2006 Flavedo A lbedo Harvest date galact osidase mann osidase galact osidase mann osidase September 0.105 a 0.871 a 0.109 a 0.443 ab October November 0.095 a 1.056 a 0.118 a 0.613 ab December January 0.037 ab 0.870 a 0.120 a 1.059 a February March 0.008 b 1.134 a 0.0 65 a 0.359 b April May 0.058 ab 0.667 a 0.042 a 0.418 ab June July 0.086 a 1.069 a 0.090 a 0.220 b 2006/2007 Flavedo Albedo Harvest date galact osidase mann osidase galact osidase mann osidase September 0.166 a 0.974 a 0.191 a 0.651 a O ctober November 0.123 ab 1.156 a 0.166 a 0.595 a December January 0.066 bc 0.710 a 0.022 b 0.127 c February March 0.059 c 0.903 a 0.024 b 0.285 bc April May 0.030 c 0.930 a 0.098 ab 0.437 ab June July Mean separation of tr eatments under each harvest date within column by DMRT ( P

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204 Table 4 2. Effect of water stress and growth regulators on the activity of glycosidases (units / g D.W.) of Marsh grapefruit peel during two seasons 2005/2006 Flavedo Albedo Month Treat. galactosidase mannosidase galactosidase mann osidase January CONT 0.037 a 0.870 a 0.120 a 1.059 a WS 0.161 a 1.054 a 0.118 a 0.541 a GR 0.090 a 1.291 a 0.070 a 0.421 a WS*GR 0.044 a 1.149 a 0.117 a 0.654 a March CONT 0.008 a 1.134 a 0.065 a 0.359 a WS 0.040 a 0.891 a 0.048 a 0.442 a GR 0 .037 a 1.018 a 0.075 a 0.407 a WS*GR 0.028 a 0.548 b 0.034 a 0.476 a May CONT 0.058 a 0.667 a 0.042 a 0.418 b WS 0.045 a 0.945 a 0.434 a 0.383 b GR 0.012 a 0.592 a 0.035 a 1.235 a WS*GR 0.054 a 0.963 a 0.020 a 0.258 b July CONT 0.086 a 1.069 a 0.090 a 0.220 a WS 0.040 a 0.852 a 0.009 a 0.183 a GR 0.053 a 0.862 a 0.044 a 0.131 a WS*GR 0.027 a 0.841 a 0.028 a 0.307 a 2006/2007 Flavedo Albedo Month Treat. galactosidase mannosidase galactosidase mannosidase January CONT 0.066 a 0.710 a 0.022 ab 0.127 a WS 0.058 a 0.789 a 0.020 ab 0.141 a GR 0.090 a 0.778 a 0.000 b 0.000 a WS*GR 0.076 a 1.035 a 0.096 a 0.502 a March CONT 0.059 a 0.903 a 0.024 a 0.285 a WS 0.093 a 0.598 a 0.129 a 0.371 a GR 0.051 a 0.869 a 0.089 a 0.408 a WS*GR 0.065 a 0.778 a 0.049 a 0.396 a May CONT 0.030 a 0.930 a 0.098 a 0.437 a WS 0.032 a 1.067 a 0.070 a 0.330 a GR 0.031 a 0.972 a 0.100 a 0.332 a WS*GR 0.042 a 0.899 a 0.087 a 0.492 a July CONT WS GR WS*GR Mean separation of treatments under each harvest date within column by DMRT ( P

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205 0.0 0.5 1.0 1.5 2.0 2.5 Sept Oct Nov Dec Jan Feb Mar Apr May Jun Harvest date Enzyme activity (unit/gDW) Figure 4 1. Effect of harvest date of Marsh grapefruit on -galactosidase and -mannosidase activity during the 2004/2005 season. Values are means S E

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206 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Sept Nov Jan Mar May Jul Harvest date Enzyme activity (unit/gDW) Figure 4 2. Effect of harvest date of Marsh grapefruit on -galactosidase and -mannosidase activity during the 2005/2006 season. Values are means S E

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207 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Sept Nov Jan Mar May Harvest date Enzyme activity (unit/gDW) Figure 4 3. Effect of harvest date of Marsh grapefruit on -galactosidase and -mannosidase activity during th e 2006/2007 season. Values are means S E

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208 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Control Jan Mar May July WS Jan Mar May July GR Jan Mar May July WS & GR Jan Mar May July Treatment & Harvest date Enzyme activity (unit/gDW) Figure 4 4. Effect of water stress and growth regulators on -galactosidase and -mannosidase activity of Marsh grapefruit peel at harvest during the 2005/2006 season. Values are means S E

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209 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Control Jan Mar May WS Jan Mar May GR Jan Mar May WS & GR Jan Mar May Treatment & Harvest date Enzyme activity (unit/gDW) Figure 4 5. Effect of field water stress and growth regulators treatments on -galactosidase and -mannosidase activity of Marsh grapefruit peel at harvest during the 2006/2007 season. Values are means S E

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210 CHAPTER 5 CHANGES IN ABSCISIC ACID LEVELS IN MARS H GRAPEFRUIT PEEL IN RELATION TO MATURITY AND THE EFFECT OF WATER STRE SS, GROWTH REGULATOR S AND DATE OF HARVEST Plant growth and development are regulated by internal signals and by external environmental conditions. One important regulator that coordinates growth and development with response to environmental stresses is the sesquiterpenoid (C15) hormone abscisic acid (ABA) (Xion and Zhu, 2003) This plant hormone in the form of S (+) (ABA) is usually present in plants at concentrations as low as 0.4 nmol/g dry weight ( Walton and Li, 1995) and modulates numerous aspects of plant growth and development including stress responses (i.e., drought, salinity and cold) and stomatal aperture (Zeevaart and Creelman, 1988 ). In plant cells, ABA is synthesized and degraded continually and catabolism can occur by several routes involving oxidation, reduction and conjugation (Cutler and Krochko, 1999). ABA levels initially are high in the ovary (2 mm fruit), and then decrease until after fruit set (about 75 days), when they begin to increase again reaching higher levels at maturity, and continue to increase as the fruit matures and progresses into senescence (Josan et al., 1999; Murti, 1988). This rapid rise in ABA level in the final stages of fruit growth have been related to the cessation of fruit growth and changes leading to ripening, senescence and abscission (Murti, 1988). Although the high level s of exogenous ABA inhibits plant growth under non-stressful conditions, an increase in ABA content is beneficial for plants under environmental stress to induce changes at the cellular and whole -plant level. ABA promotes the closure of stomata to minimize transpirational water loss; it also mitigates stress damage through the activation of many stress responsive genes that encode enzymes for the biosynthesis of compatible osmol ytes, which increase plant stress tolerance (Bray, 2002; Finkelstein et al., 2002; Hasegawa et al., 2000 ). ABA has been correlated with citrus development ( Aung et al., 1991) and involved in fruit color change (No oden, 1988).

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211 Growth regulators, such as auxins inhibit the role of ABA in fruit abscission (Taiz and Zeiger, 2002; Takahashi et al., 1975 ). F or example, application of a synthetic auxin 2,4D effectively inhibited the activity of ABA and reduced preha rvest drop of orange fruit ( Zur and Goren, 1977). GA3 depressed the level of ABA in the flavedo tissue, which has the highest ABA level compared to other fruit tissue (Goldschmidt et al., 1970) during fruit maturation and senescence. The effect of GA3 was associated with a delay in color change ( Valero et al., 1998), suggesting some link between ABA synthesis and carotenoids (Taylor, 1968). It has been reported that c onjugated ABA of fruit increased 12.6-fold at the time of chloroplast transformation to chromoplast (color break stage), which suggested a possible association of ABA with carotenoids biosynthesis ( Aung and Houck, 1993; Aung et al., 1991; Cowan and Richardson, 1997; Milborrow, 1974b ). Norman et al (1990) reported that ABA, carotenoids, and the phytol side chain of chlorophyll share mevalonate as a common precursor in the isoprenoid pathway. This mevalonate is then pyrophosphorylated, decarboxylated, and dehydrated to yield isopentenyl diphosphate (IPP) in the cytosol (Rohmer, 1999; Taiz and Zeiger, 2002). ABA is s ynthesized from carotenoids intermediates (zeaxanthin, violaxanthin, neoxanthin, and xanthoxal), and the pathway takes place in chloroplasts and other plastids beginning with IPP (Milborrow, 2001; Richardson and Cowa n, 1996; Taiz and Zeiger, 2002) Materials and Methods Field Experiments All three field experiments over three seasons 2004/2005, 2005/2006 and 2006/2007 were described before in chapter 2, pages 7375. Tissue P reparation for C hemical A nalyses At every h arvest date, tissue prepared and stored frozen at 80oC, as described before in chapter 3, page 1 39.

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212 Abscisic Acid Extraction ABA extraction was done according to the method of Miller et al. (1987) to extract IAA and the met hod of Yuan et al. (2001) with some modifications. Tis sue (3 g) was ground in liquid N2, and then 80 mL of 80% methanol was added to the powder. Samples were shaked overnight at 4oC; and the solution was filtered using filter paper (Whatman # 2, size 70 mm) with vacuum pump suction. Then, 20 ml ethanol was ad ded to the remaining particles, shaked well, and filtered again. Total volume of 100 ml was collected in a 250 ml flask, and this flask was attached to the rotoevaporator device (Rotavapor -R, Buchi Inc., Flawl, Schweiz) with water temperature near but less than 40oC, since ABA is sensitive to temperature higher than 40oC, and a vacuum of 25 mm Hg. Once the sample reached a point that 80% of methanol evaporated, and the volume of the remaining solution became 20 ml, the remaining sample was poured into anoth er flask marked Aqueous solution. Then, two drops of NaOH (0.5 N) were added to adjust pH from 5 to 8. The s ample was moved, with the same volume (about 25 ml) of ethyl acetate, to a separatory funnel (500ml): this mixture was shaked strongly by hand to facilitate the two phases separation (aqueous & organic). Sodium chloride saturated solution (2 ml) was added to the funnel to increase ionic state and hasten the separation. Aqueous phase (bottom) was separated from the ethyl acetate phase (top), and then the ethyl acetate phase was discarded. Also, acidification was accomplished by the addition of some drops of hypochloric acid to the aqueous solution to get pH = 3 to improve separation of the free form of ABA from the aqueous phase to the organic phase. This aqueous phase was moved to the separatory funnel (500ml), with the same volume of ethyl acetate, and then this mixture was shaked strongly by hand to facilitate the separation. The aqueous phase (lower) was removed and the ethyl acetate phase (top) wa s kept in a flask marked organic solution. Aqueous phase was returned again to the separatory flask with the same volume of ethyl acetate, and then this mixture was shaked

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213 strongly by hand to facilitate the two phases separation. Aqueous phase (lower) wa s removed again and the ethyl acetate phase (top) was kept in the flask marked organic solution. Sodium sulfate was added to the organic solution to eliminate residual water and clarify the solution. The sample was transferred to an evaporation flask a nd kept in ice. Samples were dried completely using rotoevaporation at temperature less than 40oC and vacuum at 30 mm Hg. Dry material was dissolved in 0.5 ml methanol and all content was collected in an eppendorf tube. With caps open, eppendorf tubes were moved to a vacuum evaporation machine (Refrigerated Condensation Trap, RT 400, Savant Instruments Inc., Farmingdale, NY, USA), and left for one hour at 35oC until contents were complete dry. Samples were stored at 20oC. ELISA Test Quantification Quantitative determination of ABA was done using Phytodetek ABA Enzyme Immunoassay Test Kit, competitive enzyme linked immunosorbent assay (ELISA), from Agdia Incorporated, Elkhart, IN, USA (PDK 09347/0096). TBS buffer and ABA standard sticks (RS 09347) were obta ined from Agdia Inc. Samples were diluted (0.25 ml of the extract + 0.975 ml of TBS buffer), and the ELISA kit protocol was followed, using 96-well plates. Plates were read using a Bench Mark Plus Microplate Spectrophotometer (BioRad, Bio Rad Lab., Hercul es, CA, USA) at wavelength 405 nm, incubator temperature range 36.837 oC. Correlation coefficients for the standard curves ranged from 0.945 to 0.957. ELISA is an effective method to measure ABA in crude extracts without excessive cleanup procedures (Norman et al., 1990). Statistical Analysis Data were analyzed by SAS 8.2 (Statistical Analysis System) using ANOVA (Analysis of Variance) and means were compared using DMRT (Duncan Multiple Range Test). Standard error of the means were also calculated by SAS ( SAS Institute Inc., 1999).

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214 Results and Discussion Effect of Harvest Date Seasonal changes of ABA are shown in Table 5 1 and Figures 51 and 5 2 for all three seasons. ABA content of both flavedo and albedo increase gradually until shown first insignificant peak in December of t he first season, then ABA level of flavedo and albedo showed the second insignificant peak in March and April, respectively During the second season, ABA level showed one peak in May and March for flavedo and albedo, respectively. However, during the thir d season, ABA of flavedo and albedo showed continuous increase from September until reaching its highest level in May. For all three seasons, it appears that ABA in both flavedo and albedo had the gradual increasing tendency. The increase in flavedo ABA le vel in December of 2004/05 and November of 2005/06 seasons may be related to and associated with the period of color break. It was reported that conjugated ABA of navel orange fruit increased 12.6 fold at the time of color break stage (Harris and Dugger, 1986), and there was a shift in free ABA at the time of chloroplast transformation to chromop last in mandarin (Lafuente et al., 1997) It was also reported that during fruit development, the activity of endogenous ABA was g reater in the peel than that in the pulp (Josan et al., 1999), and the decline of ABA levels was associated with a delay in color change ( Valero et al., 1998). The increase in ABA of the flavedo was associated with peel senescence and the conversion of chloroplasts to chromoplasts (Harris and Dugger, 1986; Martinez Romero et al., 1999; Nooden, 1988). This period may be the beginning of peel senescence due to high level of ABA However color break is te mperature dependent and does not reflect the stage of peel maturation Yuan et al (2001) found that cold weather (5 10oC) in January increased ABA level in Valencia oranges peel, and this chilling effect may be another reason for ABA increase in December of the first season of this study, when air temperat ure was around 60oF (10oC) at that time, as shown in Figure B 1, appendix. On the other hand, the

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215 second peak of ABA during the period March to April of the first season, March to May of the second season and May of the third season were apparently related to the senescent peel towards the end of the season ( Valero et al., 1998). It also reflects the difference in peel maturation rate in the third season com pared to the first two seasons. ABA found during this period may or may not be a reason of low percent of chilling injury during April -May of 2004/05 season, as shown in Table 2 13. Kawada et al. (1979) and Wang (1991) reported that ABA could increase fruit resistance to chilling injury. On the other hand, Lafuente et al. (1991) found no rela tion between ABA level and CI tolerance in cucumber. Also, tomato stored at chilling -induced temperature showed an increase in ABA level (Ludford and Hillman, 1990), although this increase was not associated with the appearance of CI symptoms ( Kubik et al., 1992). Serrano et al (1997) stated that ABA increase in mature green pepper under 2oC might be a result of the stress caused by CI, rather than a protection against CI. Senescent peel was associated with increase in color index toward the end of the season, red uction of fruit detachment force from January to March, and a gradual increase of TSS: acid ratio, in addition to increases in % decay and % water loss in storage, as shown in Table A 1, appendix. Wwn et al (2001) found that stored Ponkan mandarin yellow fruits showed significantly higher storage decay than half -yellow and green fruits, whereas green fruits had slightly lower TSS than others. Burns and Albrigo (1997) found that early in the season, grapefruit are more resistant to decay. As season progresses and the fruit senesce, th ey become more susceptible to decay and granulation. So, it is suggested that March is the beginning of ABA increase and the beginning of physiological changes that lead to peel senescence, and harvesting grapefruit before this period, maybe in January or February since ABA level is low, can reduce postharvest physiological disorders. The MarchMay period is the period of too high level of ABA, which means the period of peel senescence. So, this period

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216 should be the cut -off period of grapefruit harvest to a void peel physiological disorders after harvest. Suggested harvest date is January March period. Effect of Water Stress and Growth Regulators Results of WS and GR are shown in Table 5 2 and Figure B 6, appendix, and there were no significant differences among all treatments on the ABA level of the albedo of both seasons, and the difference was only significant for ABA of the flavedo during May of the 2005/06 season and January and May of the 2006/07 season. It is noticeable that levels of ABA in albedo ar e not that big different from flavedo during both seasons Figures 5 3, 5 4, 55 and 56 ABA level of WS -treated flavedo was high in May 2005/06 season, but t he combination of WS*GR significantly reduced the amount of ABA and increased peel turgidity comp ared to the control of the same season, Table 5 2 and Figure 5 3 and it was associated with some senescence signs, such as, significant reduction in fruit detachment force and significant increase in % decay during storage at 70oF (not at 40oF; may be due to the effect of low temperature inhibiting decay) compared to the control as shown in Table A 1, appendix The a pplication of WS and GR individually in 2005/06 season, Figure 5 3, increased and decreased the amount of ABA, respectively, compared to the control without significant differences, Table 5 2. Also, the difference was not significant between WS*GR (4.31 mg/g DW) and control (4.30 mg/g DW) in January of the 2006/07 season, but it was highly significant between WS and control (7.79 mg/g DW versus 4.30 mg/g DW), as well as GR and control (2.96 mg/g DW versus 4.30 mg/g DW) Table 5 2 and Figure 5 5 This is mainly because WS increase the level of ABA (N orman et al., 1990), and may be associated with WS effect advancing peel senescence early in the season. Also, GR, as known, retard the level of peel maturation, which associates with low level of ABA. I n Jaanuary of the 2006/07 season, t he WS treatment was associated with significantly low peel turgidity and GR treatment was associated with high significant FDF, but peel turgidity was

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217 not different from the control, Table A 1, appendix. ABA is clearly related to leaf and fruit senescence and directly i nvolved (by increasing ACC synthesis; precursor of ethylene) (Gomez cadenas et al., 2000; Goren, 1993; Goren et al., 1979; Okuda, 1999; Riov et al., 1990; Sagee et al., 1980) or indirectly (by accelerating senescenc e) (Jackson and Osborne, 1972; Lieberman et al., 1977) in increasing ethylene production that increases cellulase and polygalacturonase activity (Jackson and Osborne, 1972; Kazokas and Burns, 1998; Rasmussen, 1974) which promotes fruit softening, and leaf and fruit abscission (Lieberman et al., 1971; Rasmussen, 1974; Sagee et al ., 1980; Taiz and Zeiger, 2002). On the other hand, ethylene can be activated by water stress ( Yang and Hoffman, 1984) and may also increase free abscisic acid in the flavedo of citrus fruit enhancing senescence (Goldschmidt et al., 1973; Lafuente et al., 1997; Lafuente and Sala, 2002) During May of the 2006/07 season, Table 5 2; results were contradict ing to th ese findings because WS treatment reduced ABA levels s ignificantly compared to the control. The GR and WS*GR treatments reduced ABA levels compared to the control, but they numerically increased the ABA level compared to WS treatment. The reduction in ABA level over the 2006/07 season in WS -treated fruit, Tab le 5 2, contradict the gradual increase in ABA level with harvest date as maturation and senescence progress, Table 5 1 and Figure 5 1 I t may be possible that increase in ABA level early season after WS treatment wa s something temporary, and this effect w as vanished after the time of WS treatment wa s over in February, and then ABA returned to the normal level (which may be the normal level of ABA in senescent peel in May) and showed smaller level in May than in March. Although WS increase flavedo ABA and r educed fruit peel turgidity in January, compared to the control and GR treatment, this may reflect that WS was not effective as a tool to affect peel maturation rate, especially WS treatment did not show much difference compared to GR treatment in January and March of 2005/06 season, Table 5 2.

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218 ABA is usually produced in roots in response to environmental stress (i.e. drought ), and works as a root signal to inhibit stomatal opening (Artsaenko et al., 1995; Davies a nd Zhang, 1991; Gomes et al., 2003) by inhibiting K+ inward channels which are required for stomatal opening (Schwartz et al., 1994) functioning as an endogenous regulat or of plant transpiration, ameliorating the effects of water stress (Creelman, 1989). Free ABA is an active form of ABA localized in the cytosol, while conjugated ABA is an inactive form of ABA that covalently conjugated with another molecule, such as a monosaccharide (ABA D -glucosyl ester), whic h accumulate in the vacuoles and serve as a storage form of the hormone (Taiz and Zeiger, 2002). T his may be has some little contribution in high TSS: acid ratio in May 2005/06 season. It has been reported that increases in ABA after water stress were associated with increased sugar content of flavedo tissue of Satsuma mandarin ( Kuraoka et al., 1977; Okuda et al., 2002). This may be related to an effect of ABA in the accumulation of assimilates from the phloem into the fruit by strengt hen sink activity (Brenner et al., 1989; Kojima et al., 1994; Kojima et al., 1995; Okuda et al., 2002) This increase in ABA level could be related to the beginning of fruit senescence ( Valero et al., 1998 ). The reduction in color index of GR -treated fruits, compared to the control in May of both seasons, as in Table A 1, appendix, is mainly related to the hormonal ba lance inside the fruits. A ccording to Goldschmidt et al (1970), the hormonal balance between ABA and other plant hormones affects the intensity of senescence pheno mena (Goldschmidt et al., 1970). For example, increases in gibberellins have an inhibitory effect on ABA formation and senescent color change (Brisker et al., 1976; Goldschmidt et al., 1970; Murti, 1988). Increases in fruit AB A along with decreases in gibberellins are closely related to the onset of senescence and the appearance of puffing physiological disorder in mandarins (Goldschmidt et al., 1970; Kuraoka et al., 1977). Water stress increases the level of endogenous ABA (Lafuente

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219 and Sala, 2002) and ethylene formation (Ben Yehoshua and Aloni, 1974). Increases in ABA along with decreases in auxins induce the onset of endogenous ethylene production that influences fruit abscission in water -stressed citrus trees (Gomezcadenas et al., 2000; Guinn and Brummett, 1988; Rasmussen, 1974; Taiz and Zeiger, 2002; Takahashi et al., 1975 ), and this may be the reason of low FDF (8.43 Kg and 8.20 Kg) of water stressed fruit compared to the control (9.49 Kg and 8.56 Kg) in May of the 2005/06 and 2006/07 seasons, respectively. In this regard, IAA and ABA are antagonistic and have opposing effects (Jackson and Osborne, 1972) delaying or accelerating senescence, respectively (Osborne, 1967). In addi tion to this, GA3 is not considered a primary growth regulator in controlling fruit abscission (Gomez -cadenas et al., 2000). Moreover, Rasmussen (1981) stated that in Valencia orange, high level of GA3 in the flavedo of the fruit at the regreening of the peel may help to prevent ABA from inducing ethylene and promoting abscission, however, the effect of GA3 on the ABA cont ent was reported to be negligible (Jun et al., 2002; Zacarias et al., 1995 ). From this information, there is the possibility of using WS (based on 2006 /0 7 season data) and/or GR treatments to manipulate the physiolog ical processes in the peel of citrus fruit and advance or delay harvest time, respectively, to harvest fruit with good juice quality and less senescent peel, which means less susceptibility to postharvest problems such as, decay and physiological disorders GR (based on 2006 /0 7 season data) and WS*GR (based on 2005 /0 6 season data) treatments has more pronounced effect in this regard than WS, and data showed that the period before Ma y is the period of low ABA level, which means less maturation rate, and frui t characteristics, Table A 1, appendix, such as peel turgidity (from WS*GR in May of the 2005/06 season) and FD F (from GR in January of the 2006/07 season) w ere fairly good compared to the control. So, the suggested harvest date of Marsh grapefruit, Febr uary -March,

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220 can be advanced in January and delayed to May with the using of GR and WS*GR treatments respectively Conclusion The general trend of ABA level in both flavedo and albedo was to increase toward the end of the season. ABA increased naturally a s a result of water stress, associated with a reduction in some physical parameters such as peel turgidity which is not a result of the ABA itself; instead it is the result of other fruit maturation and senescence factors This suggests that change in ABA level may be related to changes in peel maturation Growth regulators reduce d the level of ABA compared to water stress and control, associated with reduction in color index compared to the WS treatment and the control. This may confirm the role of water stress in advancing the rate of peel maturation and senescence, as well as the opposite effect of growth regulators in delaying the se physiological processes, and hence the rate of peel senescence. Results show the possibility to use GR alone or combined w ith WS to manipulate the physiological processes in the peel of citrus fruit and advance or delay harvest time, respectively. This also suggest s that ABA can be used as a senescence indicator for citrus peel. From this, it may be possible to define physiol ogical maturity and senescence of the peel more accurately in order to harvest fruit with good juice quality and less senescent peel. The suggested harvest date according to this information wa s February March Using GR and WS*GR might allow advance ment of harvest to January or delayed to May or maybe at least April, respectively, with good fruit peel turgidity while avoid ing the high levels of ABA late in the season that were associated with peel senescence adversely affect ing fruit storability after ha rvest.

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221 Table 5 1. Effect of harvest date on ABA content (mg / g D.W.) of March grapefruit peel during t hree seasons 2004/2005 2005/2006 2006/2007 Harvest date Flavedo Albedo Flavedo Albedo Flavedo Albedo September 5.76 a 2.46 b 1.30 e 0.36 c 1. 60 c 0.75 c October 5.85 a 2.13 b November 6.62 a 4.02 ab 5.48 bc 3.83 b 2.33 bc 2.46 bc December 7.79 a 5.93 ab January 5.30 a 3.14 ab 5.37 c 5.41 ab 4.30 b 3.83 ab February 6.02 a 5.90 ab March 7.88 a 5.82 ab 6.56 ab 6.25 a 6.96 a 5.22 ab April 6.07 a 7.94 a May 6.90 a 6.14 ab 6.95 a 5.81 ab 8.97 a 6.40 a June 6.79 a 7.43 a July 3.82 d 4.33 ab Mean separation under each treatment within columns by DMRT ( P

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222 Table 5 2. Effect of water stress and growth r egulators on ABA content (mg / g D. W.) of March grapefruit peel during two seasons CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. 2005/2006 2006/2007 Month Treat. Flavedo Albedo Flavedo Albedo J anuary CONT 5.37 a 5.41 a 4.30 b 3.83 a WS 5.48 a 4.92 a 7.79 a 5.60 a GR 5.06 a 5.28 a 2.96 c 3.35 a WS*GR 4.84 a 4.93 a 4.31 b 3.73 a March CONT 6.56 a 6.25 a 6.96 a 5.22 a WS 6.99 a 4.65 a 6.14 a 2.46 a GR 6.50 a 5.96 a 5.97 a 2.98 a WS*GR 6.73 a 5.46 a 4.19 a 3.45 a May CONT 6.95 ab 5.81 a 8.97 a 6.40 a WS 7.58 a 5.96 a 3.63 b 3.84 a GR 6.16 bc 4.31 a 6.62 ab 4.04 a WS*GR 5.09 c 4.81 a 4.90 ab 4.00 a July CONT 3.82 a 4.33 a WS 4.49 a 3.80 a GR 5.50 a 3.54 a WS*GR 3.24 a 3.84 a Mean separation of treatments under each harvest date within column by DMRT ( P

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223 0 2 4 6 8 10 12 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date ABA conc. (mg/gDW) 2004/05 2005/06 2006/07 Figure 5 1. Effect of harvest date of Marsh grapefruit on ABA level of flavedo during three seasons. Values are mean SE.

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224 0 2 4 6 8 10 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date ABA conc. (mg/gDW) 2004/05 2005/06 2006/07 Figure 5 2. Effect of harvest date of Marsh grapefruit on ABA level of Albedo during three seasons. Values are mean SE.

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225 0 1 2 3 4 5 6 7 8 9 Jan Mar May Jul Harvest date ABA conc. (mg/g DW) Cont WS GR WS*GR Figure 5 3. Effect of field water stress and growth regulators treatments on ABA level in flavedo of Marsh grapefruit peel at harves t during the 2005/2006 season. Cont; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators.Values are mean SE.

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226 0 1 2 3 4 5 6 7 8 9 Jan Mar May Jul Harvest date ABA conc. (mg/g DW) Cont WS GR WS*GR Figure 5 4. Effect of field water stress and growth regulators treatments on ABA level in albedo of Marsh grapefruit peel at harvest during the 2005/2006 season. Cont; control, WS; water stress, GR; growth reg ulators, and WS*GR; water stress & growth regulators.Values are mean SE.

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227 0 1 2 3 4 5 6 7 8 9 10 11 Jan Mar May Harvest date ABA conc (mg/g DW) Cont WS GR WS*GR Figure 5 5. Effect of field water stress and growth regulators treatments on ABA level in flavedo of Marsh grapefruit peel at harves t during the 2006/2007 season. Cont; contro l, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators.Values are mean SE.

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228 0 1 2 3 4 5 6 7 8 9 10 11 Jan Mar May Harvest date ABA conc. (mg/g DW) Cont WS GR WS*GR Figure 5 6. Effect of field water stress and growth regulators treatments on ABA level in albedo of Marsh grapefruit peel at harvest during the 2006/2007 season. Cont; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators.Values are mean SE.

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229 CHAPTER 6 CHARACTERIZATION OF CITRUS PEEL MATURATI ON AND THE EFFECT OF WATER STRESS, GROWTH REGUL ATORS AND DATE OF HARVEST ON C ITRUS OIL COMPONENTS There is evidence that composition of citrus rind essential oils change during advancing maturity and senescence ( Eschinazi, 1952 ; Kesterson and Hendrickson, 1953). The use of volatile components to evaluate citrus quality, maturity and senescence is an important objective for the citrus industry. These components could help determine the best conditions for postharvest treatments or shipping that ensure optimum quality (Biolatto et al., 2002 ). Citrus peel oil is collected as a by -product of juice production and contains primarily terpenes with traces of oxygenates, sesquiterpenes, waxes, flavonoids and other non-volatile components. Valencene is the maj or hydrocarbon sesquiterpene of Valencia orange ( Choi, 2003; Tonder et al., 1998 ) and nootkatone is the major oxygenated sesquiterpene and the character impact compound of Marsh grapefruit oil but not the juice (MacLeod and Buigues, 1964). Their level in the peel, as well as juice vesicles, increases with maturation of the fruit. They are markers of economic value and reportedly are indicators of fruit ripening and senescence ( Elston et al., 2005; Wilson and Shaw, 1980). Storage temperatures, storage time and wax application affect citrus oil compositions and q uality ( Sun and Petracek, 1999). In regular commercial practice, citrus fruit are coated with waxes that provide shine and reduce water loss but also restrict gas exchange through the peel surface and thus often cause anaerobi c conditions in the fruit internal atmospheres (Davis et al., 1967; Hagenmaier, 2002; Hagenmaier and Baker, 1993) that results in the development of off -terpineol, and other off -flavor volatiles (Hagenmaier and Shaw, 2002; Ke and Kader, 1990; Porat et al., 2005 ).

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230 Since rind oil composition appears to change during m aturation and senescence, it is possible that a maturity index could be used based on evaluations of oil constituents during fruit maturation and senescence. Furthermore, it should be possible to delay senescence by the use of GA3 and obtain a reasonably a ccurate initial evaluation of the magnitude of differences that exist between fruits that are susceptible to physiological rind disorders and those that are considerably less susceptible (Coggins et al., 1969a ). Similarily, drought stress reportably effects peel oil components and reduce the amount of monoterpenes and sesquiterpenes (Hansen and Seufert, 1999) The aroma of Marsh grapefruit and V alencia orange was investigated by means of SPME/GC MS analysis of the headspace aroma of the oil to identify volatile constituents that may be considered as indicators for peel senescence. Materials and Methods Field Experiments All three field experimen ts over three seasons, 2004/2005, 2005/2006 and 2006/2007, were described before in chapter 2, pages 7375. Storage Experiment (Season 2005/2006) Fruit were stored as described before in chapter 3, page 1 39. Oil Extraction At each harvest date, as well as after 12 weeks storage starting from the January harvest date of both Marsh grapefruit and Valencia orange, ten fruits out of each replication were chosen for oil extraction and analyses. The flavedo layer was removed using an apple peeler (Back To Basics Products, Inc., Draper, UT, USA) and then using a garlic squeezer to express the oils from the flavedo. The extracted solutions were collected in 2 ml eppendorf tubes and stored in a regular freezer until analyzed.

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231 Determination of Oil Components E ach treatment was represented by three replicates in three different eppendorf tubes. The contents of the tubes were collected in a glass vial and mixed well. Sample vials were placed in a water bath at 40oF for 30 minutes to concentrate the volatile compo nents in the headspace above the solution. After cleaning at 240oC for 5 minutes, a solid phase microextraction fiber (SPME, 50/30um DVB/Carboxen/PDMS StableFlex, manual holder, gray, Supelco Inc., Bellefonte, PA, USA) ( ARTHUR and PAWLISZYN, 1990; SUPELCO, 2000) was inserted into the vial in the headspace above t he solution for 20 minutes exposure time for extraction of volatiles which were injected into the Gas chromatograph -Mass spectrometry (GC -MS, Clarus 500, PerkinElmer, Waltham, Massachusetts, USA) for analyzing (Hites, 1997) The SPME fiber was removed from the GC -MS after 5 minutes. Every sample was run twice. Statistical Analysis Data were analyzed by SAS 8.2 (Statistical Analysis System) using ANOVA (Analysis of Variance) and means were compared using DMRT (Duncan Multiple Range Test). Standard error of the means were also calculated by SAS ( SAS Institute Inc., 1999). Results and Discussion The character of the flavor is mostly dependent on oxygenated terpene derivatives, aldehydes, ketones, esters, alco hols, oxides and acids ( Braddock, 1999). Some components under some of these groups were ch osen and are discussed below. Ketones Nootkatone is the only component that was chosen to represent this group, and was only studied in Marsh grapefruit since it is the major oxygenated sesquiterpene and the character impact compound of Marsh grapefruit (MacLeod and Buigues, 1964) having a typical grapefruit aroma and low odor threshold (Berry et al., 1967).

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232 Harvest date The levels of nootkatone fluctuated early in the season, and then a pronounced increase occurred after May in the first season January in the second season, and March in the third season, Table 6 1 and Figure 6 1. An earlier report showed a similar increase in Nootkatone with maturation (Del Rio et al., 1992). This increase in nootkatone was associated with significant increases of the TSS: acid ratio from April (8.30%) to May (10.56%) of the first season, whereas nootkatone started to increase in January of the second s eason associated with a linear increase in the maturity index ( TSS: acid ratio = 8.56%), and this increase was significant (8.68% to 9.89%) after March of the third season, Table 2 1 and Figure 2 1, chapter 2. This difference between three seasons may be r elated to difference in peel maturity. It can also be noticed that pulp maturity was different among three seasons; TSS: acid ratio was 8.30% in April of the first season, whereas it was almost the same (8.56% vs. 8.58%) in January of both the second and t he third seasons, respectively. This may give some indication about the difference in peel and pulp growth rates, because in the first season Nootkatone increased late in the season where the juice quality was lower than that in the middle of the second an d third season. As known, in citrus fruit, the growth pattern of peel is different from that of pulp, and the first stage of cell division can continue in the peel until close to fruit maturation (Scott and Baker, 1947). Contrarily, Coggins (1969b ) stated that peel senescence sometimes begins before fruit has attained internal maturity. Other parameters, su ch as peel peel turgidity also changed at the same time as nootkatone and TSS: acid ratio, for instance, peel turgidity of the peel decreased significantly from April (4.73Kg) to May (4.38Kg) in the first season, from November (6.34Kg) to January (4.67Kg) in the second season, and from January (6.14Kg) to March (5.36Kg) in the third season, Table 2 5. Also, fruit detachment force decreased significantly from January (9.29Kg) to March (6.92Kg) in the third season, but it was constant from April (8.18Kg) to M ay (8.19Kg) of

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233 the first season and from November (7.73Kg) to January (7.71Kg) of the second season, Table 2 7. This increase in nootkatone associated with an increase in the maturity index ( TSS: acid ratio) was previously reported by Sawamura et al. (1989) and Biolat to et al. (2002). Also, it was reported that oils processed late in the season (May June) contained the highest nootkatone levels (0.75 0.81%) ( MacLeod and Buigues, 1964; Wilson and Shaw, 1980). S o, nootkatone might be seen as an indicator of maturity (Drawert et al., 1984 ) and quality ( Biolatto et al., 2002 ) in grapefruit. From the nootkatone data, maturity in dex of the pulp, and peel turgidity of the peel during three years, the period of Januaryearly March is a proper general time window to harvest grapefruit, but it might have little different for each season, and the harvest date adjusted within this windo w for changes in pulp maturity index because of changes in bloom dates and climate. Water stress and growth regulators Previous reports and results in chapter 2 mentioned that WS advanced peel maturation (Munne Bosch and Alegre, 2004 ; Sharon -Asa et al., 2003) and GR delayed it (Chitzanidis et al., 1988; Coggins, 1981; McDonald et al., 1997; Pozo et al., 2000; Stover, 2000) WS advances maturity and senescence of the citrus peel and has an effect on volatile compounds, which co nsists mostly of monoand sesquiterpenes (i.e. nootkatone) ( Sharon -Asa et al., 2003 ). Table 6 2 and Figures 6 2 and 6 3 showed that WS insignificantly increased and GR insignificantly dec reased the level of nootkatone compared to the control until May, then increased it after May (data of second season only) As mentioned above, nootkatone started to increase in January and March during the second and third season, respectively, Table 6 1 and Figure 6 1. WS increased the level of nootkatone significantly compared to the control in January of the second season, which perhaps shows the effect of WS in advancing peel maturity by increasing nootkatone, as a maturity indicator of grapefruit (Drawert et al., 1984 ) early in the season, however results of the third season oppose this findings since WS decreased the level of nootkatone compared to the

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234 control during the entire season. On the other hand, the GR t reatment maintained a lower level of nootkatone compared to the control during the growing season. The difference was significant in January and May of both 2005/06 and 2006/07 seasons, which indicates the role of GR in delaying the peel maturation process es and reduce the production of nootkatone until late season. It was reported that gibberellic acid delayed senescence and increased oil content of the peel (McDonald et al., 1997; Wilson et al., 1990). GA3 reportedly affects the concentrations of oil constituents, which are related to the degree of peel senescence (Coggins et al., 1969a ). GA3 was one of two growth regulators used in this study reported to reduce the rate of increase in nootkatone concentration observed with maturat ion ( Wilson et al., 1990). During the second season, the role of WS in advancing peel sene scence reflected by increase in nootkatone level in January, Table 6 2, that was associated with some changes in fruit behavior during storage. January harvested fruit from trees treated with WS and stored at 40oF for three months showed significantly high % decay (1.67%) compared to the control (0.55%), but GR treatment showed numerically l ow % decay (0.18%) compared with the control, Table A 1, appendix. Also, both groups of fruits showed almost the same percent chilling injury (38.33% for WS vs. 38.70% f or GR) compared to the control (29.44%), and this is may be WS treatment advanced the peel maturity, and hence peel reach senescence quickly (like late season fruit) and became more susceptible to chilling injury, whereas GR delay peel maturity and make the peel in juvenility stage longer (like early season fruit) and more susceptible to be injured. In Florida, citrus peel is more susceptible to chilling injury early and late in the season, but mid -season fruit are less susceptible to chilling injury (Ritenour et al., 2003) So, it can be concluded that nootkatone, as a maturity indicator reflects the peel behavior after harvest late -season fruit GR had good effect than WS adjust ing the time of harvest, based on two seas ons data because it decreases the level

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235 of nootkatone during the entire season compared to the control, which means delaying the peel maturation rate, and this was associated with reduction in % weight loss and % decay at 40oF and 70oF and higher peel tu rgidity from January to May, Table A 1, appendix. So, harvest time can be extended further within the harvest window (January-May) to get higher TSS: acid ratio, as well. Storage conditions Del Rio et al. (1992) reported that nootkatone increases with fruit maturity, and it also increases during fruit storage (Biolatto et al., 2002 ; Sawamura et al., 1989). This is noticeable in Table 6 2 by comparing fruit at harvest date with fruit stored for 3 months at 40oF at all harvest dates except for July and WS treatment in May. In general, Fruit stored at 40oF had less nootkatone compared to fruit stored at 70oF, Table 6 2 and Figures 64 and 65, respectively. It was reported that cold storage and wax application decrease nootkatone ( Sun and Petracek, 1999). Aldehydes Total aldehydes were determined in Valencia orange only. Total aldehyd es content is often used as a quality index for citrus oils ( Kesterson et al., 1971). Al dehydes such as, octanal, -sinensal are responsible for characteristic orange flavor ( Kealey and Kinsella, 1979; Moshonas and Lund, 1969; Sawamura et al., 2005). In this study, total aldehydes include the following; hexanal, heptanal, octanal, nonanal, citronellal, decanal, neral, geranial, perillaldehyde, undecanal, E,E -2,4 decadienal, and dodecanal. Harvest date Direction of changes in total aldehydes over the season differed from one season to another, Table 6 3 and Figure 6 6, but with no significant differences In the 2005/06 season,

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236 aldehydes level was the highest in March, whereas in the 2006/07 season, it was the lowest in March and the highest in May. Aldehydes level were higher in more mature peel in earlier reports, while early season oil had lower decana l, and dodecanal than late and midseason oils (Shaw and Coleman, 1974). Kesterson and Hendrickson (1958) observed an increase in the aldehyde content fr om 1.50% to 1.85% in the more mature fruit, however, the concentration of some constituents such as, geranial decreased in a fairly smooth fashion as the harvest season advanced (Coggins et al., 1969a ). So, these reports are only in agreement with data of the 2006/07 season. Water stress and growth regulators Results from use of WS and GR, Table 6 4 and Figures 6 7 and 6 8, showed a sign ificant increase from the WS*GR treatment early in January of the 2005/06 season, which may indicate the role of water stress in advancing peel maturity (Munne -Bosch and Alegre, 2004; Sharon -Asa et al., 2003) by increasing the level of aldehydes early in the season. No other differences among treatments were significant during the season except in July at a stage of fruit senescence where GR significantly reduced the aldehydes compared to the control, however there was a pronounced numerical effect WS treatment in M arch and May of 2005/06 season, as well as for WS*GR in May of 2006/07 season compared to the control and other treatments, suggesting the role of GR in delaying the effect of WS and delay the peak of aldehydes to late season. These results are in agreemen t with previous findings showed that gibberellic acid delays senescence and increases oil content of the peel ( McDonald et al., 1997; Wilson et al., 1990). Soil coverage with Tyvek to apply a severe water stress period to evaluate effects on fruit had no significant effect on aldehyde levels in either season, Table 65 and Figure 6 9 It can be concl uded that WS apparently had no effect, but GR and WS*GR treatments had some effect on aldehydes level and perhaps the maturity of the peel, however, there effect was only in one season, and the difference

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237 in aldehydes trend between two seasons make it diff icult to rely on these data to define peel maturity properly or to determine the proper harvest date using WS and/or GR. Storage conditions In general, fruit response in storage is proportional to optimum temperature in the range of 3260oF (0 15.5oC) (Grierson, 1976) depending on; cultivar, geographical area, maturity st age at harvest, storage period, susceptibility to CI, preharvest treatments, and prestorage treatment (Arpaia and Kader, 20 02; Grierson and Miller, 2006c ). All treated and untreated fruit harvested at each harvest date and stored at 40oF and 70oF for three months showed lower amounts of aldehydes compared to the fresh fruit, Table 6 4 and 6 5 and Figures 610, 6 11 and 6 12. This is a normal effect related to the temperature effect in reducing respiration rate, water loss, and other physiological processes (Grierson and Miller, 2006c ; Petracek et al., 1998), which also affect the volatile compositions of the fruit (Dou, 2003). It was shown that when oranges were kept in cold storage for periods longer than 6 weeks prior to oil extraction, the physioche mical properties of the oil changed, including a decrease in the aldehyde contents ( Wolford et al., 1971 ). Nonanal was the only component affected by the interactive effect of waxing and storage duration, where it decreas ed with long storage time (Sun and Petracek, 1999). The most pronounced difference between fruit stored at 40oF and fruit stored at 70oF was for the May -harvested fruit with a very small amount of aldehydes after storage at 40oF compared to 70oF, Table 6 4 and Figure 6 10 and 6 1 1 Also, GR treated fruit showed small amount of aldehydes at 40oF compared to 70oF. This suggests that there may be some kind of interaction between temperature and the maturity stage of the peel, as w ell as the temperature and GR treatment, because GR delay the maturation process of the peel and make the peel during May harvest in a middle stage between immaturity and senescence that make peel less susceptible to postharvest physiological disorders dur ing storage, and this was confirmed by low

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238 percentage CI during storage at 40oF in May of 2005/06 season, Table A 2, appendix. Ritenour et al. (2003) stated that in Floridas climate, fruits are most sus ceptible to CI early and late in the season. However, it is hard to rely on t his presumably interaction between temperatures maturity stage of the peel and GR treatment that produce low amounts of aldehydes to determine peel maturity stage at harvest. Es ters Geranyl acetate was the only ester determined in both Marsh grapefruit and Valencia orange. Other esters that may be important to the flavor and aroma of grapefruit oils are; octyl acetate, citronellyl acetate, and neryl acetate. These acetate est ers are among the major oxygenated components of grapefruit oil, although not of orange oil (Moshonas, 1971). Harvest date Changes in geranyl acetate with harvest date of Marsh grapefruit fluctuated over the three seasons with a general trend of increases after April of the first season March of the second season and after January of the third season, Table 6 6 and Figure 613. On the other hand, the level of geranyl acetate in Valencia orange was almost constant during the second season with a large peak after May, whereas the increasing level was very small and gradual in the third season, Table 6 6 and Figure 6 14. These trends in geranyl acetate of Marsh grapefruit were associated with significant increases in the TSS: acid maturity index after April in the fir st season and after March of both the second and third seasons, and significant decrease in peel turgidity after April of the first season, March of the second season and January of the third season, as well as a numerical drop in fruit detachment force in April of the f i rst season, and a significant drop in March of both the second and the third seasons and a significant increase in percentage decay at 40oF in March of the second season, Table 21, 2 5, 2 7 and 2 11, respectively. So, suggested harvest tim e is January March period. Regarding Valencia

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239 orange, the change in the juice maturity index, Table 22, was corresponded to changes in geranyl acetate with the same level of significance after May of the second season, but no significant difference wa s noticed in esters during the third season, Table 6 -6 and 2 2. These trends in ester content were associated with significant decrease in peel peel turgidity after May of the 2006/07 season, Table 2 6, and significant increase in TSS: acid ratio, as well as % decay and % CI at 40oF after May of the 2005/06 season, Table s 2 2 2 12 and 2 13, which may reflect a change in peel maturity stage. Hence, best harvest time should be little before May, may be April May period Not much work was done on ester s cha nges during the growing season of citrus. This is may be due to the very small concentration of esters in citrus oil, Tables 6 6 and 6 7 that make it difficult to be identified. Coggins et al. (1969a ) reported in Navel oranges that concentrations of octyl acetate and neryl acetate appeared to change little if any as the season progressed. Sawamura et al. (2005) identified the volatile constituents of peel oil of several sweet oranges in China. They reported the peak area of geranyl acetate as less than 0.005%. Choi (2005) reported very small amount of geranyl acetate in Kumquat peel oil. In another study on lime, Yadav et al. (2004) weight, which is low compared with other oil constituents. It can be concluded that there is a year to year variability in geranyl acetate level, which make it difficult to be used as indicators of peel maturity in both grapefruit and orange fruits. Water stress and growth regulators Effect of water stress and growth regulators on geranyl acetate of Marsh grapefruit peel oil was little diff erent from other oil constituents mentioned before. Data of the 2005/06 season in Table 6 8 and Figure 6 15 showed that there were no significant difference between any of the treatment and the control except in Ma y where WS and WS*GR decreased level of g eranyl acetate significantly H owever both treatments increased the level of geranyl acetate

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240 insignificantly during the 2006/07 season, and the only significant difference was the increase in geranyl acetate level by GR treatment compared to the control T able 6 8 and Figure 6 16. Level s of geranyl acetate increased after March with WS, GR and control. The same trend for the treatments and control, as well as the insignificant differences among all treatments in March, may reflect that WS and GR had no effe ct on geranyl acetate level during peel maturation. As mentioned, not much work had been done on the effect of WS and GR on esters level during fruit growth. Coggins et al. (1969a ) reported in Navel oranges treated with gibberellin that concentrations of octyl acetate and neryl acetate appeared to change little if any as the season progressed. To conclude, this variation in WS and GR affect between both seasons make it difficult to count on geranyl acetate as maturity indicator for March grapefruit. In Valencia orange, during the 2005/06 season, Table 6 9 and Figure 617, the WS and GR treatments had the same trend as the control showing an increase in geranyl acetate level after May, whereas the WS*GR treatment had an increased level of geranyl acetate after March until the end of the season. Contrarily, Table 6 9 and Figure 6 18, during the 2006/07 season, all treatme nt, but the control had decreased levels of geranyl acetate after May. Both the GR and WS*GR treatments showed increased levels of geranyl acetate compared to the control from March to May, but the difference was only significant between WS*GR and the cont rol. The only difference between WS*GR and control in one season, plus the insignificant difference of other treatments between years, make geranyl acetate not useful to be a maturity indicator component for Valencia orange. Soil coverage with Tyvek foll owed the same trend as the control, Table 6 10 and Figure 6 19, with a reduced, but insignificant, level of geranyl acetate after May, therefore severe drought stress (Tyvek) had no effect on esters level, especially as this treatment causes reduction in pulp maturity index and fruit detachment force in the 2005/06

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241 season Table A 3, appendix. So, it is not recommended as an application to determine peel maturity. Storage conditions Marsh grapefruit stored at 40oF and 70oF showed almost the same trend of geranyl acetate variation from March to July, Table 6 8 and Figures 6 20 and 6 21. The level of geranyl acetate was slightly higher at 70oF than at 4 0oF This is normal effect related to the low temperature effect in reducing respiration rate, water los s, and other physiological processes (Grierson and Miller, 2006c ; Petracek et al., 1998), which affect the volatile compositions of the fruit (Dou, 2003), however these changes in ester l evel by temperature was not much compared to fresh fruit, as shown in Figure 6 15. The small change in geranyl acetate level may be due to water loss at 70oF compared to 40oF, as reported by Yadav et al. (2004) who stated that water loss (~5%) from lime fruit caused marginal quantitative changes in composition of esters. Valencia orange also showed lower geranyl acetate level at 40oF than at 70oF, Table 6 9 and Figures 6 22 and 623. The most pronounced difference between fruit stored at 40oF and fruit stored at 70oF was in May harvested fruit where geranyl acetate was very low at 40oF compared to 70oF. This may related to the degree of peel senescence in com bination with cold temperature, as reported by Grierson and Miller (2006c ). Soil coverage with Tyvek decreased the level of geranyl acetate in fruit stored at 40oF significantly compared to the control for March harvested fruit in the 2005/06 season, Table 6 10 and Figure 624, but the situation was opposite and the difference was insignificant for fresh fruit, Table 6 10 and Figure 6-17. In July the level of geranyl acetate of the Tyvek treatment increased signi ficantly compared to the control, possibly because the peel is more senescent in July, particularly if drought stressed. Control fruit harvested in March and stored at 40oF showed higher decay and chilling injury compared to Tyvek -treated fruit, Table A 3, appendix, which suppose to be more mature than the control. All

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242 of these variations in data of storage experiment make it hard to use geranyl acetate as maturity indicator for both citrus species. Alcohols Alcohols have been identified as being the most i mportant contributor to orange flavor (Shaw, 1979). In this study, linalool is the only component studied in both Marsh grapefruit and Valencia orange. Linalool is the major alcohol in orange oils (Sawamura et al., 2005) and the most phytotoxic compound in citrus oil gland (Wild, 1992). Along with octanol and some other aldehydes, linalool is responsible for characteristic orange flavor ( Kealey and Kinsella, 1979; Sawamura et al., 2005 ). Harvest date Linalool levels in Marsh grapefruit fluctuated from one season to another Table 6 11 and Figure 6 25; in the 2004/05 season, linalool peaked in February and March and then decre ased in April and increased again toward the end of the season. I n the 2005/06 season, the peak was earlier, in November, followed by drop in January and then the level increased toward the end of the season. Unlike the second season, linalool level in 2006/07 season showed a drop in November then increased toward the end of the season. It seems that there was no specific trend in linalool levels among the three seasons. These data differ from previous reports stated that early season oil showed lower linal ool than late and midseason oils ( Shaw and Coleman, 1974 ). Also, concentrations of linalool decreased in a fairly smooth fashion as the harvest season advanced (Coggins et al., 1969a ). These results for some extent were in consisstance with the maturity index ( TSS: acid February 2004/05 season and in January of 2005/06 and 2006/07 seasons, Table 2 1, as well as the decay at 40oF started to increase significantly in February and March of the 2004/05 se ason and 2005/06 season, respectively, Table 2 11, however b ecause of fluctuation in linalool level

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243 from one harvest date to another and the difference in its trend from one season to another, it is hard to relate changes in linalool level to changes in pe el maturity, associated with changes in decay or juice quality, and use linalool as a maturity indicator for Marsh grapefruit. Like grapefruit, Valencia orange showed no specific trend in linalool level over the season during two consecutive seasons, T able 6 12 and Figure 6 26, but it appeared that linalool level fluctuated early in the season and increased significantly after May during both seasons. This increase in linalool level after May was associated with sharp increase in pulp maturity index aft er May. This increase in TSS: acid ratio in July was significant and almost doubles the amount in May of the 2005/06 season, Table 2 2, as well as significant increase in decay, Table 2 12, and chilling injury, Table 2 13, at 40oF in 2005/06 season, howeve r these changes in TSS: acid ratio, decay and chilling injury are expected to happen at the end of the season, but because the linalool level fluctuated and had no specific trend. It can be concluded that linalool can not be used as an indicator of maturit y (senescence index) of Valencia orange peel. Water stress and growth regulators During the 2005/06 season, the WS *GR treatment numerically decreased the level of linalool in the peel of Marsh grapefruit toward the end of the season, but the GR treatm ent had a wide fluctuation in linalool level from one harvest date to another with a nonsignificant peak in May, Table 6 13 and Figure 6 27. During the 2006/07 season, linalool levels fluctuated sharply from one harvest date to another, with the same trend for the WS GR treatment, Table 6 13 and Figure 6 28. In Valencia orange, Table 6 14 and Figure 6 29, during the second season, linalool levels also fluctuated between harvest dates with a high peak in Ma y for the WS -treated fruit. Only in May where thei r significant differences with the WS treatment, either alone or combined with GR, being different than the control. R esults of WS and WS*GR agreed with previous reports stating that concentrations of linalool decreased in a fairly smooth fashion as the

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244 ha rvest season advanced (Coggins et al., 1969a ). On the other hand, during the 2006/07 season, Table 6 14 and Figure 6 30, the trend in l inalool levels were completely different from that of the 2005/06 season with a significant difference only at May between the control and the WS*GR treatment H owever it is still difficult to relate this fluctuation in linalool level to peel maturity. Dro ught stress from soil coverage with Tyvek resulted in the same trend of linalool fluctuation from one harvest date to another, Table 6 15 and Figure 6 31. From all these data, it is not possible to use linalool level as an index of Marsh grapefruit or V alencia orange peel maturity. These results are in agreement with previous reports on octanol stating that octanol fluctuated widely from one harvest date to another after GA3 application, making it unlikely for use as a senescence index (Coggins et al., 1969a ). Storage conditions Fruit of Marsh grapefruit stored at 40oF showed completely opposite trend with harvest date from fruit st ored at 70oF during 2005/06 season, Table 6 13 and Figures 6 32 and 6 33. In general, the level of linalool was higher at 70oF than at 40oF Unlike the significant change at 40oF no significant reduction was noticed in March and May between WS*GR treatmen t and control at 70oF, and this is may be related to the increase in water loss from the peel at 70oF. It was reported that water loss (~5%) from lime fruit showed marginal quantitative changes in composition of alcohols (Yadav et al., 2004). It is also possible to refer the change in linalool level between the two temperatures with harvest date to an interaction between peel maturity and temperature, for instance all treat ments showed high linalool level in March and low in May at 40oF, Table 6 13 and Figure 6 32, whereas, this level was low in March and high in May at 70oF, Table 6 13 and Figure 6 33., especially levels of linalool after storage at both temperatures were v ery low compared to the original level at harvest, and more change was noticed in May at

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245 40oF and in March at 70oF. This is also may be related to some kind of effect from the preharvest treatments, especially WS showed numerically lower values than GR, Ta ble 6 13. A similar trend was also noticed with Valencia orange with a significant difference between WS*GR and control for the May harvest stored at 40oF, but not at 70oF, Table 6 14 and Figures 6 34 and 6 35. Also, fruit treated with Tyvek and stored at 40oF and 70oF, Table 6 15 and Figure 6 36, showed no specific trend at 70oF, but the level of linalool decreased numerically toward the end of the season at 40oF with a higher level for the treated fruit compared with the control. Moreover, there were n o consistent relationship with physical characteristics of the peel and the maturity index of the pulp, Table A 3, appendix with the linalool level. From all of these data, it still appears that linalool cannot be used as an indicator of peel senescence o f March grapefruit or Valencia orange. Terpenes The prominent chemical classes present in citrus oil are terpenes, and the hydrocarbon, d limonene, is the major constituent, which provides little to no direct aroma impact to citrus (Braddock, 1999). In this study, both -pinene and myrcene were determined in both Marsh grapefruit and Val encia orange oils, which are rich in both components (Choi et al., 2001; Sawamura et al., 2005). Also, Valencene was determined in Valencia orange only, because it is the character impact compound of Valencia orange (Choi, 2003; Tonder et al., 1998). Harvest date -pinene and myrcene fluctuated from one harvest date to another in all three season with no specific trend, Table 6 16 and Figures 6 -37 and 6 38. The pinene and myrcene were exactly the same during the first two seasons of the study, -pinene. The peak in their level was in February of the first -pinene increased in

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246 March, but myr cene increased in November. This increase in February of 2004/05 and November of the 2005/06 season were associated with higher values of peel peel turgidity in February (4.92 Kg) and November (6.34 Kg), respectively, compared to other harvest dates, Table 2 5 In addition, TSS: acid ratio; 8.05 in February 2004/05 and 7.62 in November 2005/06 (Table 2 1), was acceptable as the legal maturity of grapefruit starts at 7:1 (Soule and Grierson, 1978; Wardowski et al., 1995) The level of these two terpenes in grapefruit decreased in the 2004/05 season after March for a period of one month (March April) but during the 2005/06 season, the level was almost constant for two months (January March). These results may reflect that terpenes increase in less mature peel, and this is agrees with previous report stated that limonene (as acyclic terpene) decreas ed with maturity of grapefruit (Shaw et al., 1991) however, this finding can not be pinene and myrcene increased again in -pinene and myrcene as indicators for maturity and s enescence index of the peel, because of variation and inconsistency in their level from one harvest date to another over a period of three seasons. No previous -pinene or myrcene with maturation. pine ne and myrcene were higher than that of valencene i n Valencia orange during the 2005/06 and 2006/07 season, Table 617 and Figures 6 39 and 6 -40. Valencene is present in small quantity in orange, but plays an important role in the overall flavor and arom a of orange fruit ( Vora et al., 1983; Weiss, 1997) pinene increased gradually toward the end of the season, but myrcene increased late season whereas valencene levels increased mid season and decreased again toward the end of the 2005/06 season, Figure 6 -39, but levels were almost constant in the 2006/07 season, Figure 6 40. These results are also hard to relate w ith a

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247 senescence index of Valencia orange peel, because values increased and decreased, and did not match (may be due to experimental difference trees grow in different environmental condition, etc) with previous reports such as those stating that valen cene formed in the flavedo and increased as the fruit matured (Coggins et al., 1969a ; Del Rio et al., 1992 ; Elston et al., 2005; Maccarone et al., 1998 ; Sharon -Asa et al., 2003; Shaw and Coleman, 1974 ). Sharon -Asa et al (2003) examined the expression of the Cstps1 gene, a key gene in the production of vale ncene, and found that it is developmentally regulated and encoded to valencene synthase that accumulates only towards fruit maturation, corresponding well with the time of valencene accumulation H owever results of valencene during 2006/07 season are in a greement with findings of Del Rio et al. (1991) who stated that valencene can be detected 9 weeks after anthesis and reache s its maximum level 19 weeks after anthesis, then decreases and becomes stable between weeks 20 and 53. The reduction in valencene late 2005/06 season may be related to some other physiological processes happened at that time of the year, such as increase in chlorophyll content due to natural regreening that may hide/reduce valencene level, especially it is naturally very low compared to other terpenes. Natural regreening of ripe citrus fruit happen at warm temperature during late spring and summer season, and considered a real problem in Valencia orange in southern California and Florida (Hsu et al., 1989; Thomson et al., 1967) In Table 2 4 there was a reduction in color index of Valencia orange late in the season. It is still -pinene, myrcene and valencene to identify senescence index of the peel based on this study. Water stress and growth regulators In M arsh grapefruit, the terpenes level fluctuated from one harvest date to another, Table 6 18 and Figure 6 41 and 6 42, in the 2005/06 season. The most pronounced effect was that the WS treatment increased levels of terpenes significantly in March compared with the control and

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248 GR treatment. Also, GR increased the levels of terpenes in May compared to WS. The level of terpenes was numerically higher in May with GR than in March with WS. As known, WS advance the maturity of the peel (Sharon -Asa et al., 2003), but GR delay it ( Coggins, 1981). This is confirm the findings stated that terpenes level increase in less mature peel, and this is in agree with previous report sta ted that limonene (as acyclic terpene) decreased with maturity of grapefruit (Shaw et al., 1991) Wilson et al. (1990) and McDonald et al. in ( 1997) stated that treatment with GA3 generally increased peel oil concentration. Drought stress advances maturity and senescence of the citrus peel and may have effect on volatile compounds, which consists mostly of monoand sesquiterpenes (the major components of citrus oil). Drought stress can be a factor affects the overall emission of terpenes from citrus tree. Severe and mild stresses reduce the amount of emitted sesquiterpenes, but no effect was noticed from slight stress. Also, the effect of water stress was closely related to the surrounding temperature. Mild drought stress induced a decrease in trans -caryophyllene showed no response (Hansen and Seufert, 1999) Effects of stress on volatiles were confirmed in Mediterranean Cypress (Yani et al., 1993), Holm Oak (Bertin and Staudt, 1996), and Aleppo pine (Ormeno et al., 2007) This data suggests that water stress may directly reduce volatiles by influencing peel maturity. Low ter penes in the peel of WS treated fruit in May compared to March of the 2005/06 season, Table 6 18 and Figure 6 41 and 6 42, when the temperature was higher, Figure B 1, appendix, may be a direct effect of stress or an effect on peel maturity in lowering the content of volatile components of the fruit. Since drought stress causes a restriction in carbon acquisition through -caryophyllene and trans -ocimene, which represent about 82% of the total terpenoid emission from citrus leaves, and

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249 reduce to 6% under severe drought stress (Hansen and Seufert, 1999) This study on leaves may indicate that fruit peel could also be affected by water stress. -pinene was completely opposite to that of myrcene with the control and all treatments during the 2006/07 season, Table 6 18 and Figure 6 43 and 6 44, with a significant difference in -pinene level between WS and contr ol in January of the 2006 / 0 7 season, as well as significant increase in GR and WS*GR compared to the control in January of the same season. These data did not show a specific trend in terpenes level that can be used to define peel senescence accurately (fo r instance, WS and GR have almost the same effect on -pinene and Myrcene level s in January 2006/07 season). In Chapter 2, it was suggested that the cut -off point to harvest Marsh grapefruit should be after March, because peel start to be senescent after this period and this was associated with change in s ome physical characteristics, such as a significant reduction in fruit detachment force in March of both the 2005/06 and 2006/07 seasons, Table 2 7. According to terpenes level, it should be de creasing after March rather than in creasing if senescence relat ed. So, it is suggested that it is hard to count on these two terpenes as maturity indicators of Marsh grapefruit peel. In Valencia orange all treatments and the control generally increased the level of -pinene, followed by valencene that was very small during the 2005/06 season, Table 6 -pinene fluctuated from one harvest date to anoth er with the same trend Figures 6 45 and 6 46, but valencene increased until Ma y then decreased in July, Figure 6 47. The most pronounced effect was for the WS*GR treatment that increases the level -pinene in May compared to the control bu t this effect was for WS that increase the level of valencene in May. Also, in May, GR treatment numerically decreased level -pinene, but not the valencene. The level of valencene in GR -treated fruit

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250 increased with the growing season, and t hen decreased late in July. A p revious report stated that the concentrations of many oil components, such as valencene was less affected by GA3 compared to the control (Coggins et al., 1969a ). Valencene concentration was concomitant with biochemical and physiological changes associated with senescence in a fairly small peak compared to the control and possibly could be used as an indicat or of the maturity of oranges (Lewis et al., 1967). Table 6 19 shows that the level of valencene in GR treated fruit was almost the same like the control fruit in Ma y then became less than the control in July with no significant difference, and this is in agree with Coggins et al. (1969a ) who stated that the magnitude of change in valencene was less in GA3pinene, myrcene or valencene level of GR treated fruit was noticed in the 2006/07 season either, Table 6 19 and Figures 6 48, 6 49 and 6 50. Based on the previous reports about terpenes as well as the physiologic al rind disorder data, Table A 2, data of all three terpenes in WS and/or GR treated fruit during 2005/06 season show that it is hard to relat their level to the degree of peel senescence. The general trend of terpenes level, except valencene was to incr ease toward the end of the season with severe water stress application by soil coverage with Tyvek, Table 6 20 and Figure 6 51, 6 52 and 653, for both seasons of study in Valencia orange. This is in agreement with previous reports stating that drought s tress advances maturity and senescence of the citrus peel -pinene and myrcene) and sesquiterpenes (e.g. valencene) (Sharon -Asa et al., 2003). Level of -pinene increased with severe stress (Tyvek) treatment significantly compared to the control in both seasons, but the level fluctuated later with maturation. Also, the effect of severe water stress was only noticeable on myrcene during the 2005/06 season. Severe and mild stresses reduced the amount of emitted sesquiterpenes (e.g.

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251 valencene) (Hansen and Seufert, 1999) and this was noticeable in the 2005/06 season only. So, it is not possible to apply severe water stress and use these terpenes that changes with peel maturation and senescence as indicators to define senescence index of Valencia orange peel. Storage conditions pinene and myrcene in Marsh grapefruit during storage, Table 618, were higher at 70oF, Figures 6 54 and 655, than that at 40oF, Figures 6 56 and 6-57, respectively with -pinene at both storage temperatures. T he most noticeable change was for higher level of terpenes than the control (numerically) and GR -treated (significantly) for the WS treated an d WS*GR treated fruit harvested in May of the 2005/06 season and stored for three months at 70oF, Table 6 18 and Figures 6 54 and 655 Fresh fruit at the May harvest had high levels of terpenes in the GR treatment, Table 6 18 and Figures 6 41 and 6 42, b ut it was significantly the lowest (at P after storage at 70oF, Table 6 18 and Figure 654 and 6 55. This change in fruit behavior after storage is possibly related to an interaction between fruit waxing and storage temperature. Wax application to grapefruit before storage increased level s of -phellandrene, 3 carene, and ocimene as temperature increased during storage ( Sun and Petracek, 1999). It may also be related to the effect of GR in delaying the physiological changes in the peel. Because of the inconsistency of the terpenes levels between the two storage temperatures, st pinene and myrcene, and hence, these components may not be considered as candidates for senescence indicators. pinene, myrcene and valencene in Valencia orange peel were fluctuate d after storage for all treatment compared to the control with no specific trend at both temperatures, Table 6 19 and Figures 6 58 to 6 63. Th e reduction in terpene level of WS and GR treated fruit after March compared with the control during storage Fi gures 6 58 to 6 6 0 was associated with

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252 a numerical reduction in chilling injury at 40oF for March and May harvested fruit, Table A 2, appendix. Data of Tyvek during storage, Table 6 20 and Figures 6 64 to 6 66, did not show any specific trend at both storage temperatures So, all three terpenes can not be used as indicator of peel senescence for Valencia orenage. Conclusion The quality of citrus oil is dependent on several factors, such as soil, climate, method of extraction, fruit variety, and fruit ma turity ( Wolford et al., 1971). Concentrations of many rind oil constituents vary during maturation and senescence (Scora and Newman, 1967 ). Quantitative analyses of these constituents are important, particularly in relation to qual ity control methods of the oil (Wilson and Shaw, 1980). Nootkatone increased with fruit maturity during all three seasons and was associated with significant increases of TSS: acid ratio and signif icant decreases of peel peel turgidity WS increased the level of Nootkatone at the beginning of the 2005/06 season only, and GR decreased the level of nootkatone compared to the control from January to May of both seasons The period of JanuaryMarch is a normal time window to harvest grapefruit, and WS and GR could be used to adjust the time of harvest. GR has a good effect in this regard that can extend this window until April or May Nootkatone reflected the peel maturity stage at harvest, and its behav ior after harvest, and it might be seen as an indicator of peel maturity for Marsh grapefruit. Aldehydes fluctuated from one harvest date to another with no significant difference or any specific trend, but it was usually higher in more mature (senesc ent) peel based on data of the 2006/07 season. March is the beginning of peel maturity of Valencia orange, and this can be adjusted by WS, which increased the level of aldehydes early in the 2005/06 season only, while adjusting the maturity index of the peel: increasing the color index and decreasing fruit detachment force, as well as increasing chilling injury at 40oF. GR also had some effect in this

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253 regard and it decreased aldehydes level late in July of the 2005/06 only However because of aldehydes fluctuation from one harvest date to another and little re lationship to the peel maturity stage and peel behavior after harvest, they probably have little use as indicator s of maturity of Valencia oranges. Esters, such as geranyl acetate fluctuated over the three seasons with a general trend of increase toward the end of the season in Marsh grapefruit, but in Valencia orange, geranyl acetate level increased very little over time, and was associated with some small changes during storage, such as incre ase in pulp maturity index and % decay and % CI at 40oF, which is consider ed to happen as a storage effect rather than because of peel maturity status, and therefore reflect s no change in peel maturity stage in January -April period for Marsh grapefruit a nd in April May period for Valencia orange. WS and GR had no significant effect on geranyl acetate while adjusting harvest date and maturity index of the peel. Overall, harvest data indicate that geranyl acetate can not be used as a candidate to determ ine peel maturity index. Alcohols, such as linalool fluctuated from one season to another, and there were no specific trend in all seasons for both Marsh grapefruit and Valencia orange Linalool increase d significantly after May; the end of the seas on, but only reached the same level as earlier in the harvest season. Although there were some changes in linalool levels after application of WS and GR, there were no significant difference among all treatments and control at all harvest dates. These chan ges in linalool level from one harvest date to another were associated with almost the same values of pulp maturity index, peel color index and peel turgidity at each harvest date. So, it is not possible to use linalool as a senescence index of Marsh gra pefruit or Valencia orange peels.

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254 -pinene and myrcene in grapefruit fluctuated from one harvest date to another in all seasons with no specific trend. Their levels increased in less mature peel, in November based on data of the second and third seasons, as well as in senescent peel after March based on data of the first and third seasons -pinene and myrcene as indicators for a maturity and senescence index of Marsh grapefruit peel. In Valencia or -pinene and myrcene were higher than that of valencene. Level of pinene increased gradually toward the end of both season s but myrcene only showed a general trend of increasing toward the end of both seasons whereas valencene level inc reased mid season and decreased again toward the end of 2005/06 season and remain constant in 2006/07 season These levels were hard to correlate with the pulp maturity indices or to a peel senescence index of -pinene and myr cene fluctuated in Marsh grapefruit from one harvest date to another after application of WS and GR, with a trend towards higher levels in less mature peel for both treatments. So, there is no specific trend in terpenes level that can be used to define peel senescence accurately based on WS and GR In Valencia orange, all pinene myrcene and valencene also fluctuated from one harvest date to another. The most pronounced effect was for the WS *GR -pinene in Ma y and WS treatment increased the level of valencene in May. Also, in May, the GR -pinene, but not of valencene, however comparing these data with previous reports about terpenes and fruit behavior with maturation and during storage confirm the difficulty of using these three t erpenes as indicator s of senescence index of Valencia orange peel. Volatile component results showed differences among all components in response to harvest date, water stress and growth regulators. For all studied components, except nootkatone

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255 in Mars h grapefruit, these differences in the level and the trend with fruit maturation showed too many variations that can not be used to define peel senescence index accurately. More research is required on the qualitative and quantitative changes in oil compo sition with harvest date to evaluate changes with physiological age of the peel. How changes in volatiles can be altered by adjusting maturation and senescence of the peel with the application of water stress and growth regulators may still have value in f urther studies to find consistent changes in volatiles with peel maturation.

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256 Table 6 1. Changes of Marsh grapefruits nootkatone peak area (millions) with harvest date over three years Date 2004/2005 2005/2006 2006/2007 September NA 1.901 b October NA November 0.176 b 0.059 b 0.016 b December 1.441 b January 1.385 b 1.139 b 1.369 b February 2.677 b March 1.528 b 7.151 ab 2.442 b April 3.228 b May 2.971 b 11.865 a 10.668 a June 12.022 a July 12.708 a Means with the same letters in each column are not significantly different ( P

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2 57 Table 6 2. Effect of water stress and growth regulators on March grapefruits nootkatone peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, WS; water stress, GR; growth regula tors, and WS*GR; water stress & growth regulators. 2005/2006 2006/2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 1.139 b 1.886 b 5.864 a 1.369 a WS 1.701 a 6.927 a 3.960 a 0. 463 bc GR 0.625 c 1.373 b 4.687 a 0.449 c WS*GR 1.141 b 2.588 b 4.058 a 0.933 ab Mar CONT 7.151 a 7.470 a 5.637 b 2.442 a WS 4.202 a 5.026 a 9.988 a 1.631 a GR 3.784 a 5.646 a 5.204 b 1.678 a WS*GR 3.981 a 6.406 a 9.247 ab 1.485 a May CONT 11.865 a 11.924 a 27.836 a 10.668 a WS 10.373 a 8.881 ab 23.180 a 8.278 b GR 4.112 b 11.613 a 24.543 a 5.033 c WS*GR 3.536 b 6.762 b 32.725 a 11.357 a Jul CONT 12.708 a 9.672 b 19.887 ab WS 17.678 a 13.73 6 a 25.800 a GR 16.463 a 12.658 ab 14.580 b WS*GR 17.688 a 10.396 ab 27.257 a Mean separation of treatments under each harvest date within column by DMRT ( P Table 6 3. Changes of Valencia oranges aldehydes peak area (millions) with harvest date over two years. Date 2005/2006 2006/2007 January 206.4 a 214.7 a March 222.4 a 186.4 a May 193.3 a 295.9 a July 197.5 a 275.1 a Means with the same letters in each column are not significantly different ( P

PAGE 258

258 Table 6 4. Effect of water stress and growth regulators on Valencia oranges aldehydes peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season.CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. 2005/2006 2006/2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 206.4 b 97.2 a 26.1 a 214.6 a WS 244.1 b 73.2 ab 82.9 a 141.2 a GR 213.6 b 77.6 ab 91.1 a 210.1 a WS*GR 289.6 a 51.5 b 21.3 a 196.2 a Mar CONT 222.4 a 83.1 a 104.9 a 186.4 a WS 932.1 a 88.2 a 79.4 a 197.6 a GR 298.4 a 28.5 a 116.9 a 230.4 a WS*GR 253.2 a 89.3 a 84.6 a 206.7 a May CONT 193.3 a 4.1 b 46.6 ab 295.9 a WS 228.8 a 8.6 b 32.9 b 215.9 a GR 186.8 a 4.2 b 73.5 a 222.0 a WS*GR 229.7 a 17.6 a 27.5 b 404.4 a Jul CONT 197.5 a 39.6 a 38.7 b 275.1 a WS 120.3 bc 34.3 a 49.2 b 212.7 a GR 49.5 c 33.7 a 104.9 a 260.8 a WS*GR 173.1 ab 22.4 a 81.8 a 217.3 a Mean separation of treatments under each harvest date within column by DMRT ( P

PAGE 259

259 Table 6 5. Effect of soil coverage with Tyvek on Valencia oranges aldehydes peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, Tyvek; soil covered with Tyvek. 2005/2006 200 6/2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 206.4 a 97.2 a 26.1 a 128.5 a TYVEK 215.7 a 18.1 b 24.1 a 495.8 a Mar CONT 222.4 a 83.1 a 104.9 a 170.7 a TYVEK 229.5 a 65.2 a 77.2 a 121.9 a May CONT 193.3 a 4. 1 a 46.6 a 134.9 a TYVEK 171.2 a 50.6 a 32.3 a 320.2 a Jul CONT 197.5 a 39.6 a 38.7 a 240.8 a TYVEK 226.3 a 131.8 a 157.1 a 312.2 a Mean separation of treatments under each harvest date within column by DMRT ( P Table 6 6. Changes of Marsh grapefruits geranyl acetate peak area (millions) with harvest date over three years. Date 2004/2005 2005/2006 2006/2007 September 2.7 b 9.2 b 7.4 a October 13.4 ab November 1.2 b 9.7 b 6.9 a Dece mber 8.9 ab January 6.6 ab 16.8 ab 7.1 a February 24.1 ab March 40.3 a 9.6 b 11.4 a April 5.7 ab May 8.5 ab 26.1 ab 12.4 a June 15.6 ab July 29.7 a Means with the same letters in each column are not significantly different ( P

PAGE 260

260 Table 6 7. Changes of Valencia oranges geranyl acetate peak area (millions) with harvest date over two years. Date 2005/2006 2006/2007 January 0.35 b 0.22 a March 0.50 b 0.32 a May 0.49 b 0.81 a July 8.20 a 0.94 a Means with th e same letters in each column are not significantly different ( P Table 6 8. Effect of water stress and growth regulators on March grapefruits geranyl acetate peak area (millions) at harvest over two years and during storage for 12 weeks duri ng the 2005/06 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. 2005/2006 2006/2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 16.8 a 13.2 ab 13.8 a 7.1 b WS 18.3 a 2 5.8 a 11.7 b 6.6 b GR 13.5 a 15.5 ab 8.4 c 5.6 b WS*GR 21.8 a 12.4 b 6.6 d 13.5 a Mar CONT 9.6 a 6.6 ab 6.7 ab 11.4 a WS 9.7 a 3.4 b 7.4 a 10.2 a GR 11.8 a 10.7 a 6.1 b 11.1 a WS*GR 16.5 a 7.7 a 6.1 b 9.6 a May CONT 2 6.1 a 12.9 ab 23.7 b 12.4 b WS 9.9 b 9.9 b 15.6 d 14.7 ab GR 19.6 ab 16.6 a 33.8 a 21.8 a WS*GR 4.5 b 9.7 b 19.5 c 17.7 ab Jul CONT 29.7 a 18.1 b 18.2 c WS 37.3 a 21.4 ab 27.8 b GR 30.8 a 20.1 ab 20.6 bc WS*GR 21.2 a 31.4 a 64.7 a Mean separation of treatments under each harvest date within column by DMRT ( P

PAGE 261

261 Table 6 9. Effect of water stress and growth regulators on Valencia oranges geranyl acetate peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, WS; water stress, GR; growth re gulators, and WS*GR; water stress & growth regulators. 2005/2006 2006/2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 0.35 b 1.57 a 4.87 a 0.22 a WS 0.34 b 1.49 a 10.85 a 1.06 a GR 0.36 b 1.88 a 3.30 a 0.73 a WS*GR 0. 87 a 1.05 b 2.87 a 0.47 a Mar CONT 0.50 b 2.09 b 1.43 c 0.32 b WS 1.66 a 1.22 c 4.41 ab 1.02 ab GR 1.44 a 3.21 a 2.98 bc 0.74 ab WS*GR 0.73 b 1.46 c 5.20 a 1.20 a May CONT 0.49 a 0.26 b 2.66 a 0.81 b WS 0.89 a 0.59 ab 2.92 a 0.99 b GR 0.64 a 0.42 b 4.29 a 1.30 b WS*GR 2.21 a 1.03 a 2.07 a 3.30 a Jul CONT 8.20 a 4.84 a 1.09 c 0.94 a WS 1.83 b 4.83 a 2.88 b 0.69 a GR 6.73 a 3.02 ab 4.28 a 0.76 a WS*GR 6.14 a 2.45 b 3.66 ab 1.19 a Mean separation of treatments under each harvest date within column by DMRT ( P

PAGE 262

262 Table 6 10. Effect of soil coverage with Tyvek on Valencia oranges geranyl acetate peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, Tyvek; soil covered with Tyvek. 2005/ 2006 2006/2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 0.35 a 1.57 a 4.87 a 0.65 a TYVEK 0.29 a 1.94 a 2.62 a 0.09 a Mar CONT 0.50 a 2.09 a 1.44 a 0.37 a TYVEK 0.95 a 0.82 b 0.85 a 0.23 a May CONT 0.49 a 0.26 a 2.66 a 0.41 a TYVEK 0.27 a 0.47 a 1.35 a 0.82 a Jul CONT 8.20 a 4.84 b 1.09 a 1.64 a TYVEK 4.66 a 11.56 a 3.87 a 0.79 a Mean separation of treatments under each harvest date within column by DMRT ( P Table 6 11. Changes of Marsh grapefruits linalool peak area (millions) with harvest date over three years. Date 2004/2005 2005/2006 2006/2007 September 51.2 abc 40.8 a 40.1 a October 76.3 abc November 7.5 c 72.4 a 8.7 b Dece mber 71.6 abc January 35.0 c 38.9 a 34.5 a February 116.6 a March 113.5 ab 51.2 a NA April 37.8 bc May 55.1 abc 54.9 a 54.4 a June 56.4 abc July NA Means with the same letters in each column are not significantly different ( P 0.05)

PAGE 263

263 Table 6 12. Changes of Valencia oranges linalool peak area (millions) with harvest date over two years. Date 2005/2006 2006/2007 January 127.9 a 183.6 c March 136.1 a 166.4 d May 61.3 c 189.3 b July 106.9 b 258.0 a Means with th e same letters in each column are not significantly different ( P Table 6 13. Effect of water stress and growth regulators on March grapefruits linalool peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, WS; water stress, GR; growth reg ulators, and WS*GR; water stress & growth regulators. 2005/2006 2006/2007 Month Treatment Harvest date After 40 o F After 70 o F Harvest date Jan CONT 38.9 a 7.7 c 17.9 c 34.5 a WS 81.6 a 10.2 b 25.4 b NA GR 37.0 a 8.2 c 32.2 a 101.2 a WS*G R 106.5 a 14.9 a 26.7 b 17.6 a Mar CONT 51.2 a 8.5 c 21.2 a NA WS 52.9 a 11.6 bc 15.6 a 2.2 a GR 37.6 a 15.7 ab 18.6 a 2.2 a WS*GR 48.6 a 17.7 a 20.2 a NA May CONT 54.9 a 6.9 a 36.4 a 54.4 a WS 28.9 a 3.0 b 34.4 a 48.1 a b GR 89.3 a 5.0 ab NA 42.2 b WS*GR 40.4 a 3.1 b 28.3 a 29.3 c Jul CONT NA 7.6 a 22.3 b WS 34.9 a 7.1 b 20.9 b GR 40.7 a 5.5 c 30.6 a WS*GR 25.5 a 7.3 ab NA Mean separation of treatments under each harvest date within column by DMRT ( P

PAGE 264

264 Table 6 14. Effect of water stress and growth regulators on Valencia oranges linalool peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, WS; water stress, GR; growth regulato rs, and WS*GR; water stress & growth regulators. 2005/2006 2006/2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 127.9 a 92.9 a 118.8 ab 183.6 a WS 133.7 a 83.7 a 277.9 a 6.9 a GR 127.9 a 79.2 a 191.4 ab 85.7 a WS*GR 1 39.9 a 96.1 a 70.8 b 101.6 a Mar CONT 136.1 a 76.4 a 85.5 a 166.4 a WS 316.7 a 66.9 a 77.9 a 99.2 a GR 159.3 a 65.7 a 84.6 a 2.1 a WS*GR 138.8 a 55.9 a 79.9 a 2.8 a May CONT 61.3 b 19.3 b 165.7 a 189.3 a WS 105.9 a 13.4 c 145.4 a 214.1 a GR 58.2 b 9.9 c 81.6 a 178.3 a WS*GR 102.5 a 36.7 a 108.6 a 3.1 b Jul CONT 106.9 a 32.0 a NA 258.0 a WS 56.9 a 68.5 a 21.2 a 158.9 a GR 109.2 a 26.9 a 1.3 a 98.1 a WS*GR 85.2 a 77.5 a 110.7 a 190.0 a Mean separation of treatments under each harvest date within column by DMRT ( P

PAGE 265

265 Table 6 15. Effect of soil coverage with Tyvek on Valencia oranges linalool peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, TYVEK; soil covered with tyvek 2005/2006 2006 /2007 Month Treatment Harvest date 40 o F 70 o F Harvest date Jan CONT 127.9 a 92.9 a 118.8 a NA TYVEK 189.7 a 132.3 a 198.2 a NA Mar CONT 136.1 a 76.4 a 85.5 a 123.5 a TYVEK 233.6 a 98.6 a 313.9 a 85.4 a May CONT 61.3 a 19.3 a 165.6 a NA TYVEK 102.5 a 47.5 a 184.4 a 1.9 Jul CONT 106.9 b 32.0 NA NA TYVEK 230.9 a NA NA NA Mean separation of treatments under each harvest date within column by DMRT ( P Table 6 16. Changes of Marsh grapefruits terpenes peak area (millions) with harvest date over three years Date 2004/2005 2005/2006 2006/2007 pinene Myrcene pinene Myrcene pinene Myrcene September 115.0 cd 367.1 de 90.9 b 342.0 ab 93.5 b 318.1 a October 230.5 bc 841.9 bcd November 21.8 d 87.5 e 485.1 a 1490.5 a 182.5 b 453.1 a December 350.5 b 1223.9 ab January 114.9 cd 399.7 cde 93.6 b 348.5 ab 216.6 b 222.2 a Februar y 596.7 a 1679.2 a March 303.6 bc 1022.1 b 93.9 b 350.8 ab 1067.6 a 169.9 a April 273.2 bc 895.0 bc May 407.9 b 1297.6 ab 278.1 ab 1068.1 ab 121.9 b 289.1 a June 343.0 b 1231.1 ab July 22.6 b 5.1 b Means with the same letters in each column are not significantly different ( P

PAGE 266

266 Table 6 17. Changes of Valencia oranges terpenes peak area (millions) with harvest date over two years. Date 2005/2006 2006/2007 pinene Myrcene Valencene pinene Myrcene Valencene January 50.7 b 227.3 b 2.9 b 261.0 a 401.5 ab 2.6 a March 47.0 b 256.2 b 46.0 a 218.3 a 334.3 b 0.4 b May 64.8 b 219.5 b 34.8 a 272.3 a 4.5 c 1.6 ab July 135.8 a 529.7 a 8.5 b 551.2 a 580.2 a 1.2 ab Means with the same letters in each column are not significantly different ( P Table 6 18. Effect of water stress and growth regulators on March grapefruits terpenes peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, WS; water stress, GR; growth regul ators, and WS*GR; water stress & growth regulators. pinene Myrcene 2005/06 2006/07 2005/06 2006/07 Month Treat. Harvest 40 o F 70 o F Harvest Harvest 40 o F 70 o F Harvest January CONT 93.6 a 41.5 b 42.4 bc 216.6 c 348.5 b 213.3 b 212.9 b 222.2 a WS 16.9 a 7.7 d 36.6 c 332.4 b 613.8 b 61.7 c 251.9 ab 49.4 a GR 80.8 a 32.7 c 69.2 a 307.6 b 332.9 b 197.1 b 302.6 a 34.4 a WS*GR 43.0 a 68.2 a 50.0 b 425.9 a 1062.8 a 281.9 a 231.6 b 360.5 a March CONT 93.9 b 49.1 bc 56 .3 a 1067.6 a 350.8 b 190.1 b 242.6 a 169.9 a WS 292.2 a 59.6 ab 59.7 a 518.5 a 1041.9 a 240.0 ab 235.6 a 21.3 a GR 80.5 b 36.9 c 66.4 a 648.1 a 344.4 b 201.1 b 261.6 a 156.3 a WS*GR 173.4 ab 74.4 a 72.3 a 793.3 a 646.1 ab 281.9 a 283.5 a 70.7 a May CONT 278.1 ab 66.5 a 154.4 a 121.9 b 1068.1 ab 233.7 a 726.1 a 289.1 a WS 71.5 b 52.9 a 224.4 a 241.5 a 283.3 b 168.9 a 941.6 a 648.3 a GR 595.9 a 71.4 a 2.3 b 168.5 ab 1860.5 a 247.0 a 66.5 b 470.4 a WS*GR 50.5 b 50.2 a 221.3 a 115.9 b 250.9 b 160.0 a 825.1 a 372.9 a July CONT 22.6 a 98.0 b 95.8 b 5.1 b 212.3 a 393.2 b WS 223.2 a 75.8 c 102.1 b 379.9 a 346.4 a 598.7 a GR 275.1 a 66.7 c 116.0 b 473.9 a 293.8 a 1. 2 d WS*GR 108.3 a 130.2 a 176.1 a 220.4 a 257.7 a 29.2 c Mean separation of treatments under each harvest date within column by DMRT ( P .

PAGE 267

267 Table 6 19. Effect of water stress and growth regulators on Valencia oranges terpenes peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. pinene 2005/06 2006/07 Month Treat. Harvest 40 o F 70 o F H arvest January CONT 50.7 a 93.9 a 62.3 b 261.0 a WS 63.0 a 69.9 ab 415.7 a 360.1 a GR 48.5 a 79.9 ab 162.8 b 323.9 a WS*GR 62.9 a 49.4 b 62.8 b 275.9 a March CONT 47.0 a 181.9 a 77.4 a 218.3 b WS 256.9 a 138.3 a 63.1 a 345 .3 ab GR 101.9 a 125.1 a 69.6 a 252.2 b WS*GR 100.6 a 88.4 a 67.2 a 716.5 a May CONT 64.8 bc 166.3 a 214.2 a 272.4 a WS 92.3 ab 63.3 b 420.0 a 498.9 a GR 53.4 c 39.2 b 143.1 a 50.0 a WS*GR 101.3 a 152.1 a 338.2 a 518.9 a July CONT 135.8 a 153.7 b 975.3 a 551.2 a WS 154.0 a 158.1 b 90.5 b 267.0 a GR 163.6 a 115.5 b 129.6 b 263.2 a WS*GR 134.2 a 353.9 a 167.4 b 553.1 a Myrcene 2005/06 2006/07 Month Treat. Harvest 40 o F 70 o F Harvest Jan uary CONT 227.3 a 343.2 a 290.5 b 401.5 a WS 249.3 a 277.6 a 1835.6 a 4.8 a GR 203.4 a 295.8 a 602.1 ab 437.5 a WS*GR 281.8 a 232.5 a 258.7 b 146.6 a March CONT 256.2 a 681.4 a 289.9 a 334.3 a WS 1154.9 a 503.7 a 238.8 a 3. 4 a GR 383.0 a 460.9 a 274.9 a 110.3 a WS*GR 410.5 a 314.9 a 257.3 a 426.8 a May CONT 219.5 b 564.0 a 1021.3 a 4.4 a WS 348.0 a 238.2 b 284.3 a 495.1 a GR 191.5 b 174.2 b 485.8 a 423.5 a WS*GR 400.3 a 506.0 a 794.8 a 23 5.1 a July CONT 529.7 a 372.3 a 1497.1 a 580.2 a WS 388.3 a 293.2 a 384.5 a 702.7 a GR 353.4 a 561.8 a 217.7 a 317.8 a WS*GR 509.9 a 4.8 a 695.0 a 144.7 a

PAGE 268

268 Table 6 19. Continued Valencene 2005/06 2006/07 Month Treat. Harvest 40 o F 70 o F Harvest January CONT 2.9 a 10.3 a 61.7 a 2.6 a WS 4.0 a 1.7 a 208.0 a 5.5 a GR 2.9 a 2.1 a 79.8 a 5.1 a WS*GR 2.2 a 5.9 a 46.1 a 2.2 a March CONT 46.0 a 20.8 a 104.9 a 0.4 b WS 30.8 a 24.8 a 98.8 a 3.4 a b GR 3.9 a 34.3 a 152.9 a 0.9 ab WS*GR 16.0 a 18.9 a 87.9 a 7.9 a May CONT 34.8 ab 0.4 b 8.5 a 1.6 a WS 50.9 a 0.7 b 4.8 a 1.8 a GR 37.2 ab 0.8 b 9.6 a 1.3 a WS*GR 20.1 b 18.2 a 0.7 a 3.0 a July CONT 8.5 a 3.9 a 6.8 a 1.2 a WS 2.7 a 0.8 a 11.4 a 1.4 a GR 4.4 a 0.9 a 11.3 a 1.7 a WS*GR 2.1 a 0.3 a 9.6 a 1.6 a Means with the same letters in each column are not significantly different ( P

PAGE 269

269 Table 6 20. Effect of soil coverage with Tyve k on Valencia oranges terpenes peak area (millions) at harvest over two years and during storage for 12 weeks during the 2005/06 season. CONT; control, TYVEK; soil covered with tyvek. pinene 2005/06 2006/07 Month Treat. Harvest 40 o F 70 o F Harvest J anuary CONT 50.7 b 93.9 a 62.3 a 296.2 b TYVEK 62.6 a 128.7 a 154.5 a 794.9 a March CONT 47.0 a 181.9 a 77.4 a 374.3 a TYVEK 98.1 a 154.1 a 498.4 a 246.4 a May CONT 64.8 a 166.3 a 214.2 a 675.7 a TYVEK 86.1 a 129.7 a 206.6 a 391.4 b July CONT 135.8 b 153.7 b 975.3 a 1555.4 a TYVEK 243.1 a 685.1 a 658.7 a 1197.3 a Myrcene 2005/06 2006/07 Month Treat. Harvest 40 o F 70 o F Harvest January CONT 227.3 b 343.2 a 290.5 a 642.5 a TYVEK 277.5 a 437.9 a 615.6 a 627.3 a March CONT 256.2 a 681.4 a 289.9 a 125.3 a TYVEK 412.6 a 514.0 a 1650.0 a 382.9 a May CONT 219.5 a 564.0 a 1021.3 a 174.9 a TYVEK 303.8 a 512.3 a 873.9 a 494.2 a July CONT 529.7 a 372.3 a 1497.0 a 334.3 b TYVEK 471.6 a 410.3 a 617.3 a 975.7 a Valencene 2005/06 2006/07 Month Treat. Harvest 40 o F 70 o F Harvest January CONT 2.9 10.3 a 61.7 a 1.6 a TYVEK NA 3.8 a 31.8 a 1.8 a March CONT 46.0 a 20.8 a 104.9 a 0.4 a TYVEK 41.9 a 11.4 a 26.5 a 0.4 a May CONT 34.8 a 0.4 a 8.5 a 0.7 a TYVEK 10.1 a 0.3 a 0.7 a 1.3 a July CONT 8.5 a 3.9 a 6.8 a 2.1 a TYVEK 0.9 a 3.2 a 12.1 a 2.1 a Means with the same letters in each column are not significantly different ( P

PAGE 270

270 0 2 4 6 8 10 12 14 16 18 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date Peak area (millions) 2004/05 2005/06 2006/07 Figure 6 1. Effect of harvest date of Marsh grapefruit on nootkatone peak area (millions) during three season s Values are mean SE.

PAGE 271

271 0 5 10 15 20 25 Sept Nov Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 2. Effect of field water stress and growth regulators treatments on nootkatone of Marsh grapefruit peel during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 272

272 0 2 4 6 8 10 12 14 Sept Nov Jan Mar May Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 3. Effect of field water stress and growth regulators treatments on nootkatone of Marsh grapefruit peel during the 2006/2007 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 273

273 0 5 10 15 20 25 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 4. Changes in nootkatone of water stress and growth regulators treated Mar sh grapefruit during storage at 40oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 274

274 0 5 10 15 20 25 30 35 40 45 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 5. Changes in nootkatone of water stress and growth re gulators treated Marsh grapefruit during storage at 70oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 275

275 0 50 100 150 200 250 300 350 400 Jan Mar May Jul Harvest date Peak area (millions) 2005/06 2006/07 Figure 6 6. Effect of harvest date of V alencia orange on aldehydes during two years Values are mean SE.

PAGE 276

276 0 200 400 600 800 1000 1200 1400 1600 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 7. Effect of field water stress and growth regulators treatments on aldehydes of Valencia orange peel during the 2005/2006 season. CONT; control, WS; water stress, GR; growt h regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 277

277 0 100 200 300 400 500 600 700 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 8. Effect of field water stress and growth regulators treatments on aldehydes of Valencia orange peel during the 2006/2007 season. CONT; control, WS; water str ess, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 278

278 0 100 200 300 400 500 600 700 800 900 Jan Mar May Jul Harvest date Peak area (millions) Control 2005/06 Tyvek 2005/06 Control 2006/07 Tyvek 2006/07 Figure 6 9 Effect of soil coverage with Tyvek on aldehydes of Valencia orange during the 2005/06 and 2006/07 seasons. Values are mean SE.

PAGE 279

279 0 20 40 60 80 100 120 140 160 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 10. Changes in aldehydes of water stress and growth regulators treated Valencia orange during storage at 40oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are me an SE.

PAGE 280

280 0 20 40 60 80 100 120 140 160 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 1 1 Changes in aldehydes of water stress and growth regulators treated Valencia orange during storage at 70oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regu lators. Values are mean SE.

PAGE 281

281 0 20 40 60 80 100 120 140 160 180 200 Jan Mar May Jul Harvest date Peak area (millions) Control @ 40F Tyvek @ 40F Control @ 70F Tyvek @ 70F Figure 6 12. Effect of soil coverage with Tyvek on aldehydes of Valencia orange during storage at 40oF and 70oF during the 2005/2006 season. Values are mean SE.

PAGE 282

282 0 10 20 30 40 50 60 70 80 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date Peak area (millions) 2004/05 2005/06 2006/07 Figure 6 13. Effect of harvest date of Marsh grape fruit on geranyl acetate during three years of experiments. Values are mean SE.

PAGE 283

283 0 1 2 3 4 5 6 7 8 9 Jan Mar May Jul Harvest date Peak area (millions) 2005/06 2006/07 Figure 6 14. Effect of harvest date of Valencia orange on geranyl acetate during two years of experiments. Values are mean SE.

PAGE 284

284 0 10 20 30 40 50 60 Sept Nov Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 15. Effect of field water stress and growth regulator treatments on levels of geranyl acetate of Marsh grapefruit at harvest during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 285

285 0 5 10 15 20 25 Sept Nov Jan Mar May Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 16. Effect of field water stress and growth regulator treatment on levels of geranyl acetate of Marsh grapefruit at harvest during 2006/2007 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regul ators. Values are mean SE.

PAGE 286

286 0 1 2 3 4 5 6 7 8 9 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 17. Effect of field water stress and growth regulator treatments on levels of geranyl acetate of Valencia orange at harvest during 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*G R; water stress & growth regulators. Values are mean SE.

PAGE 287

287 0 1 1 2 2 3 3 4 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 18. Effect of filed water stress and growth regulator treatments on levels of geranyl acetate of Valencia orange at harvest during 2006/2007 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 288

288 0 1 2 3 4 5 6 7 8 9 Jan Mar May Jul Harvest date Peak area (millions) Control 2005/06 Tyvek 2005/06 Control 2006/07 Tyvek 2006/07 Figure 6 19. Effect of soil coverage with Tyvek on geranyl acetate of Valencia orange during 2005/06 and 2006/07 seasons. CONT; control, WS; water stress, GR; gr owth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 289

289 0 5 10 15 20 25 30 35 40 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 20. Changes in geranyl acetate of water stress and growth regulators treated Marsh grapefruit during storage at 40oF during the 2005/2006 season. CONT; contr ol, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 290

290 0 10 20 30 40 50 60 70 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 21. Changes in geranyl acetate of water stress and growth regulators treated Marsh grapefruit during storage at 70oF during the 2 005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 291

291 0 1 2 3 4 5 6 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 22. Changes in geranyl acetate of water stress and growth regulators treated Valencia oranges during storage at 40oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 292

292 0 2 4 6 8 10 12 14 16 18 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 23. Changes in geranyl acetate of water stress and growth regulators treated Valencia oranges during storage at 70oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 293

293 0 2 4 6 8 10 12 14 Jan Mar May Jul Harvest date Peak area (millions) Control @ 40F Tyvek @ 40F Control @ 70F Tyvek @ 70F Figure 6 24. Changes in geranyl acetate of Tyvek treated Valencia oranges during storage at 40oF and 70oF in 2005/2006 season. Values are mean SE.

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294 020 40 60 80 100 120 140 160 180 200 SepOct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date Peak area (millions) 2004/05 2005/06 2006/07 Figure 6 25. Effect of harvest date of Marsh grapefruit on linalool during three years of experiments. Values are mean SE.

PAGE 295

295 0 50 100 150 200 250 300 Jan Mar May July Harvest date Peak area (millions) 2005/06 2006/07 Figure 6 26. Effect of harv est date of Valencia orange on linalool during two years of experiment Values are mean SE.

PAGE 296

296 0 20 40 60 80 100 120 140 Sept Nov Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 27. Effect of field water stress and growth regulators treatments on linalool of Marsh grapefruit peel during the 2005/2006 season. CONT; control WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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297 0 20 40 60 80 100 120 140 Sept Nov Jan Mar May Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 28. Effect of field water stress and growth regulators treatments on linalool of Marsh grapefruit peel during the 2006/2007 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 298

298 050 100 150 200 250 300 350 400 450 500 JanMar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 29. Effect of field water stress and growth regulators treatments on linalool of Valencia orange peel during the 2005/ 2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 299

299 0 50 100 150 200 250 300 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 30. Effect of field water stress and growth regulators treatments on linalool of Valencia orange peel duri ng the 2006/2007 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 300

300 0 50 100 150 200 250 300 Jan Mar May Jul Harvest date Peak area (millions) Control 2005/06 Tyvek 2005/06 Control 2006/07 Tyvek 2006/07 Figure 6 31. Effect of soil coverage with Tyvek on linalool of Valencia orange peel during the 2005/06 a nd 2006/07 seasons. Values are mean SE.

PAGE 301

301 0 5 10 15 20 Jan Mar May Jul Harvest time Peak area (millions) Control WS GR WS*GR Figure 6 32. Changes in linalool of water stress and growth regulators treated Marsh grapefruit after 12 weeks storage at 40oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 302

302 0 5 10 15 20 25 30 35 40 45 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 33. Changes in linalool of water stress and growth regulators treated Marsh grapefruit after 12 weeks storage at 70oF during the 2005/2006 season. CONT; control, WS; water s tress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 303

303 0 20 40 60 80 100 120 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 34. Changes in linalool of water stress and growth regulators treated Valencia oranges after 12 weeks storage at 40oF during the 2005/2006 season CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 304

304 0 50 100 150 200 250 300 350 Jan Mar May Jul Harvest date Peak area (millions) Control WS GR WS*GR Figure 6 35. Changes in linalool of water stress and growth regulators treated Valencia oranges after 12 weeks storage at 70oF during the 2005/2006 season. CONT; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 305

305 0 50 100 150 200 250 300 350 400 450 Jan Mar May Jul Harvest date Peak area (millions) Control @ 40F Tyvek @ 40F Control @ 70F Tyvek @ 70F Figure 6 36. Effect of soil coverage with Tyvek on linalool of Valencia orange peel after storage at 40oF and 70oF during the 2005/2006 season. Values are mean SE.

PAGE 306

306 0 400 800 1200 1600 2000 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date Peak area (millions) 2004/05 2005/06 2006/07 Figure 6 -pinene in Marsh grapefruits peel oil during three years of experiments. Values are mean SE.

PAGE 307

307 0 400 800 1200 1600 2000 Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Harvest date Peak area (millions) 2004/05 2005/06 2006/07 Figure 6 38. Effect of harvest date on myrcene in Marsh grapefruits peel oil during three years of experiments Values are mean SE.

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308 -20 80 180 280 380 480 580 680 780 Jan Mar May July Harvest date Peak area (millions) a-pinene Myrcene Valencene Figure 6 39. Effect of harvest date on terpenes in Valencia oranges peel oil during the 2005/06 season. Values are mean SE.

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309 -20 80 180 280 380 480 580 680 780 Jan Mar May July Harvest date Peak area (millions) a-pinene Myrcene Valencene Figure 6 40. Effect of harvest date on terpenes in Valencia oranges peel oil during the 2006/07 season. Values are mean SE.

PAGE 310

310 0 400 800 1200 1600 2000 Sept Nov Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 pinene of Marsh grapefruit peel oil during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 311

311 0 400 800 1200 1600 2000 Sept Nov Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 42. Effect of field water stress and growth regulators treatments on myrcene of Marsh grapefruit peel oil during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 312

312 0 200 400 600 800 1000 1200 Sept Nov Jan Mar May Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 pinene of Marsh grapefruit peel oil during the 2006/2007 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 313

313 0 200 400 600 800 1000 1200 Sept Nov Jan Mar May Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 44. Effect of field water stress and growth regulators treatments on myrcene of Marsh grapefruit peel oil during the 2006/2007 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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314 0 50 100 150 200 250 300 350 400 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 45. Effect of field water stress and growth regulators treatments on a -pinene of Valencia orange peel oil during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are me an SE.

PAGE 315

315 0 200 400 600 800 1000 1200 1400 1600 1800 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 46. Effect of field water stress and growth regulators treatments on myrcene of Valencia orange peel oil during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 316

316 0 10 20 30 40 50 60 70 80 90 100 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 47. Effect of field water stress and growth regulators treatments on valencene of Valencia orange peel oil during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 317

317 0 100 200 300 400 500 600 700 800 900 1000 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 pinene of Valencia orange peel oil during the 2006/2007 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regu lators. Values are mean SE.

PAGE 318

318 0 100 200 300 400 500 600 700 800 900 1000 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 49. Effect of field water stress and growth regulators treatments on myrcene of Valencia orange peel oil during the 2006/2007 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 319

319 0 1 2 3 4 5 6 7 8 9 10 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 50. Effect of field water stress and growth regulators treatments on valencene of Valencia orange peel oil during the 2006/2007 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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320 0 400 800 1200 1600 Jan Mar May Jul Harvest date Peak area (millions) CONTROL 2005/06 TYVEK 2005/06 CONTROL 2006/07 TYVEK 2006/07 Figure 6 -pinene of Valencia orange peel oil during the 2005/06 and 2006/07 seasons. Values are mean SE.

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321 0 400 800 1200 1600 Jan Mar May Jul Harvest date Peak area (millions) CONTROL 2005/06 TYVEK 2005/06 CONTROL 2006/07 TYVEK 2006/07 Figure 6 52. Effect of soil coverage with Tyvek on myrcene of Valencia orange peel oil during the 2005/06 and 2006/07 seasons. Values are mean SE.

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322 -10 0 10 20 30 40 50 60 Jan Mar May Jul Harvest date Peak area (millions) CONTROL 2005/06 TYVEK 2005/06 CONTROL 2006/07 TYVEK 2006/07 Figure 6 53. Effect of soil coverage with Tyvek on valencene of Valencia orange peel oil during the 2005/06 and 2006/07 seasons. Values are me an SE.

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323 0 50 100 150 200 250 300 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 -pinene of water stress and growth regulators treated Marsh grapefruit during storage at 70oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

PAGE 324

324 0 200 400 600 800 1000 1200 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 55. Changes in myrcene of water stress and growth regulators treated Marsh grapefruit during storage at 70oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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325 0 20 40 60 80 100 120 140 160 180 200 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 -pinene of water stress and growth regulators treated Marsh grapefruit during storage at 40oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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326 0 50 100 150 200 250 300 350 400 450 500 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 57. Changes in myrcene of water stress and growth regulators treated Marsh grapefruit during storage at 40oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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327 0 50 100 150 200 250 300 350 400 450 500 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 -pinene of water stress and growth regulators treated Valencia oranges during storage at 40oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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328 0 100 200 300 400 500 600 700 800 900 1000 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 59. Changes in myrcene of water stress and growth regulators treated Valencia oranges during storage at 40oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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329 0 5 10 15 20 25 30 35 40 45 50 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 60. Changes in valencene of water stress and growth regulators treated Valencia oranges during storage at 40oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*G R; water stress & growth regulators. Values are mean SE.

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330 0 100 200 300 400 500 600 700 800 900 1000 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 -pinene of water stress and growth regulators treated Valencia oranges during storage at 70oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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331 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 62. Changes in myrcene of water stress and growth regulators treated Valencia oranges during storage at 70oF for 12 weeks during the 2005/2006 season. WS; wate r stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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332 0 50 100 150 200 250 300 350 400 Jan Mar May Jul Harvest date Peak area (millions) CONTROL WS GR WS*GR Figure 6 63. Changes in valencene of water stress and growth regulators treated Valencia oranges during storage at 70oF for 12 weeks during the 2005/2006 season. WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. Values are mean SE.

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333 0 100 200 300 400 500 600 700 800 900 1000 Jan Mar May Jul Harvest date Peak area (millions) CONTROL @ 40F TYVEK @ 40F CONTROL @ 70F TYVEK @ 70F Figure 6 -pinene of Valencia orange peel during storage at 40oF and 70oF for 12 weeks during the 2005/2006 season. Values are mean SE.

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334 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 Jan Mar May Jul Harvest date Peak area (millions) CONTROL @ 40F TYVEK @ 40F CONTROL @ 70F TYVEK @ 70F Figure 6 65. Effect of soil coverage with Tyvek on myrcene of Valencia orange peel during storage at 40oF and 70oF for 12 weeks during the 2005/2006 season. Values are mean SE.

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335 0 30 60 90 120 150 Jan Mar May Jul Harvest date Peak area (millions) CONTROL @ 40F TYVEK @ 40F CONTROL @ 70F TYVEK @ 70F Figure 6 66. Effect of soil coverage with Tyvek on valencene of Valencia orange peel during storage at 40oF and 70oF for 12 weeks during the 2005/2006 season. Values are mean SE.

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336 CHAPTER 7 CHARACTERIZING CITRU S PEEL MATURATION AN D HANDLING PROBLEMS MULTIVARIATE AND REGRESSION ANALY SES A unique aspect of citrus is that the maturity and quality of the peel are likely distinctly independent from maturity and quality of the internal edible portions. Further, the edible quality is a human perception and may not be ti ghtly linked to the physiological maturity of the peel External peel characterization may or may not follow internal grade standards of the pulp. It is essential to make sure that citrus fruit meet the internal maturity standards when harvested to meet re gulatory requirements ; all fruit must be mature, of similar varietal characteristics and be free from bruises, cuts not healed, decay, growth cracks and insect larvae (Taylor et al., 1994) but it is also important to characterize peel matur ity to determine the peels susceptibility to physical, physiological, or pathological injury during harvesting, postharvest handling storage and marketing. Understanding peel maturity can facilitate harvest at the optimum time and allow adjustment of harvest and postharvest procedures to minimize fruit losses during times of vulnerable peel condition. R esults of previous chapters show ed that the suggested harvest window for Marsh grapefruit to avoid storage problems is January March, and for Valencia orange is March May Currently, fresh citrus harvest begins early in the season based on internal quality (edibility) and minimum external color for fresh market Fruit harvesting for fresh use stops when internal ratio of TS S: acid ( e.g. insipid ly sweet orange fruit due to acid drop to 0.4% ) and market condition problems become high. Peel changes have not been related to optimal harvest time. Several physical parameters and chemical parameters were measured at several harve st dates over the season to determine if peel maturation and senescence can be monitored by some combination of factors in order to minimize fruit disorders resulting from immature or senescent peel or unusual stress levels that the peel may have been subj ected to. Some factors (water stress

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337 and growth regulators) were introduced to induce differences in peel condition or maturity (advance or delay, respectively) to test if any physical or chemical measurements showed corresponding changes in amount or rate so that they could be used to indicate stage of peel development (immature, mature, or senescent). For the purposes of these studies, peel maturity is defined as the physiological state of the peel that relates to maximum peel health and quality retentio n during common commercial handling, storage and marketing conditions. Thus, a mature peel would be expected to maintain peel quality longest after harvest, whereas fruit with immature or senescent peel would develop decay or peel disorders much soo ner. In the current studies, fruit weight loss and the development of decay or chilling injury (CI) after harvest common factors that limit the marketable life of citrus fruit were chosen as indicators of peel maturity. This work hypothized that f ruit we ight loss, and the development of decay and chilling injury after harvest common factors that limit the marketable life of citrus fruit, were chosen as indicators of peel maturity and the goal was to find a combination of peel physical (color, peel turgi dity and fruit detachment force) and chemical (peel sugars, glycosidases, abscisic acid and volatile components) measurements that as they changed indicated the maturing and senescing process of the peel The hypothesis of this work was that as harvest dat es advance, citrus fruit peel will change from immature to mature to senescent, and peel physical, metabolic, or enzymatic changes will reflect th ese changes The second goal was to study some factors or condition such as water stress and growth regulators that might affect citrus peel maturity, which in turn might affect storability of citrus fruits. The hypothesis was that the use of growth regulators and/or water stress in the modeling of citrus peel maturation would presumably alter peel development or aging and be reflected in the various measurements made over the harvest season

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338 that provide useful information in assessing peel development. These treatments are not normally applied to induce maturation changes and could lead to results that may agree or disagree with previous findings. To achieve these two goals, the current study utilized multivariate analyses; principal component analysis (PCA) and multiple stepwise regression analysis (MSR) comparing post storage problems such as weight loss, decay and chilling injury to peel physical and chemical measurements. These comparisons were done to understand variations in two citrus cultivars, in regards to harvest times and treatments, so that a broad picture of peel maturity and senescence could be obta ined to discuss the optimal harvest time that minimize fruit loss and extend shelf life. For each cultivar, PCA was run for all data sets in individual and in combined years to see if any difference in data trends occurred within and among seasons. The MSR was done for each one of the postharvest peel characteristics (dependent variables weight loss, decay and chilling injury) versus other physical and chemical characteristics (independent variables) to find any relationships. Simple regression (SR) analy sis and correlation matrices were also created for grapefruit data, orange data and the combined data of both species to find relationships between post storage problems and any of the physical or chemical measurements that might be used as peel maturity i ndex in both species. Materials and Methods Data for the PCA, MSR and SR were obtained from field and storage experiments on both Marsh grapefruit in t wo seasons (2004/2005 and 2005 /200 6 ) and Valencia orange in the 2005/2006 season chapter 2, pages 69 75. Fruit were stored at two different temperatures; 40oF (4.5oC) and 70oF (2 0oC), as described in chapter 2, page 76. A t harvest the following analyses were done as described in chapter 2, pages 7677; TSS: acid ratio, peel color, peel peel turgidity fr uit detachment force (FDF), and postharvest peel characteristics ( weight loss, decay and

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339 chilling injury ) were determined after 12 weeks storage At e ach harvest date, flavedo and albedo tissues were prepared and stored, as described in chapter 3, page 139 until chemical analyses, which include d extraction and determination of soluble sugars, chapter 3, pages 139 -mannosidase, chapter 4, pages 169 172 and ABA, chapter 5, pages 188189. V olatile components were extracted and measu red as described in chapter 6, pages 204 205. Principle component analysis (PCA) was done, using Unscrambler X, version 10.1, multivariate data analysis software, CAMO Software, Inc., Woodbridge, NJ, USA, for all physical variables (weight loss, decay, chi lling injury, color, peel turgidity detachment force) and chemical variables (peel sugar, glycosidases, ABA and volatile components, and juice TSS: acid ratio) with the control and all treatments (water stress, growth regulators and the combined treatment ) to see if there were any trend s in the data and if there were any difference among early, mid and late season data PCA was also run for individual year and combined years. Scores plot show the the trend and the grouping of similar data point (harvest date treatment) and loadings plot show bloom date and all physical and chemical variables. Specific variables in loading plot will best fit with specific groups in scores plot based on the weight or loading value of the variable to a specific group. Mul tiple stepwise regression (MSR) analyses, simple regression (SR) analyses and correlation matrices were run, using SAS 9 .2 (Statistical Analysis System) ( SAS Institute Inc., 2008). MSR was run for each one of the postharvest peel physical characteristics; weight loss, decay, chilling injury versus other physical characteristics; color, peel turgidity and detachment force and chemical parameters; sugars, glycosidases, ABA and volatile components and juice TSS: acid ratio SR was also run for each one of the postharvest peel physical characteristics versus same physical and chemical characteristics, except glycosidases and ABA, nootkatone,

PAGE 340

340 aldehydes and valencene. Differences in R2 between significant variables were evaluated based on how much amount of variance the variable is accounted for out of the total variance MSR w as run for individual year and combined years. SR was run for grapefruit data, orange data and the combined data of both species. All PCA M SR and SR were run with and without volatile components, since there were only two replicates of the volatile components, whereas the other variables allowed use of three replicates. Results and Discussion Marsh Grapefruit at 40oF (4.5oC) Results of pre vious chapters show ed that grapefruit optimal harvest dates are during mid season to ensure good juice quality ( TSS: acid 7:1) and minimum post storage problems S uggested storage temperature for Marsh grapefruit harvested during this window of of January March should be stored at 40oF to reduce storage problems During this mid-season period, moderate weight loss (2.6 2 .7 %) and decay (2.2 9.2%) and least chilling injury (27.5 28%) were found after 12 weeks of storage, since most of the storage problems are more related to early and late harvest; periods that presumably represent immature and senescent peel respective ly This optimal harvest period was also associated with good juice TSS: acid ratio at harvest ranged between 8.2 8.5 from January to March. To obtain a broader picture about grapefruit peel maturation required for optimal harvest dates selected physic al characteristics that represent postharvest problems and limit the marketable life of the fruit such as weight loss, decay and chilling injury were analyzed with other physical and chemical variables using PCA so that trends and covariance could be disc erned. Score for each data point (harvest date treatment) as well as variables that were used to create data trends for the first season (2004/2005) are represented in Figure 7 1, A and B,

PAGE 341

341 respectively. A combined view of both scores and loadings plots i s represented in Figure B 7 Appendix. The first two components of this PCA explained 48% of the total variance, with the first and second PC accounting for 34% and 14%, respectively. The low variance (48%) resulted from the low degree of clustering and ma y also be due to the overlap between samples from early season and mid -season, as well as between mid -season and late season samples (Fig. 7 1, A) H owever, away from harvest date overlap areas, the observed proximity of some given samples can explain the ir load for specific variables, for instance in Figure 7 1, were three separated clusters consisting of a cluster for early season fruit harvested in September and October (4SC and 4OC) that includes fruit with the highest -galactosidase in both flavedo a nd albedo, compared to the second cluster of mid -season samples harvested in March (5MrC) that have the highest color index, sucrose content in both flavedo and albedo flavedo reducing sugars and albedo ABA The third cluster wa s for fruit harvested late season between April and May (5AC and 5MyC) that have the highest scores on PC 1 and PC 2, and was differentiated by albedo reducing sugars, weight loss decay TSS: acid ratio and days from bloom date Also, this third cluster has the lowest score in PC 1 and PC 2 in the dimension of FDF, since it is located on the opposite side of this cluster. Among the three groups, harvest dates were not separated with respect to the rest of physical or chemical variables. This is probably because the other factors had very little variation in values throughout the harvest season. Results of MSR (Table 7 1) showed that percentage weight loss was significantly related to detachment force, and this relationship was negative with FDF which accounted for 43 % of the varian ce in weight loss value. Percentage decay was significantly related to 2 variables accounting for 56% of the variance. Flavedo -mannosidase accounted for the majority ( 41% ) of the variance (but can not be related to any of harvest dates since it is projec ted toward the center

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34 2 of PCA plot, as shown in Figure 7 1, and this was also confirmed before in Table 4 1 and Fig. 4 1 that showed insignificant fluctuation of flavedo -mannosidase throughout the season) and albedo ABA related to another 15% of the varia nce in decay. The total increase in peel ABA throughout the season, especially ABA of the flavedo (Table 5 1) was associated with peel senescence and the conversion of chloroplasts to chromoplasts (Harris and Dugger, 1986; Martinez -Romero et al., 1999; Nooden, 1988), and this increase in ABA was a ssociated with the increase in fruit decay with the progress of the harvest season (Table 211), but the low amount of variance (15%) did not make albedo ABA as a good harvest predictor. However, flavedo mannosidase and albedo ABA are related to 56% of t he variation, therefore their combination has potential, but neither could easily be measured for a commercial test for maturity. Chilling injury was not significantly related to any variable measured at harvest (Table 7 1). Less than half the variance ( 45%) also was accounted for from the first two components of PCA of the second season data (2005/2006) (Fig. 72, A and B) but less overlap was found among early, mid and late season samples (Fig. 7 2, A). Early harvested fruit in September (5SC) was differentiated by flavedo -galactosidase. Mid -season harvested fruit in January and March were differentiated by ABA level of flavedo and albedo, flavedo reducing sugars, albedo mannosidase and firmness. Fruit harvested in March have the highest values of f lavedo reducing sugars and showed the lowest score in PC 1 and PC 2 in dimension of chilling injury since they are located on the opposite side of this cluster L ate harvested fruit in May and July were differentiated by post storage variables ( weight los s decay and chilling injury ) plus other variables measured at harvest such as; flavedo sucrose, albedo sucrose, albedo reducing sugars, peel color, juice TSS: acid ratio and days from bloom date (Fig. 7 2, A and B). Fruit harvested in

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343 July showed the high est weight loss and decay, and decay was positively co related to days from bloom date ; the latest the harvest date from bloom date, the higher % decay. MSR results (Table 7 1) confirm PCA results showing that % decay was positively related to bloom date w hich related to 41% of the total variance of decay values Table 2 11 showed that decay increased gradually with the progression of the season. Chilling injury was significantly correlated with both weight loss and decay (Table 7 1) since injured fruit are more susceptible to decay and water loss (Cohen et al., 1990), however these post storage variable are not useful as a harvest predictor of handling ability The incidence of chilling injury wa s nega tively correlated w ith flavedo reducing sugars content that related to 31% of the total variance in chilling injury values (Table 7 1) High levels of reducing sugars in grapefruit peel are considered to indicate greater resistance to chilling injury in grapefruit peel at mi dseason (FebruaryMarch in Florida) (Purvis and Grierson, 1982; Purvis et al., 1979). Means comparison by DMRT showed that chilling injury changed significantly during the 2005/2006 se ason; it was high in early season harvests (November (65.78%)), and low in mid-season (March (26.67%)), then increased again in late season harvests (July (52.41%)) (Table 2 13 and Figure 2 25) This was associated with significant increase in reducing sug ars from November (39.33 g /mg D.W. ) to March (195.06 g /mg D.W. ) followed by a significant reduction in July (109.23 g /mg D.W. ) (Table 3 1 and Fig. 3 2). The reciprocal significant relationship among weight loss, decay and chilling injury (decay & chi lling injury, chilling injury & decay, and weight loss & chilling injury) and the significant relationship between decay and the interval between bloom and harvest dates (Table 7 1) indicat e that days between bloom date and harvest date may be use ful as a way to determine the level of peel maturity Maturity of the peel relates to its susceptibility to postharvest problems C onsidering the period of less chilling injury during mid-season as a period of

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344 mature peel therefore the two periods of high susce ptibility to chilling injury; early and late season can be viewed as periods of immature and senescent peel, respectively. This can be done by counting days from bloom to the beginning of early harvest where chilling injury is high in November (234 days) then decrease by mid -season period in March (352 days) and same when chilling injury start to increase again in July (474 days) with late season harvest. This suggest s that harvest could be in the period of minimum chilling injury (Table 2 13) from De cember (264 days from bloom date) to April (382 days from bloom date) (Table A 6 Appendix) Increase in weight loss and decay with the progress of harvest season, as well as the susceptibility of early season and late season fruit to chilling injury have been previously reported by Coggins et al. (Coggins et al., 1969a ) and Ritenour et al. (Ritenour et al., 2003) Combination of two seasons data in Figure 7 3 reduced the explained variance in PCA analysis to 41% and increased the overlap among sampling times (Fig. 7 3, A) Early harvested fruit in September and October (4SC and 4OC) were differentia ted by flavedo galactosidase. Mid -season harvested fruit overlapped between early and late -harvests, and were not differentiated by any variable. Late harvested fruit in July was differentiated by flavedo reducing sugars and days from bloom date. C ombining two seasons MSR (Table 7 1) showed that g rapefruit % weight loss at 4 0oF was significantly related to 2 variables measured at harvest ; TSS: acid ratio and albedo sucrose, which related to 34% and 4% of the weight loss variation respectively TSS: acid ratio is a maturity index for the pulp, and the low variance accounted for by albedo sucrose does not make it a good candidate to measure peel maturity as defined by weight loss Percentage decay was significantly related to 3 variables measured at harvest; albedo ABA 16%, FDF 8% and flavedo sucrose 3% of the variance, a combined 27 % of the variance. The combined seasons results were almost the same for variance accounted for by

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345 albedo ABA (16% compared to 15 % in the 2004/2005 season).Again, this low percentage of v ariance did not make albedo ABA a good candidate to predict harvest. When volatile components were incorporated into the variables matrix and the number of replicates decreased from three to two replicates (Figs. 7 4 to 76), less overlap among sampling p eriods resulted and the total variance explained by the PC 1 and PC 2 increased to 52% for the 2004/2005 season (Fig. 7 4) and 53% for the 2005/2006 season (Fig. 75) H owever, the total variance remained the same, 41% for the combined seasons (Fig. 7 6). This addition of volatile components increased the total number of significant variables in MSR of both seasons. FDF was now significant along with days from bloom date and flavedo reducing sugars for the post storage variables (weight loss, decay and chil ling injury, respectively) in two seasons rather than one season (Table 7 2). Figure 7 4 shows that during 2004/2005 season, early harvested fruit in September and October (4SC and 4OC) were differentiated using flavedo and albedo galactosidase. Mid sea son samples (5FC and 5MrC) were the highest in albedo sucrose and reducing sugars and all volatile components except nootkatone. Late season samples (5AC and 5MyC) appeared to be differentiated by the amount of sucrose and reducing sugars of the flavedo, c olor, nootkatone, juice TSS: acid ratio and time from bloom date, albedo ABA, % weight loss and % decay. The late season samples had a negative relationship with FDF since they were located diagonally in two different quadrant of the PCA plot. MSR (Table 7 2) showed that during the 2004/2005 season, g rapefruit % weight loss at 4 0oF was significantly related to 2 variables that accounted for 49 % of the variance in weight loss values. FDF accounted for most of the multiple linear regression equation (30 % ), and confirm the negative correlation of the PCA Flavedo reducing sugars accounted for an other 19

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346 % of variation Percentage decay was significantly related to 4 variables accounting for 82% of the variance. Days from bloom date measured at harvest account ed for most of the variance (55%), and TSS: acid ratio, geranyl acetate and flavedo sucrose accounted for another 27% of the total variance. Percentage chilling injury at 4 0oF was significantly related to 5 variables accounted for 92 % of the variance in c hilling injury values in the following order: Albedo ABA 25%, nootkatone 22%, flavedo reducing sugars 24%, myrcene 11% and albedo sucrose 10%. Second season data (Fig. 7 5) showed that early harvested fruit could not be differentiated by any variable. Mid -season samples overlapped with early and late harvested samples, and samples harvested in January were differentiated by albedo -mannosidase, linalool and myrcene. Late harvested samples were differentiated by juice TSS: acid ratio, nootkatone, geranyl a cetate, flavedo sucrose, albedo reducing sugars, days from bloom date, weight loss, decay and chilling injury. MSR (Table 7 2) showed that weight loss was significantly related to 2 variables; FDF accounted for the most variance (41%) and linalool added 8 % to the variance. FDF was significantly related with weight loss in both seasons, but the amount of variance that it accounted for out of the total variance was moderate (30% and 41% in the first and second season, respectively), but still might be a usef ul variable as a harvest predictor, except it related negatively to weight loss in the first season and positively in the second season (Fig. 7 4 and 7 5, and Table 7 2). Percentage decay was only significantly correlated with days from bloom date, which accounted for 40% of the total variance (Table 7 2). Percentage decay increased significantly toward the end of the season as shown in Table 211. Days from bloom date are quick and easy to calculate for predicting harvest date. They accounted for 55% and 40% of the total variance of

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347 decay values during the first and second season, respectively. This amount of variance accounted for is less than half the total, but the relationship might improve with additional tests over more than two seasons. The interval from bloom date to harvest may be a good measurement to predict acceptable harvest dates, particularly for stopping late harvest, as it is easy to measure compared to most chemical and many physical peel measurements. Chilling injury was significantly re lated to flavedo reducing sugars accounting for 33% of the variance, and peel color accounted for another 11% of the total variance (Table 7 2). The relationship between % chilling injured fruit and the level of flavedo reducing sugars in the 2004/2005 sea son (Fig. 7 4) and 2005/2006 season (Fig. 7 5) was also interesting because this relationship shows more susceptibility to chilling injuries with the beginning of the increase in flavedo reducing sugars which preceded color change (Fidelibus et al., 2008; Pourtau et al., 2006; Price et al., 2004; Rolland et al., 2006) For two seasons, significant increases in flavedo sucrose from September (0.00 & 6.55 g/mg D.W ) to November (37.24 & 26.80 g /mg D.W. ) and reducing sugars from September (17.01 & 32.79 g/mg D.W ) to November (45.33 & 39.33 g /mg D.W. ) (Table 3 1) were associated with a significant increase in color index from September ( 9.47 & 10.85) to November ( 5.92 & 3.33) (Table 2 3) and significant increases in chilling injury from September (12.41% & 43.33%) to November (41.85% & 65.78%) (Table 213) during the first and second seasons, respectively, as reported in Chapter 2. MSR showed that the relationship between chilling injury and flavedo reducing sugar s was significant in both seasons, but the relationship with color change was significant only during the 2005/2006 season, and the combined seasons (Table 7 2). These results agree with a previous finding by Kawada (1980) who found that immature grapefruits were more resistant to CI th an those har vested after color break in November as reported by Sauls (1998) and shown in chapter 2

PAGE 348

348 (Table 3 2 and Fig. 3 2) Also, Lafuente et al. (1997) reported that chlorophyll increases chilling injury resistance for early harvested fruit (September and October, Table 2 13 and Fig. 2 25). It was previously reported tha t chilling injury is related to oxidative stress (Haray adi and Punkin, 1991; Wise and Taylor, 1987) and that chlorophyll demonstrated a protect ive role in plants against oxidat ive damage ( Larson, 1988). These data show that to some extent under subtropical condition, like Florida, peel color index may be used as a quick and easy tool t o determine the level of peel maturity, however it would not be a very accurate tool because color change is temperature dependent (Young and Erickson, 1961), but at least as previously reported it coincides with the beginning of peel maturity (Erickson, 1960) from the chilling injury perspective that increased in November then decreased during mid-season (Table 2 13). Also, comparing these results with previously mentioned result s about the role of flavedo reducing sugars in chilling injury resistance for the mid -season fruit may reflect that changes in level of flavedo reducing sugars from September (32.79 g/mg D.W ) to November (39.33 g /mg D.W. ) may not be enough to resist chi lling injury but the beginning of increase in reducing sugars by January (89.19 g/mg D.W ) followed by the tremendous change in March (195.06 g /mg D.W. ) (Table 3 1) increased peel resistance during January March period (Table 2 13). This change in leve l of flavedo reducing sugars may make them good candidate as harvest predictor of grapefruit, especially with the moderate amount of variance (33%) that flavedo reducing sugars contribute to the regression equation in both seasons. The combined seasons plot (Fig. 7 6) confirms the results of each season and showed the possibility of differentiating late season samples for both seasons by the same variables. Table 7 2 showed that grapefruit % weight loss at 4 0oF was significantly related to 2 variables measu red at harvest ; juice TSS: acid ratio and peel color accounted for 34% and 6% of the variance in

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349 weight loss values, respectively. Percentage decay was significantly related to flavedo sucrose confirming the first season result, and accounted for 26% of the variance. As in the second season results, chilling injury was significantly related to peel color but accounted for only 7% of the variance. F ruit become less firm with maturation, and senescing peel has an albedo layer with small cells of low cytoplas mic content, low metabolic activity, larger intercellular spaces and weakened cell wall, which break easily ( Coggins, 1969b ). Firmness could be a good candidate to represent fruit maturation and reduction of shelf life, however in this study figures 7 1, 7 2, 7 4 and 7 6 show that in PCA, firmness separated samples in the overlap area between mid and late season harvested fruit, but it was not related statistically to these samples because it had low variance values in either PC 1 or PC 2 Moreover, the MSR did not show any significant relationship between firmness and any postharvest variables (Ta ble 7 1 and 72). Volatile components, such as nootkatone, myrcene and geranyl acetate showed significant relationships with postharvest variables only in one season but may be good candidates to identify peel maturity, if the one positive result were conf irmed by results in a more substantial study In general, results of individual years and combined years showed that FDF, interval between bloom and harvest date and flavedo reducing sugars were three good candidates to predict acceptable harvest periods for storage at 40 oC. Days from bloom date and FDF could be a quick practical method compared to measuring reducing sugars, but using FDF requires more data from more than two seasons and testing fruit from different location on the tree which probably va ry in FDF. Results of the 2005/2006 and 2006/2007 seasons in chapter 2 showed that FDF respectively decreased significantly from September (10.59 Kg & 12.65 Kg) to November (7.73 Kg & 9.59 Kg), and remained the same until January (7.71 Kg & 9.29 Kg) and de creased

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350 significantly in May (3.89 Kg & 6.29 Kg) (Table 2 7). This may indicate that November may be a transition period from immaturity to maturity and March may be the transi t ion period from maturity to senescence of the fruit. This reduction in FDF afte r November was associated with moderate weight loss (Table 2 9) and low chilling injury (Table 2 13) during storage after November until spring when decay (Table 2 11) and weight loss increased. Average of the 2004/2005 and 2005/2006 seasons data from chapter 2 showed that fruit harvested in early season (September October period) showed fairly high % weight loss (3.48%) that decreased gradually until it was 2.36% in the December January period, when it starts to increase again to 3.65% in May (Table 2 9). Decay increased gradually with the progress of the season; it was 1.48% in September, then 2.23% in January and 6.48% in May. Chilling injury was high in early season harvests, November, (40.4%), and low for mid-season in February (26.8%), then increas ed again with late season harvest in May (32.6%) (Table 2 13 and Figure 225). Results of FDF showed continous decreasing with the progress of the season, which might be hard to differentiate the three different stages of peel development (immaturity, matu rity and senescence), and chilling injury showed the same trend from early season to mid-season, but it increased again lates season, so both variables fit only at 2 points of the season, wich can be used to differentiate the beginning of the third period of peel development, senescene based on the increase in chilling injury. According to the results of FDF, reducing sugars and post storage variables for the period of this study, harvest season should start sometime after November at 263276 days from blo om date (sometime in December) (Table A 6 Appendix) where moderate weight loss and decay and least chilling injury found, and the cut -off point of harvested Marsh grapefruit to be stored at 40oF should not be later than late March at 372377 days from bloom date (average of two

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351 seasons, Table A 6 Appendix), or possibly early April (382384 days from bloom date, Table A 6 Appendix) since weight loss, decay and chilling injury were more related to May and July harvested fruit that may represent the peri od of senescent peel. The start date of harvest in December was at FDF = 8.7 Kg (average of two seasons, Table 2 7) and flavedo reducing sugar content = 42.3 g/mg D.W (average of two seasons, Table 3 1) and the cut -off date was at FDF = 6.4 Kg (average of two seasons, Table 2 7) and flavedo reducing sugar content = 146.7 g/mg D.W (average of two seasons, Table 3 1). Marsh Grapefruit at 70oF (21oC) Since no chilling injury occurs at 70oF only weight loss and decay were postharvest problems and are dis cussed in relation to other physical and chemical variables measured at harvest. Collectively, r esults of previous chapters show ed that the suggested harvest window of Marsh grapefruit to be stored at 70oF was a period from December to March with moderat e incidence of weight loss ( 5.5%) and decay (28.4%) after 12 weeks of storage associated with good juice quality (average TSS: acid ratio was 8.0 in December and 8.6 in March) at harvest. W eight loss and decay were analyzed with other physical and chemic al variables using PCA and MSR. In PCA, the first two components of the PCA plot for the 2004/2005 season explained 50% of the total variance, with the first and second PCs accounting for 37% and 13%, respectively (Fig. 7 7). Like fruit stored at 4 0oF fr uit harvested early in September and October (4SC and 4OC) for storage at 7 0oF had the highest -galactosidase for both flavedo and albedo tissues. These were the only two variables that differentiated this early harvest group. High galactosidase activity at the first sampling date, September in both flavedo and albedo tissues w as report ed in cha pter 4 for all three seasons (Table 4 1 and Figs. 4 1, 4 2 and 4 3 ). galactosidase has been associated with actively growing tissue, where high levels of this enzyme

PAGE 352

352 activity have been correlated with cell wall loosening mechanisms responsible for growth (Labrador and Nicolas, 1984). These 4SC and 4OC samples showed low values of PC1 and PC 2 in dimension of flavedo ABA since they are located diagonally in two different quadrant, however flavedo ABA was not particularly related to e arly harvested fruit in December (4DC) or mid -season harvested fruit in January (5JaC) since it was projected toward the center of the PCA plot. Mid -season samples (harvested in January March period) were scattered and overlapped with early and late seas on samples. February samples (5FC) had the highest albedo reducing sugars and March samples (5MrC) were high in flavedo sucrose. Late season fruit harvested in April and May (5AC and 5MyC) of the 2004/2005 season were differentiated by days from bloom date flavedo reducing sugar and decay (Fig. 7 7). Data analysis using MSR (Table 7 3) showed that weight loss was significantly related to 3 variables measured at harvest during the 2004/2005 season and these variables accounted for 62% of the variation in w eight loss with flavedo sucrose associated with most of the variance (41%), flavedo -galactosidase accounted for 13% and flavedo ABA accounted for 8%. Decay was significantly related to the interval between bloom and harvest dates which related to 41% of the variation in decay values. Although overlap among the three harvesting periods (early, mid or late harvests) was less during the 2005/2006 season, the explained variance by the two PCA components was less (45%) (Fig.7 8). Early season samples in September and November (5SC & 5NC) could not be compared with respect to flavedo or albed o -galactosidase Mid -season harvested fruit in January were differentiated by flavedo and albedo content of -galactosidase and -mannosidase. However, Table 4 1 shows no significant differences in glycosidases levels between fruit harvested in November and January of the 2005/2006 season. Mid-season samples harvested in

PAGE 353

353 March were comparable with respect to firmness and the high level of flavedo and albedo ABA compared to other harvest dates. Late season harvested fruit in May were differentiated by flaved o reducing sugars and fruit harvested in July were differentiated by flavedo sucrose, juice TSS: acid ratio, time from bloom date, FDF, weight loss and decay (Figs. 7 -8 and B 14, appendix). Results of MSR of the 2005/2006 season (Table 7 3) showed that wei ght loss was significantly related to 3 variables accounting for 59% of the total variance of weight loss values. These variables were flavedo reducing sugars accounting for 37%, albedo ABA for 12% and flavedo sucrose accounting for 10% (total = 59%). Deca y was significantly related to flavedo sucrose that accounted for 12% of the variance, FDF accounted for 8%, firmness accounted for 7% and flavedo -galactosidase accounted for 6% (total = 33%). The negative relationship between decay and firmness are in agreement with previous reports; as fruit become older, susceptibility to postharvest diseases increases because peel becomes less firm and hence le ss force is required by the pathogens to puncture the peel (Coggins et al., 1969a ). The PCA reduced the amount of variance for the com bined season data matrix to 41% (Fig. 7 9) and showed that early harvested fruit in September (4SC & 5SC), October (4OC) and November (5NC) were not differentiated by any variables. Flavedo -mannosidase differentiates mid -season harvested fruit in January, and firmness and albedo reducing sugars differentiated fruit harvested in March. Late season harvested fruit, April, May and July, were differentiated using juice TSS: acid ratio, ABA level in flavedo and albedo, peel color and days from bloom. Percentag e weight loss was projected toward the center of the PCA plot, and decay was located in the overlap area between mid and late season harvested fruit. In spite of weak PCA relationships, MSR analysis (Table 7 3) showed that weight loss was significantly co rrelated to 4

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354 variables measured at harvest and accounted for 62% of the total variance of weight loss values. Flavedo sucrose and reducing sugars accounted for most of this variance (55%), and albedo sucrose and flavedo -galactosidase accounted for 4% an d 3%, respectively. Decay was significantly correlated to TSS: acid ratio and FDF, which accounted for 28% and 9% of the variance, respectively (Table 7 3). This overview of PCA and MSR results showed that % weight loss after 12 weeks of storage during the 2004/2005 season was significantly related to flavedo galactosidase that were initially high in September (0. 213 units/g DW) (Table 4 1) and probably represent s the beginning of stage 3 of fruit growth, that is characterized by cession of cell division in the flavedo epidermis, as the fruit approaches to ma turity (Bain, 1958; Monse lise, 1986) The level of enzyme started to decrease after September (0. 213 units/g DW) with a significant reduction by November (0. 108 units/g DW) (Table 4 1) associated with the beginning of color break (significant change in color index from September [ 9.47] to November [ 2.40] (Table 2 3) and reduction of whole fruit firmness (significant change in peel turgidity from September [4.45Kg] to November [4.01Kg] (Table 2 5) that may represent the beginning of stage 3 of fruit development. After the drop in enzyme level in November, the level of enzyme became more or less constant with the progress of harvest dates until a significant drop in activity again in the March sample (0.042 units / g DW) Data for the combined seasons had the same trend of signific ant reduction in flavedo galactosidase level from September (0.159 units/g DW) to November (0.102 units/g DW) and another significant drop in March (0.025 units/g DW) (Table 4 1). In both the 2004/2005 and 2005/2006 seasons, changes in color index from September ( 9.47 & 10.85) to November ( 2.40 & 3.33) (Table 2 3) were also preceded by significant increases in flavedo sucrose from September (0.00 & 6.55 g/mg D.W ) to November (37.24 &

PAGE 355

355 26.80 g /mg D.W. ) and reducing sugars from September (17.01 & 32.79 g/mg D.W ) to November (45.33 & 39.33 g /mg D.W. ) of both seasons (Table 3 1). The total increase in peel ABA throughout the season, especially ABA of the flavedo (Table 5 1) reportedly is associated with peel senescence and the conversion of chloroplasts to chromoplasts (Harris and Dugger, 1986; Martinez Romero et al., 1999; Nooden, 1988). Flavedo ABA level increased during the 2004/2005 season from 5.76 mg/g DW in September to 6.62 mg/g DW in November, with a significant increase in albedo ABA during the 2005/2006 season from September (0.36 mg/g DW) to November (3.83 mg/g DW) which was also associated with a significant increase in flavedo ABA from September (1.30 mg/g DW) to November (5.48 mg/g DW) The significant results between FDF and decay in the second season and combined season data (Table 7 3) may make FDF a candidate to determine the level of peel maturity and predict good harvest dates for storage. FDF decreased significantly from September (10.59 Kg) to November (7.73 Kg) of the 2005/2006 season (Table 2 7), but the low variance accounted for by FDF (Table 7 3) may requires more work on detachment force with more fruit samples and more than two seasons to prove its usefulness as a peel maturity index and harvest predictor. Use might still be limited because of the narrow range of detachment forces found, the limit of minimum detachment needs more investigation and FDF is affected by several factors, such as tree moisture level Less force wa s required to remove oranges following rains and early in the morning when the fruit wa s in a turgid condition ( Coppock, 1961). Detachment force and percent age plugged Marsh grapefruit decreased with greater detachment angles (Coppock et al., 1 969) Also, FDF of Navel orange increased with increasing fruit size and larger stem/peduncle diameter (Hield et al., 1967; Kender and Hartmond, 1999). At early maturity citrus fruit are firmly attached to the stem, and FDF is extremely variable within the tree

PAGE 356

356 generally being higher in the top and exterior parts of the canopy where fruit are more exposed to the sun and develop stronger stems ( Kender and Hartmond, 1999). In general, wounding or mechanical injuries of the peel reduce FDF due to triggering of internal ethylene which initiat es formation of the abscission zone. The closer the location of the wound to the calyx abscission zone, the larger the reduction in FDF (Kostenyuk and Burns, 2004) Fruit detachment force is positively correlated with the ratio of endogenous IAA to ABA or endogenous IAA, but negatively to endogenous ABA in the fruit abscission zone (Rasmussen, 1973; Yuan et al., 2001) This relationship between endogenous hormones may give some indication about using water stress and/or grow th regulators to adjust the internal level of ABA, which can be used as an indicator of peel maturity, especially with the second season data (Fig. 7 -8) where mid -season samples from WS GR and the combined treatments (6MrW, 6MrG and 6MrWG) and control (6M rC) were identified by flavedo and albedo ABA levels. MSR show ed a significant and negative relationship between weight loss and flavedo ABA in 2004/2005 season and albedo ABA in 2005/2006 season (Table 7 3) However one seasons results are not enough to suggest using ABA as an indicator of peel maturity especially since there were no significant differences in albedo ABA among all treatments and the control in March of 2005/2006 seasons (Table 5 2); perhaps because March is still in the harvest window of grapefruit, and might not be a critical month for ABA level in the fruit. Table 5 2 also showed that in May, Flavedo ABA decreased significantly in WS*GR treatment (5.09 mg/g DW ) compared to the control (6.95 mg/g DW ). This may be the result of WS increas ing the level of ABA (Norman et al., 1990), and may be associated with WS effect advancing peel senescence early in the season. T his rapid rise in ABA level in the final stages of fruit growth have been related to the cessation of fruit growth and

PAGE 357

357 changes leading to ripening, senescence and abscission ( Murti, 1988), and this is obvious in lower FDF of water stressed fruit compared to the control by May of 2005/2006 season (Fig. 238) This is also confirmed by the negative relationship between flavedo and albedo ABA, and FDF in 2005/2006 season (Fig.78, B). There was a significant relationship between decay and bloom date during the first season that accounted for 45% of the total variance (Table 7 3) at 70oF indicating that time from bloom date could be a useful variable to determine the time when increases in fruit decay may coincide with peel senescence, and may be a good candidate for evaluating storage potential. As the growing season progressed (days from bloom date increased), the internal fruit maturity ( TSS: acid ratio) increa sed, but firmness and FDF decreased gradually and susceptibility to decay increased (Juste et al., 1988; Ladaniya, 2008 ). This was also reported in chapter 2 However one seasons significant data of bloom date, FDF and firmness may not support using them as quick and easy methods to determine the time at which decay will significantly increase in the fruit during handling and storage, which perhaps reflecting the level of peel maturity. The addition of volatile components and re duction of replicates number in the matrix pool of the variables improved the amount of variance explained by PCA components one and two to 53% in the 2004/2005 season (Fig. 7 10) but not in the 2005/2006 season (Fig. 711) nor in the combined seasons plot (Fig. 7 12). Also, the number of significant variables decreased in the comparison of % decay to harvest variables in the 2005/2006 season (Table 7 4). In the 2004/2005 season, flavedo and albedo -galactosidase appeared to differentiate early season samp les (4SC and 4OC) while volatile components (except nootkatone) and albedo sucrose and reducing sugars differentiated mid -season samples (5FC and 5MrC). B loom date, nootkatone, color, juice TSS: acid ratio, flavedo sucrose and reducing sugars, weight loss and decay

PAGE 358

358 differentiated late season samples (5AC and 5MyC). Table 7 4 showed that during the 2004/2005 season, g rapefruit % weight loss at 70oF was significantly related to 4 variables accounted for 87 % of the variance i n weight loss values. Flavedo redu cing sugars accounted for most of the multiple linear regression equation for weight loss (55%) Flavedo galactosidase and myrcene additionally accounted for 14% and 13%, respectively. Decay was significantly related to the interval between bloom and har vest date, which accounted for 57% of the total variance of decay values. Figure 7 11 showed that during the 2005/2006 season, early harvested fruit could not be identified by any variables, mid -season harvested fruit in March were identified by the ABA l evels of flavedo and albedo. Fruit harvested late season in May and July were identified with days from bloom date, juice TSS: acid ratio, nootkatone, geranyl acetate, flavedo sucrose and decay. MSR showed that weight loss was significantly related to 3 va riables measured at harvest and accounted for 72% of the total variance in weight loss. These variables were TSS: acid days from bloom date and FDF accounting for 57%, 10% and 5% of the total variance, respectively (total = 72%). Decay was significantly r elated to flavedo sucrose but only 12% of the total variance of decay values was accounted for. When the two seasons were combined for PCA analysis (Fig.7 12), overlap among harvest periods increased and only two harvest period groups could be identified ; a group of early harvested fruit in September and October (4SC, 5SC and 4OC) was separated by flavedo galactosidase, and the second group for fruit harvested late season in July was identified by days from bloom date, TSS: acid ratio and nootkatone. MS R data (Table 7 4) showed that both flavedo reducing sugars and -galactosidase were accounting for the variation in weight loss as in the first season. No variables accounted for the variation in decay in the combined data.

PAGE 359

359 The significant relationship between juice TSS: acid ratio and post storage variables (weight loss and decay) along with changes in other harvest variables may indicate that internal and external maturity changes are somewhat synchronized and using the internal changes in TSS: acid ity to predict the time at which peel becomes more susceptible to storage problems may indicate transition into peel senescence. Similarly, TSS: acid ratio significantly changes from September (6.7; average of two seasons) to November (7.6; average of two seas ons) (Table 2 1) along with significant changes in color, FDF, firmness, sugars, -galactosidase and ABA may indicate transition into best harvest period. Bloom date was related significantly to % decay in the 2004/2005 season (Table 7 3 and 7 4) and to % weight loss in the 2005/2006 season (Table 7 4). So, time from bloom may provide an estimate about the level of peel maturity associated with the susceptibility of the fruit to decay and weight loss and this is suggest that harvesting by late Novem ber (25 4 268 days from bloom; average of two seasons) or early December (276 days from bloom; average of two seasons) is the beginning of grapefruit harvest for storage at 70oF. Generally, the significant data of days from bloom date and FDF to weight loss and decay, and the significant data of firmness to decay in only one year are not enough to use them as peel maturity ind ices From these few significant results for fruit stored at 70oF, and in reference to chapter 2, % weight loss and % decay for fruit harvested and stored at 40oF and 70oF have almost the same trend during the season with higher post storage disorder rate s at 70oF, with weight loss and decay increas ing toward the end of the season. Figure 7 6 show s that at 40oF fruit had more decay from mid -season harvest in March until late season in May, but high % weight loss was more related to May and July harvest s whereas at 70oF (Fig. 7 12), high % decay was more related to mid season fruit harvested in March thru late season in July, but

PAGE 360

360 weight loss was already high in early season samples harvested in September into mid -season fruit harvested in January These conditions suggest that March (352 357 days from bloom; average of two seasons) could be the cut off point for grapefruit harvest for stora ge at 70oF The suggested harvest window of fruit stored at 70oF is shorter than that for fruit stored at 40oF, and perhaps the storage period should be shorter as well since the biochemical changes th e deterioration rate of the peel and water loss are fa ster at higher temperatures (Grierson, 1974; Grierson and Miller, 2006c ; Petracek et al., 1998) To extend the harvest window past Ma rch fruit should be harvested, handled, shipped and marketed quickly with no storage. Valencia Orange at 40oF (4.5oC) A storage experiment with Valencia orang e was run during the 2005/2006 season. Results of previous chapters indicated that fruit should be harvested during March May; a period of moderate weight loss (3.7 %) and least decay (4.2%) and chilling injury (7.22%) after 12 weeks of storage, since most of the storage problems are more related to early and late harvest; periods that presumably represent immature and senescent peel. This intermediate period was associated with juice TSS: acid ratio at harvest ranged from 15.8 in March to 25.9 in May. This high ratio of 25.0 in May represent insipid juice, which may be related to the senescence of the pulp. However, these data only represent one seasons results and should be evaluated with caution The data for harvest sample s and the 40oF storage data were better related than for grapefruit in terms of good clustering and minimum overlap among the three harvest periods: early, midand late season (Fig. 7 13). The first two components of PCA explained 67% of variance with PC 1 and PC 2 explaining 40% a nd 27% of the total variance, respectively. The early harvested fruit in January were differentiated by reducing sugars of flavedo and albedo. Mid-season harvested fruit were not separated by any variables. The third group of fruit samples could be divided into late harvest ed samples and very late harvested

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361 samples. Late harvested fruit in May was separated by FDF and flavedo sucrose. The very late season harvested samples in July were separated by albedo sucrose, color, TSS: acid days from bloom date, wei ght loss, decay and chilling injury (Figs. 7 13 and B 19, Appendix). Results of MSR, Table 7 5 showed that Valencia orange % weight loss at 40oF was significantly related to 3 variables measured at harvest account ing for 30 % of the variance in weight loss values. Peel color accounted for 22% of the weight loss variation. Days from bloom date and TSS: acid ratio accounted for the other 8 % of variation, 4% each Percentage decay was significantly related to 2 variables account ing for 35% of the variance Days from bloom date to harvest is accounted for 3 0 % of the variance, and peel color accounted for another 5 % of the variance. Oranges are less susceptible to chilling injury compared to grapefruit (Pantastico et al., 1968; Ritenour et al., 2003) but chilling injured fruit w ere significantly related to 2 variable s that accounted for 60% of the total variance. TSS: acid ratio accounted for most of the variance (5 6 % ) while flavedo sucrose accounted for only 4% of the variance The low variance of flavedo sucrose (Table 7 5) and the nonsignificant change in these suc rose values throughout the season (Table 3 2) discount flavedo sucrose as a good candidate for harvest prediction. Peel color is related significantly to weight loss and decay (Table 7 5) and they all increase with the progress of the season (Table 2 4, 2 10 and 2 12). Measuring fruit color is a quick and easy method to be done in the field to indicate the proper time of fruit harvest, for instance, Table 2 4 showed better coloration in March (color index = 3.22) than in January (color index = 1.43), whic h means postpone fruit harvest from January to March, especially with better juice quality in March ( TSS: acid ratio = 15.77) than in January ( TSS: acid ratio = 10.49), as in Table 2 2. However, peel color is a temperature related variable (Young and Erickson, 1961) especially in Florida, and may be difficult to relate to level of peel maturity at harvest in some years.

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362 The time from bloom and harvest date was significant ly correlated with weight loss and decay and seems to be an easy way to determine the time when the peel becomes more susceptible to postharvest probl ems and may give some indication about peel maturity For instance, % weight loss increased significantly from March (3.64%) to July (5.03%) (Table 2 10) % decay increased significantly from May (5.18%) to July (9.81%) (Table 2 12) and % chilling injury increased significantly from May (5.37%) to July (17.59%) (Table 2 13). These data were also associated with results of peel color and juice TSS: acid ratio and together may indicate that fruit should be harvested sometime from the beginning of March until not later than late May, since postharvest problems (weight loss, decay and chilling injury) were more related to very late harvest in July (Fig. 7 13). When volatile components were incorporated and the replicates number decreased in the data matrix for PCA the trend of clusters and overlap did not change very much but the total variance explained by PC 1 and PC 2 decreased to 57% (Fig. 7 14). Also, the total number of significant variables decreased with the addition of volatile components (Table 7 5) Early harvested fruit in January were differentiated by reducing sugars of flavedo and albedo. Peel peel turgidity (firmness) identified both early and mid -season harvested fruit. Mid-season harvested fruit were not separated by any other variables. The third group of fruit samples could again be divided into late harvest ed samples and very late harvested samples. Late harvested samples in May were separated by flavedo sucrose, FDF and valencene. The very late -season harvested samples in July were charact erized by albedo sucrose, color, TSS: acid ratio, geranyl acetate, days from bloom date, weight loss, decay and chilling injury (Figs. 7 1 4 and B 20, Appendix). Other volatiles, such as linalool, aldehydes, myrcene and pinene were not associated with any of the groups.

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363 Results of MSR (Table 7 5) showed that w eight loss at 40oF was significantly related to 2 variables measured at harvest and accounted for 63 % of the variance in weight loss values. TSS: acid accounted for most of the multiple linear regre ssion equation (54% ) and a lbedo reducing sugars accounted for an other 9 % of variation. D ecay was significantly related to 2 variables account ing for 42% of the variance ; l inalool (34% ) and firmness (8% ). Chilling injury was significantly related to TSS: a cid ratio accounting for 57% of the variance in chilling injury. There was a reciprocal significant relationship between % decay and % chilling injury, but these two variables were post storage and cannot be used to identify the best harvest period. Lina lool is the major alcohol in orange oil ( Sawamura et al., 2005). Along with octanol and some aldehydes, it is responsible for characteristic orange flavor ( Kealey and Kinsella, 1979; Sawamura et al., 2005). It was negatively related to decay and decreased with the progress of the season until a sharp and significant increase occurred from May (peak area = 61.3 millions ) to July ( peak area = 106.9 millions) (Table 6 12). The time of this change was associated with a significant increase in decay from Ma y (5.2 %) to Jul y ( 9.8 %) (Table 2 12) and in chilling injury from May (5.4%) to July (17.6%) (Table 213). The increase in decay after May was associated significantly with a reduction in peel firmness from March (9.71 Kg) to May (7.85 Kg) (Table 2 6) All of these ch anges in peel firmness, linalool content and decay incidence by May reflect that the period after May might be a period of peel senescence, and no harvest of Valencia orange should be later than May. Although firmness is an easy and practical method that can be done in the field and give some indication about harvest time, more research on peel firmness may determine if with good control of sampling to avoid within tree and environmental variability it may be a useful tool of indicating maturation Even t hough results showed that firmness was significantly related to

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364 decay, and only 8% of the variation in decay was accounted for. P eel firmness is related negatively to water content of the peel (Oberbacher, 1965). Climate affects peel thickness; mild winter nights promote thin -skinned fruit, and cold winter nights result in thick -skinned fruit (Wutscher, 1976). Mature rind does contain lower concentrations of cellulose, hemicellulose, and pectic substances than immature rind (Eaks and Sinclair, 1980), which should alter firmness. Also, the polysaccharide fractions of flavedo tissue repor tedly decreases as maturation proceeds (Muramatsu et al., 1999). TSS: acid ratio was significantly related to both weight loss and chilling injury with over 50% of the variation in each accounted for by this internal juice quality ratio. The significant changes in weight loss, chilling injury and even decay after May were associated with a magnification in TSS: acid ratio from May (25.9) to July (49.3) that mainly related to sugar accumulation and acid consumption toward the end of season ( Samson, 1986). This may give some indication that TSS: acid ratio reflects the time at which post storage peel problem start confirming its importance as a current harvest predictor. Although TSS: acid ratio is not an index of peel maturity but rather an index of internal maturity and edibility of the fruit, this data again suggests it may give an indication of coincidence between peel and pulp maturity. Albedo reducing sugar was significantly related to weight loss (Table 7 5). Sugars increase gradually during maturation, reaching their peak then start declining gradually toward the en d of the season while the fruit remain on the tree ( Bartholomew and Sinclair, 1951). Table 3 2 showed that albedo reducing sugars in the 2005/2006 season reached their maximum level in March (157.4 g/mg D.W ) then start to decline again when the temperature start ed to increase above 65oF ( Fig B 1 A ppendix) and significant ly declined in July (85.3 g/mg D.W ). These results are in agreement with Grierson (2006a ), and were associated with a significant increase in

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365 weight loss from March (3.6%) to July (5 %) (Table 2 10). However, the low association (r2 = 9%) be tween albedo reducing sugars and % weight loss may not make reducing sugars a good candidate to predict harvest. Valencene is the character impact compound of Valencia orange and could be related to peel maturity (Choi, 2003; Tonder et al., 1998). It is formed in flavedo and increases as the fruit matures (Coggins et al., 1969a ; Del Rio et al., 1992; Elston et al., 2005; Maccarone et al., 1998; Sharon -Asa et al., 2003; Shaw and Coleman, 1974 ). Figure 7 14 showed that valencene could be used to define mid-season harvested fruit in March but it had a low loading value in PC 1, and was not significantly correlated to any of the post storage variables (weight loss, decay or chilling injury) in MSR analysis (Table 7 5).Also, it would be hard to measure because it is present in very low quantities in oranges ( Vora et al., 1983; Weiss, 1997) compared to other terpenes (Table 6 17), and would require a better detection method as some samples were below current detection levels. These results on Valencia orange stored at 40oF showed that some physical measurements like peel color and firmness could be used as harvest predictors with some limitations because they are environmentally related v ariables and more evaluation would be needed. Chemical constituents such as, linalool and albedo reducing sugars showed good results, but their use as harvest predictor is limited by the complication of measurements. However, these data for one season show ed a significant change in color index and albedo reducing sugars from January to March and March may be the beginning of a good harvest period for Valencia orange intended for storage. Juice TSS: acid ratio changed from January to March at a similar time to color change. Although TSS: acid ratio is an easy maturity index for the pulp, it still may give some indication about peel maturity if peel and pulp changes are synchronized.

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366 The time between bloom and harvest date was significantly correlated with we ight loss and decay and would be an easy way to determine the time when the peel becomes more susceptible to postharvest problems. This time interval should relate to peel maturity and be useful if the year to year variation due to environmental factors c an be accounted for. Susceptibility of the fruit to post storage problems can be used to express the level of peel maturity, for instance, % weight loss increased significantly from March (3.64%) to July (5.03%) and the difference was not significant betw een March and May (3.77%) (Table 2 10), % decay increased significantly from May (5.18%) to July (9.81%) and no differences between harvest dates before May (Table 2 12) and % chilling injury increased significantly from May (5.37%) to July (17.59%) with n o differences before May (Table 2 13). These post storage changes were associated with a reduction in peel firmness from March (9.71 Kg) to May (7.85 Kg), and changes in some chemical characteristics of the peel, such as a significant changes in albedo red ucing sugars from March (157.4 g/mg D.W) to July (85.3 g/mg D.W) and a significant increase in linalool level from May (peak area = 61.3 millions) to July (peak area = 106.9 millions). Although these chemical measurements are not quick and practical way to measure peel maturity, t hese data indicate that fruit should be harvested sometime at the beginning of March (355 days from bloom date, Table A 6 Appendix) and the harvest window should not be later than late May, or possibly early June (440 to 450 day s from bloom date, Table A 6 Appendix), since postharvest problems (weight loss, decay and chilling injury) were more related to very late harvest in July that represent a period of peel senescence. Valencia Oranges at 70oF (21oC) Only weight loss and decay were postharvest problems at 70oF, and are discussed in relation to other physical and chemical variables. In a storage experiment on Valencia orange run during the 2005/2006 season r esults in previous chapters showed that for storage at 70oF,

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367 fru it should be harvested some time between March and May to avoid excessive water loss ( 5 %) (Table 2 10) and loss of fruit marketability by late harvest in May, especially as fruit showed significant decay incidence if harvested in May (46.3%) (Table 212) Usually oranges start showing shrinkage at weight loss of 2.5%, and become unsalable after 5% of the original weight is lost under normal handling conditions (Grierson and Wardowski, 1978). Th e harvest window was associated with good juice quality ( TSS: acid ratio ranged between 17 and 25) (Table 2 2) However these data only represent one seasons results and should be evaluated with caution. The data from harvest sample s and the 7 0oF storage data (Fig. 7 15) were better related than for grapefruit (Fig. 7 8 ) in terms of good clustering and minimum overlap among the three harvest periods ( early, mid and late season ). They had the same clustering trend by harvest date as oranges stored at 40oF (Fig. 7 13) and the total variance (67%) explained by the first tw o components of PCA was the same. Figure 7 15 showed that early harvested fruit (January) with storage data at 70oF were differentiated by firmness and reducing sugars of flavedo and albedo, while FDF and weight loss differentiated mid -season harvested fru it in March from other dates. Late harvested fruit in May were differentiated by flavedo sucrose and decay, and very late harvested fruit in July were differentiated by albedo sucrose, peel color, juice TSS: acid ratio and days from bloom date. MSR (Table 7 5) showed that Valencia orange % weight loss at 7 0oF was significantly related to FDF but with low accounting for variance in weight loss (7 % ). Percentage decay was significantly related to 4 variables accounted for 72% of the variation in decay. Peel color accounted for most of the variation (50%), TSS: acid ratio accounted for 11%, while flavedo reducing sugars and albedo sucrose accounted for another 11% of the total variance.

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368 FDF decreased gradually during the season and was significantly lower in March (8.42 Kg) compared to January (11.5 Kg) and May (10.56 Kg) (Table 2 8). It has been speculated that endogenous hormones from young tissues reduce the abscission of mature fruit expressed as higher values of FDF The increase of FDF after March, towa rd the end of the growing season, may have be en associated with the later time of harvest coinciding with new flushes, young fruit development for the following seasons crop, and more root grow. These young growing tissues are rich sources in endogenous p lant hormones (Goldschmidt, 1976; Hofman, 1990; Plummer et al., 1991). This is mainly related to the high ratio of IAA to ABA at the abscission zone in the calyx (Rasmussen, 1973; Yuan et al., 2001) FDF is an easy and quick method to be applied in the field t o predict harvest date, however it is an environmentally related variable and research in more than one season would be needed, especially as only 7 % off the variation in weight loss was accounted for. Color was significantly related to % decay with 50% of the variation in decay values was accounted for by color. A significant increase in color index from January (1.43) to March (3.22) (Table 2 4) was not associated with any significant changes in % decay (Table 2 12). Color index did not significantly change after March until the end of the harvest season in July, but the % decay significantly increased after March (24.4%) to the end of the season (May = 46.3% and July = 52.2%). This stabilization in color before decay increased may indicate the beginning of a good harvest period in March, especially with the increase in TSS: acid ratio (15.8) by March compared to January (10.5). A reduction in color by July (3.67) compared to May (4.01) (Table 2 4) was mainly due to the natural regreening of the fruit by the end of the season associated with the beginning of the Spring new flush (Sauls, 1998) Similarly to the report of Huff (1984), this regreening was also preceded by significant reduction in flavedo reducing suga rs (Huff,

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369 1984) from May (57.5 g/mg D.W ) to July (26.7 g/mg D.W ) (Table 3 2). This period of new flush, fol lowed by new bloom in March 2006 (Albrigo, 2006) may reflect the beginning of senescence of the current crop by May (fruit age 14 months; March 2005 May 2006) (Albrigo, 2005) suggesting that the acceptable harvest window should end before May (may be by mid to late April), especially since a significant increase in decay occurred by May. The changes in this one season should be evaluated with caution, and more than one seasons data should be considered to confirm these results, especially with the low variation in % decay accounted for (8%) by flavedo reducing sugars. Addition of volatile components to the PCA data matrix of Valencia oranges harvested and stored at 70oF (Fig. 7 16) resulted in similar PCA plots as for fruit stored at 40oF (Fig. 7 14) in terms of the same trend for separation of harvest samples (early, mid and late season) and variables, with similar explained variance by PC 1 and PC 2 (57% at 40oF vs. 58% at 70oF ). The only difference in PCA plot between fruit stored at 4 0oF (Fig. 7 1 4 ) and fruit stored at 7 0oF (Fig. 7 1 6 ) was that weight loss differentiated very late harvest in July for fruit stored at 40oF (Figs. 7 14) but it differentiated the late harvest ed samples by May for fruit stored at 70oF (Figs. 7 16). This suggests that fruit held at 70oF should be harvested earlier than if stored at 40oF confirm ing the pre viously mentioned cut -off point for 70oF harvested fruit to be no later than late April, especially with the significant increase in decay (46.3%) by May (Table 2 12). T he number of significant variables was increased in MSR compared to the 40 oC storage (Table 7 5). Percentage weight loss at 70oF was significantly related to 4 variables that accounted for 8 2 % of the variance in weight loss values. TSS: acid accounted for most of the variance ( 63% ) of weight loss va lues, and suggests using internal maturi ty to determine peel maturity if both peel and pulp changes are synchronized Bloom, firmness and FDF accounted

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370 for the other 19 % of variation (16%, 2% and 1%, respectively) Weight loss was mostly a concern related to May or later harvested fruit (Fig. 7 16) at 414 444 days from bloom date (Table A 6 Appendix). It also was significantly and negatively correlated with firmness (Table 7 5). This negative relationship between weight loss and firmness using MSR was positive using PCA (Fig. 7 16), which may justified according to the findings of Goodner et al. (2001) who stated that some false positive correlations can occur in multivariate analyses of large and diverse data sets. The low variance accounted for by fruit firmness in relation to weight loss (2%) (Table 7 5) did not make firmness a good predict or of peel maturity and optimal harvest date, although firmness decreased significantly from March (9.71 Kg) to May (7.85 Kg). Also, although FDF identified late season harvested samples in May (Fig. 7 16), the very low amount of variance accounted for (1%) (Table 7 5) makes it an unsuitable candidate to predict harvest. Firmness and FDF are affected by environmental factors and new growth, and therefore, may not be good candidates to predict the level of peel maturation Their role as harvest predictors ma y be developed with research in more than one season. Percentage decay was significantly related to 3 variables that accounted for 68% of the variance. Albedo sucrose accounted for 48% of the variance in decay, TSS: acid ratio accounted for 9% and a ldehyde s accounted for another 1 1 % of the total variance (total = 68%) (Table 7 5). The significant increase in % decay from March (24.4%) to May (46.3%) (Table 2 12) was associated with a significant increase in albedo sucrose from March (34.9 g/mg D.W ) to May (90.7 g/mg D.W ) (Table 3 2). This increase in albedo sucrose toward late season may represent a period of peel senescence and may be related to findings of Sinclair and Crandall (1949) about the physiological breakdown of the fruit while on the tree that may lead to a significant reduction in peel firmness from March (9.7 Kg) to May (7.8 Kg) (Table (2 6), which can be one reason for

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371 the increase in decay (Coggins et al., 1969a ). Another factor in sugar changes is that a lbedo tissue may serve as a transit sink or reservoir for sucrose enroute by symplastic pathway via plasmodesmata (Garcia Luis et al., 1991) to juice sacs during late -season fruit dev elopment (Koch, 1984; Koch and Avigne, 1990; Yen and Koch, 1990) These changes in peel sugars are promising, but require confirmation with more research on more than one season. However the complication of sugar m easurements does not make peel sugar a practical method to predict harvest. Oxygenated compounds, main ly alcohols (octanol & linalool) and aldehydes (octanal, -sinensal) are important for characteristic orange aroma ( Kealey and Kinsella, 1979; Sawamura et al., 2005 ). Decay was significantly related to aldehyde s, but the low variance accounted for by aldehydes (11%) (Table 7 5) and the nonsignificant change in them throughout the season (Table 6 3) did not make aldehydes a good candidate for harvest prediction. Results indicate d that March was the suggested s tart point for Valencia orange harvested for 70oF storage. The increase in weight loss ( 5.3%) associated with a significant increase in % decay (46.3%) by May (Table 2 12) suggests that the harvest period should have ended by late April ( 404 414 days from bloom date, Table A 6 Appendix) before fruit became unsalable ( 5% weight loss (Grier son and Wardowski, 1978)). Co unting the days from bloom date to harvest is a quick and easy way to predict the level of peel maturation that reflected fruit problems related to weight loss and decay, and to ensure good juice quality during this period (T SS: acid ratio 25:1) (Table 2 2) To extend the harvest window after May, fruit should be harvested, handled, shipped and marketed quickly with no storage. This one season data should be confirmed by testing in another season. This suggested harvest window is based on low percentage of unmarketable fruit found during these periods, as previously mentioned in chapter

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372 2. Firmness, FDF and color for some extend can be used as harvest predictors and they are easy and quick methods that can be applied in the f ield. More research would be desirable because they are mostly affected by environmental factors and new growth, and are likely to change out of synchronization with other peel measurements from year to -year. Chemical constituents, such as albedo sucrose and reducing sugars, and linalool showed some promising results that need to be confirmed by results from more than one season to ensure their possible roles as harvest predictors, but the disadvantage of using them is the complications of time and methods required for chemical measurements. Time from bloom date is the easiest method to perform. Predicting optimal harvest date this way may indicate stage of peel maturity related to avoiding postharvest storage problems in an acceptable manner. Harvest wind ow of Valencia oranges for 70oF storage can be from early March ( 355 360 days from bloom date, Table A 6 Appendix) and no later than late April ( 404 414 days from bloom date, Table A 6 Appendix) to avoid storage problems (weight loss ( 5.3%) a nd decay (46.3%)) if harvesting in May. Also, these post storage problems were associated with reduction flavedo reducing sugars from March (106.06 g/mg D.W.) to May (57.48 g/mg D.W.) and increase in albedo sucrose from March (34.93 g/mg D.W.) to May (9 0.67 g/mg D.W.). Simple Regression vs. Multiple Regressions of Marsh Grapefruit and Valencia Orange Principal component analysis (PCA) was run separately for grapefruit and orange to study the trend of harvest dates (scores plot) and their relatio n to physical and chemical characteristics of the peel, post storage variables (weight loss, decay and chilling injury), juice TSS: acid ratio and days from bloom to harvest. Thereafter, multiple stepwise regressions (MSR) analysis was run to see any signi ficant relationship between post storage variables and other variables. All significant relationships that resulted from MSR are summarized in Table 7 6. S ome variables may be biologically related, but account for little of the variance in post storage

PAGE 373

373 me asurements making them of little use, such as, albedo reducing sugars, flavedo mannosidase, geranyl acetate, myrcene, nootkatone and aldehydes. These variables showed some promising results and they may be used to identify peel maturity and predict the o ptimal harvest window to harvest the fruit with the least post storage problems. Although some of these comparisons are results from only one season, their trend and significant relationship with post storage variables have potential and need confirmation with additional research in additional seasons. Before doing further research or making a conclusion about the relationship of these variables with peel maturity, it should be noted that some of them may not be directly related, but are related significan tly to other variables that may make the target variable significantly related to the post storage variable (weight loss, decay, or chilling injury). Post storage variables (weight loss, decay and chilling injury) of Marsh grapefruit stored at 40oF or 70oF (two seasons; 2004/2005 and 2005/2006), Valencia orange stored at 40oF or 70oF (one season; 2005/2006) and the combined data of both species at 40oF or 70oF (three seasons; 2 for grapefruit and 1 for orange) were analyzed versus other physical and che mical variables, juice TSS: acid ratio and days from bloom date (measured at harvest) using simple regression (SR) analysis to confirm the significant relationships and to find if there were any common variables between grapefruit and orange, or even betwe en the two storage temperatures that can be used to detect optimal harvest date based on maturity level of the peel at harvest that related to the least post storage problems. Marsh grapefruit and Valencia orange stored at 40oF Table 7 6 shows from M SR that there were some common variables that might be used to determine level of peel maturity that then relates to optimal harvest date for least storage problems at 40oF for both Marsh grapefruit and Valencia orange. These variables are the

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374 interval between bloom and harvest date, peel color and juice TSS: acid ratio. The significant relationship between bloom date, color or TSS: acid ratio and any of the post storage variables in MSR is affected by other variables in the correlation matrix. The importance of SR analysis is to test all possible correlation between all independent variables and each of the three dependent variables (weight loss, decay and chilling injury); one variable at a time (rather than multiple variables at a time as in MSR) to c onfirm usage of these variables as harvest predictor for both grapefruit and orange that may be applied for any other citrus variety in the future. Fruit stored at 40oF (Table 7 7 ) showed significant results in all three matrices (grapefruit, orange and c ombined data) between the number of days from bloom date and both weight loss and decay. Chilling injury was also related to days from bloom date in both grapefruit and orange matrices, but the combined data did not show any significance. These results con firm previously mentioned data of MSR about calculating days between bloom and harvest date to predict the optimal harvest time related to having the least post storage problems. Also, these results also support using the bloom date as a quick and practica l way to harvest both grapefruit and orange. Peel color also showed significant simple relationships with weight loss, decay and chilling injury at 40oF in orange and the combined data matrices (Table 7 7 ), but not in the grapefruit matrix. This is a contr adicting result to MSR (Table 7 6) about the significant relationship between color and weight loss or chilling injury of grapefruit stored at 40oF. This is may be due to that color came as a second variable in the MSR equation, which means it is a residua l variable rather than a primary variable. Also, because in SR variables analyzed one at a time in relation to the dependent variable compared to MSR that analyze multiple variables at a time, and once a variable is added, it change the relationship of the second variable to the dependent

PAGE 375

375 variable to give a different result than SR. This means that color may be an important harvest predictor of Valencia orange, but it is not so important for grapefruit However these results should be managed with caution, because they only represent one season data for Valencia orange, and its usage required more research for more seasons, especially color is environmentally related variable. Significant correlations occurred between juice TSS: acid ratio and weight lo ss in grapefruit, orange and combined matrices (SR, Table 7 7 ). TSS: acid ratio does not appear to be directly related to peel maturity. Instead, it is an internal index, but this result may indicate that peel and pulp maturity may be reasonably synchroniz ed The significant relationships in all three matrices between TSS: acid and peel color (except grapefruit) may reflect the coexistence of peel maturity and pulp maturity However i t still can t say that peel maturity and pulp maturity changes occur simu ltaneously during the season, because it reflects the fruit edibility, and changes consistently and responds to some weather changes different from color as happen in tropics where fruit can reach their maturity while the external color remain green, and also the difference between California oranges (well colored and less sweet) and Florida oranges (sweeter and less color) and Florida oranges It does change smoothly and similarly from one year to another and increases during the season (Fig. 2 1 and 2 2) and relates to immature (sour) to mature (edible) and senescent (insipid) pulp. Simple regression analysis (Table 7 7 ) showed that albedo sucrose measured at harvest has promising results in all three matrices, (grapefruit, orange and combined) in relat ion to decay and chilling injury. This variable showed a significant relationship with weight loss in orange and combined matrices, but not in the grapefruit data matrix, although the relationship was significant in MSR (Table 7 6). This significant variab le that was previously added by MSR and

PAGE 376

376 confirmed by SR is mainly related to late season harvest and may be a results of physiological breakdown of the cell walls while on the tree ( Sinclair and Crandall, 1949) that represent a period of senescent peel. The significant incre ase in percentage chilling injury of grapefruit from March (26.67%) to May (46.67%) (Table 2 12) was associated with significant increase in albedo sucrose from March (34.9 g/mg D.W ) to May (90.7 g/mg D.W ) (Table 3 2) of the 2005/2006 season. Overall, d ata of MSR and SR indicate that the interval between bloom and harvest date and juice TSS: acid ratio, are potential harvest predictors of both grapefruit and orange for 40oF storage. Peel color can be a harvest predictor for Valencia orange with some li mitations based on one season data and the environmental relationship. Albedo sucrose is a potential harvest predictor for grapefruit and orange. Flavedo reducing sugars gave good results in MSR mainly with grapefruit, but SR showed significant results wit h orange, so this variable might be used for both grapefruit and orange. Marsh grapefruit and Valencia orange stored at 70oF Like fruit stored at 40oF, results of fruit stored at 70oF were run for MSR and SR to test which variables are significant and if there are any common variables that can be used to predict optimal harvest date of grapefruit and orange with least storage problems at 70oF. Table 7 6 showed that there are some common variables that can be used to predict optimal harvest date for bo th Marsh grapefruit and Valencia orange. These variables are the interval between bloom and harvest date, FDF, flavedo reducing sugars and juice TSS: acid ratio. The significant relationship between each of bloom date, FDF, flavedo reducing sugars and TSS: acid and any of post storage variables in MSR was tested using SR analysis to test for individual relationships (Table 7 8 ).

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377 Simple regression analysis for fruit stored at 70oF (Table 7 8 ) showed significant results in all three matrices (grapefruit orange and combined data) between the number of days from bloom date and decay, but no significant results with weight loss, and these results are different from MSR that showed a significant relationship between days from bloom date and weight loss at 7 0oF for both grapefruit and orange (Table 7 6). Also, SR showed a significant relationship between days from bloom and percentage decay in orange accounting for 23 % of the variance in decay (Table 7 8 ), but it was not significant in MSR (Table 7 6). These results confirm using days between bloom and harvest date to predict optimal harvest time related to least decay at 70oF storage. Therefore, counting days from bloom date may be a quick and practical way to establish harvest dates for both grapefruit and orange for both storage temperatures. Fruit detachment force (FDF) also was significantly related with weight loss at 70oF in grapefruit, orange and combined data matrices, but only for grapefruit with regards to decay (Table 7 8 ), which were the same res ults from MSR (Table 7 6). Also, data of SR suggested looking at FDF as a harvest predictor, because the amount of variance between FDF and weight loss at 70oF increased from 1% in MSR (Table 7 5) to 32% in SR (Table 7 8 ). This means that FDF may be a good harvest predictor of Marsh grapefruit and Valencia orange However these results require more research with more seasons since there are some limitations because detachment force to some extend is environmentally related. Significant results were als o noticed between juice TSS: acid ratio and % decay in grapefruit, orange and combined matrices (Table 7 8 ). Significant results were also noticed between TSS: acid ratio and percentage weight loss in orange and combined data matrices, but not in the grape fruit matrix. This non significant relationship with weight loss in grapefruit was significant in MSR (Table 7 6) accounting for 32% of the variation (Table 7 4). This indicates

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378 the importance of SR indicating some relationships that did not appear in MSR. The significant relationships in all three matrices between TSS: acid and bloom date with regards to decay), FDF with regards to weight loss), color (with regards to decay), flavedo reducing sugars and albedo reducing sugars (only in grapefruit matrices) reflect the coexistence of peel maturity and pulp maturity. TSS: acid ratio is still a quick and practical way to harvest both grapefruit and orange for both storage temperatures. Flavedo reducing sugars were significantly related in MSR with grapefruit we ight loss and orange decay when stored at 70oF (Table 7 6). Results showed that these reducing sugar levels might be used as a common harvest predictor mainly for grapefruit (relatively good variance accountability with weight loss = 37%, Table 7 3 and 55%, Table 7 4) but only 8 % for orange (Table 7 5). Results of SR confirmed this relationship with weight loss of grapefruit, but not in orange confirming using flavedo reducing sugars to predict harvest date in Marsh grapefruit with some limitations due t o the complication of chemical measurements. Simple regression analysis (Table 7 8 ) adds another variable to the list of common variables for grapefruit and orange. This variable peel color was not a commonly used variable for grapefruit and orange stored at 70oF in MSR (Table 7 6), but it was significant in SR with percentage decay for grapefruit, orange and combined data matrices (Table 7 8 ), and it accounted for 25 % of the variation in decay of orange (Table 7 5). Color can be used as a harvest predict or of both grapefruit and orange, but with caution since it is an environmentally related variable, and its increase with decay does not mean it is simply related to level of peel maturity since in some climates like the tropics peel may reach its senescen ce while it is still green.

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379 Overall, data of MSR and SR indicate that the interval between bloom and harvest date, juice TSS: acid ratio FDF and color are potential harvest predictors of both grapefruit and orange for 70oF storage. Particularly peel color still needs more research before using it as harvest predictor. Combination of MSR and SR The high significan t relationships ( r P between physical or chemical measurements and postharvest variables in SR at 40oF (Table 7 7) or 70oF (Table 7 8) were compared with MSR results (Table 7 6 ) to see if the y were significant in MSR or not. In case o f a variable did not show a significant relationship in MSR (Table 7 6) and it showed significant relationship in SR (Table 7 7 and 78), t his variable (for example, firmness, albedo sucrose, geranyl acetate and linalool for fruit stored at 40oF ) will be added to the list of measurements that can be used to predict harvest as summarized in Table 7 -9. T he interval between bloom date to harve st date (calendar date), TSS: acid ratio, FDF and firmness are potential harvest predictors of both Marsh grapefruit and Valencia orange to store fruit at any of the two storage temperatures (Table 7 9). Conclusion This research is based on a differen t approach to understand the role of peel maturation in citrus as it relates to postharvest handling and keeping quality of the fruit. Currently, citrus is harvested based on internal quality ( TSS: acid ratio ). Peel changes have not been related to best ha rvest time. Peel physical and chemical characteristics may be useful to identify peel maturity more accurately. Physical (color, FDF and firmness) and chemical (flavedo sucrose, reducing sugars and -galactosidase, albedo sucrose and ABA, linalool, and TSS : acid ratio) characteristics of the fruit for both grapefruit and orange showed significant relationships with some post storage related variables (weight loss, decay and chilling injury) These physical or

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380 chemical variables may be good candidates to pre dict the level of peel maturation with some caution, because physical variables, such as color, firmness and detachment force may give an idea about peel maturation and are easier to determine, but may have accuracy issues partly because they are environmentally affected variables. However, more work might develop these variables into reliable predictors of peel maturity if weather effects (i.e., temperature, day length, precipitation) could be factored into the relationships between these variables and ha rvest date, increasing their usefulness in monitoring maturity. Also, chemical variables are usually not practical for quickly and easily predicting maturation level, but they may help refine more practical variables by also measuring and correlating with them in further verification studies. The goal of using principal component analysis (PCA) and multiple stepwise regression (MSR) analysis were to obtain a broader picture about the window of peel maturity that is related to common postharvest problems, w eight loss, decay and chilling injuries. Results suggested that most peel problems happened early and late in the harvest season; two periods that probably represent the immature and senescence stages of the peel, respectively, based on the development of significant percentage of unmarketable fruit. This is the reason for suggesting the mid -season harvest period as the best period for harvest for storage. It seems that there is no specific variable that can be used to identify peel maturity that indicate the beginning of harvest accurately, because biochemical and physical changes that relate to postharvest peel characteristics that affect shelf life happen at a slow and gradual rate; increasing or decreasing during the growing season in a specific trend that may differ in rate or level from one season to another, which may or may not be associated with difference in peel maturity from one season to another H owever, the simple regression (SR) analysis was done after MSR on two seasons of grapefruit studie s on grapefruit and the one season on orange to see

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381 if other variables that were not used in MSR might relate to post storage variables. Both MSR and SR came up with some promising results from the interval between bloom and harvest date, peel color, detachment force, flavedo reducing sugars, albedo sucrose and juice TSS: acid ratio, which is mainly an index of pulp maturity, but still seems to indicate similar changes in rate of peel and pulp maturation. It is hard to predict a specific harvest time that has no problems later in storage H owever, based on the lowest postharvest problems developed during storage, suggested harvest window of Marsh grapefruit for storage at 40oF storage starts in December (263 267 days from bloom date) at FDF = 8.7 Kg and flavedo reducing sugar = 42.3 g/mg D.W and end in March (352 377 days from bloom date) or early April (382384 days from bloom date) at FDF = 6.4 Kg and flavedo reducing sugar = 142.7 g/mg D.W Such grape fruit harvested for 70oF storage should be harves ted in l ate November (254258 days from bloom date) or early December (263267 days from bloom date) and discontinued by March (352357 days from bloom date). Valencia orange harvest should be starting in March (355 360 days from bloom date) for both 40oF and 70oF storage at peel turgidity = 9.71 Kg, flavedo reducing sugars = 106.1 g/mg D.W. and albedo sucrose = 34.9 g/mg D.W., and harvest end by May (440 445 days from bloom date) or early June (445 450 days from bloom date) for 40oF storage and by late April (440 445 days from bloom date) for 70oF storage at peel turgidity = 7.85 Kg, flavedo reducing sugars = 57.5 g/mg D.W. and albedo sucrose = 90.7 g/mg D.W. The most important thing that should be considered from these results that suggested harvest window is mainly based on best storage data (less percentage of unmarketable fruit), and to increase the harvest window, fruit shold be harvested, handeled, shipped and marketed quickly with no storage.

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382 Days from bloom date to harvest date (calendar date), TSS: acid ratio, FDF and firmness are potential harvest predictors for both grapefruit and orange. These variables are good candidates for peel maturity indices or a matrix index of two or more of them. They are quick practical methods that can be done i n the field (with reasonable number of fruit from different levels of the tree and different locations in the field considering the variability among fruits ), Chemical measuremnts, such as albedo sucrose and linalool are good candidates but they are not p ractical methods because they require multiple steps of lab analyses. Using any of the physical or chemical measurements as peel maturity indices would require more research over more than two seasons to collect chemical and physical peel characteristics t ogether in one data matrix for a recommended harvest period.

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383 Table 7 1 Stepwise regression of physical characteristics and chemical characteristics (except volatile components) of Marsh grapefruit (3 rep. / treat.) harvested and stored at 40oF for 12 weeks during t wo different seasons ( P Depend. Variable 2004/2005 (N = 27) 2005/2006 (N = 54) 2 Combined Years (N = 81) Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F W eight L oss (%) FDF 0.45 0.43 0.0030 % chill. Inj. 0.03 0.43 0.0001 TSS:acid 0.25 0.34 <.0001 % chill. Inj. 0.02 0.41 0.0027 % decay 0.04 0.45 0.0235 Alb. Sucrose 0.01 0.49 0.0187 Decay (%) Flav mano 27.66 0.41 0.0040 Bloom 0.03 0.41 0.0003 Alb. ABA 1.65 0.16 0.0002 Alb. ABA 1.68 0.56 0.0125 % chill. Inj. 0.09 0.49 0.0092 % chill. Inj. 0.21 0.25 0.0033 % weight loss 2.27 0.35 0.0008 FDF 1.15 0.43 0.0027 Flav. Sucrose 0.09 0.46 0.0386 Chilling Injury (%) Flav Red Sug 0.11 0.31 0.0010 % decay 0.88 0.38 < .0001

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384 Table 7 2 Stepwise regression of physical characteristics and chemical characteristics ( with volatile components) of Marsh grapefruit (2 rep. / treat.) harvested and stored at 40oF for 12 weeks during t wo different seasons ( P Depend. Variable 2004/2005 (N = 18) 2005/2006 (N = 36) 2 Combined Years (N = 54) Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F W eight L oss (%) FDF 0.37 0.30 0.0179 FDF 0.25 0.41 0.0019 TSS:acid 0.34 0.34 <.0001 Flav Red Sug. 0.01 0.49 0.0345 % chill. Inj. 0.02 0.49 0.0342 % chill. Inj. 0.02 0.42 0.0106 Linalool 1.44 0.57 0.0215 Color 0.16 0.48 0.0173 % decay 0.03 0.52 0.0491 Decay (%) Bloom 0.43 0.55 0.0005 Bloom 0.03 0.40 0.0028 Fla v. Sucrose 0.21 0.26 <.0001 TSS:acid 14.37 0.65 0.0482 % chill. Inj. 0.11 0.34 0.0160 Geranyl 2.30 0.74 0.0421 Flav. Sucrose 0.35 0.82 0.0259 Chilling Injury (%) Alb. ABA 2.71 0.25 0.0327 Flav Red Sug. 0.14 0.33 0.0210 % decay 0.85 0.34 0.0271 Nootkatone 0.00 0.47 0.0267 Color 3.21 0.44 0.0194 Color 2.37 0.41 0.0169 Flav Red Sug. 0.43 0.71 0.0041 Myrcene 1.46 0.82 0.0144 Alb. Sucrose 0.18 0.92 0.0031

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385 Table 7 3 Stepwise regression of physical characteristics and chemical characteristics (except volatile components) of Marsh grapefruit (3 rep. / treat.) harvested and stored at 7 0oF for 12 weeks during t wo different seasons ( P Depend. Variable 2004/2005 (N = 27) 2005/2006 (N = 54) 2 Combined Years (N = 81) Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F W eight L oss (%) Flav. Sucrose 0.09 0.41 0.0003 Flav. Red. Sug 0.01 0.37 <.0001 Flav. Sucrose 0.05 0.38 <.0001 Flav. galact 16.31 0.54 0.0136 Alb. ABA 0.51 0.49 0.0011 Flav. Red. Sug 0.02 0.55 <.0001 Flav. ABA 0.45 0.62 0.0418 Flav. Sucrose 0.03 0.59 0.0013 Alb. Sucrose 0.02 0.59 0.0081 Flav. galact 9.07 0.62 0.0304 Decay (%) Bloom 0.13 0.45 0.0001 Flav. Sucrose 0.37 0.12 0.0115 TSS:acid 5.30 0.28 <.0001 FDF 5.01 0.20 0.0280 FDF 5.35 0.37 0.0018 Firmness 10.69 0.27 0.0271 Flav. galact 98.15 0.33 0.0370

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386 Table 7 4 Stepwis e regression of physical characteristics and chemical characteristics ( with volatile components) of Marsh grapefruit (2 rep. / treat.) harvested and stored at 7 0oF for 12 weeks during t wo different seasons ( P Depend. Variable 2004/2005 (N = 18) 2005/2006 (N = 36) 2 Combined Years (N = 54) Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F W eight L oss (%) Flav Red Sug. 0.10 0.55 0.0004 TSS:acid 1.52 0.57 0.0033 Flav. Sucrose 0.08 0.44 <.0001 Flav. galact 17.3 5 0.69 0.0212 Bloom 0.03 0.67 0.0047 Flav Red Sug. 0.01 0.61 <.0001 Myrcene 1.60 0.82 0.0078 FDF 0.36 0.72 0.0228 Flav. galact 11.97 0.66 0.0133 % decay 0.04 0.87 0.0442 Flav. manno 2.30 0.68 0.0440 TSS:acid 0.28 0.69 0.03 64 % Decay Bloom 0.16 0.57 0.0003 Flav. Sucrose 0.36 0.12 0.0387 TSS:acid 5.69 0.29 0.0006 FDF 5.86 0.38 0.0117

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387 Table 7 5 Stepwise regression of physical characteristics and chemical characteristics ( with and without volatile components) of Valencia orange harvested and stored at 40oF and 7 0oF for 12 weeks during 2005/2006 season ( P Depend. Variable Without volatile components ( 3 rep. / treat. & n = 60) With volatile components ( 2 rep. / treat. & n = 40) 40 o F 70 o F 40 o F 70 o F Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F Indep Var Slope R 2 P R > F W eight L oss (%) % chill. Inj. 0.03 0.46 <.0001 FDF 1.33 0.07 0.0459 TSS:acid 0.10 0.54 <.0001 TSS:acid 0.24 0.63 0.0105 Color 0.62 0.68 0.0003 Alb Red Sug 0.01 0.63 0.0015 Bloom 0.06 0.79 <.0001 Bloom 0.02 0.72 0.0088 Firmn ess 0.64 0.81 0.0155 TSS:acid 0.06 0.76 0.0025 FDF 0.23 0.82 0.0354 Decay (%) Bloom 0.10 0.30 0.0045 Color 4.72 0.50 <.0001 Linalool 5.26 0.34 0.0161 Alb Sucrose 0.36 0.48 <.0001 % chill. Inj. 0.35 0.41 0.0019 TSS:acid 0.83 0.61 0.0002 F irmness 1.83 0.42 0.0301 TSS:acid 1.11 0.57 0.0081 Color 2.46 0.46 0.0304 Flav Red. Sug 0.18 0.69 0.0003 % chill. Inj. 0.30 0.54 0.0040 Aldehydes 3.06 0.68 0.0407 Alb. Sucrose 0.20 0.72 0.0166 Chilling Injury (%) TSS:acid 0.81 0.56 < .0001 TSS:acid 0.93 0.57 <.0001 % decay 0.53 0.64 0.0007 % decay 0.54 0.68 0.0011 % weight loss 2.49 0.66 0.0388 Flav Sucrose 0.15 0.70 0.0181

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388 Table 7 6 Summary of physical and chemical characte ristics that showed significant relationship with weight loss, decay and chilling injury in MSR of Marsh grapefruit and Valencia orange for storage at 40oF and 70oF. Each variable is accounted for a specific amount of variance ( 11-30%, 31-50%, or ) out of the total variance of postharvest variable. S torage at 40 O F S torage at 70 O F W eight loss (%) D ecay (%) C hilling injury (%) W eight loss (%) D ecay (%) V ariable G rap e fruit O range G rap e fruit O range G rap e fruit O range G rap e fruit O range G rap e fruit O range B loom 55% & 40% (30%) 10% 16% 57% TSS: ACID 34% 54% (5%) 10% 57% (56%) 57% & 1% 63% 29% 9% (11%) C olor 6% (22%) (5%) 11% & 7% (50%) FDF 30% & 41% 5% 1% (7%) 9% (8,9%) F irmness 8% 2% F la v R ed S ug. 19% 24% & 33% 55% & 17% (8%) F lav. S ucrose 8% & 26% (4%) 44% ( 41 ,10, 38% ) 12% A lb. R ed S ug 9% A lb. S ucrose 10% 48% (3%) F lav galact. 14% & 5% F lav manno. (8%) F lav. ABA 15% & 16% 25% A lb. ABA 8% 34% L inalool 9% G eranyl 11% 13% M yrcene 22% Nootkatone 11% Aldehydes 55% & 40% (30%) 10% 16% 57%

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389 Table 7 7 Comparison of simple regression ( r and P value) of grapefruit, orange and combined data in regards to physical and chemical characteristics (with volatile components) at 4 0oF for 12 weeks ( P Bold numbers are for significant relationships Underlined variables are potential harvest indicators Y; significant in MSR and N; not significant in MSR. Variable Weight loss (%) Decay (%) Chilling injury (%) TSS: acid ratio Grapefruit Oran ge Combined Grapefruit Orange Combined Grapefruit Orange Combined Grapefruit Orange Combined Bloom 0.51 N <.0001 0.42 0.0075 0.51 N <.0001 0.34 0.0129 0.45 0.0034 0.38 0.0002 0.35 0.0100 0.51 N 0.0008 0.06 0.5530 0.90 <.0001 0.78 <.0001 0.58 <.0001 FDF 0.24 0.0823 0.29 0.0701 0.35 0.0006 0.19 0.1766 0.10 0.5404 0.15 0.1585 0.01 0.9380 0.08 0.6260 0.17 0.0932 0.03 0.8361 0.22 0.1746 0.29 0.0052 Firmness 0.14 0.3159 0.08 0.6112 0.35 0.0004 0.21 0.1294 0.26 0.1018 0.12 0.2508 0.31 0.0214 0.13 0.4275 0.51 N <.0001 0.09 0.5249 0.21 0.1820 0.27 0.0080 Color 0.03 0.8167 0.59 Y <.0001 0.45 <.0001 0.23 0.0914 0.35 0.0271 0.21 0.0371 0.22 0.1123 0.32 0.0408 0.47 <.0001 0.43 0.0010 0.56 0.0002 0.58 <.0001 TSS: acid 0.58 Y <.0001 0.72 Y <.0001 0.68 Y <.0001 0.09 0.5255 0.45 0.0034 0.32 0.0016 0.46 0.0005 0.75 Y <.0001 0.18 0.0768 1.00 1.00 1.00 Flavedo sucrose 0.23 0.1006 0.32 0.0422 0.32 0.0016 0.51 Y <.0001 0.23 0.1535 0.38 0.0001 0.14 0 .2946 0.08 0.6266 0.07 0.4724 0.42 0.0014 0.32 0.0451 0.30 0.0028 Flavedo red. S ug 0.15 0.2829 0.23 0.1542 0.11 0.2690 0.06 0.6389 0.42 0.0074 0.13 0.2145 0.05 0.7234 0.411 0.0081 0.16 0.1147 0.37 0.0064 0.64 <.0001 0.36 0.0003 Albedo sucrose 0.25 0.0701 0.57 N 0.0001 0.24 0.0171 0.40 0.0025 0.48 0.0015 0.25 0.0165 0.37 0.0054 0.45 0.0033 0.48 <.0001 0.31 0.0201 0.68 <.0001 0.21 0.0449 Albedo red. sugars 0.24 0.0764 0.08 0.6230 0.13 0.1999 0.04 0.7625 0.22 0.1767 0.13 0.2072 0.06 0.6635 0.45 0.0033 0.33 0.0013 0.53 <.0001 0.52 0.0006 0.44 <.0001 Geranyl 0.21 0.1257 0.39 0.0116 0.11 0.2732 0.03 0.8164 0.24 0.1397 0.00 0.9923 0.26 0.0563 0.64 N <.0001 0.59 N <.0001 0.41 0.0020 0.76 <.0001 0.21 0.0451 Linalool 0.35 0.0101 0.22 0.1779 0.08 0.4295 0.09 0.5381 0.43 0.0053 0.16 0.1306 0.19 0.1602 0.16 0.3202 0.54 N <.0001 0.40 0.0025 0.43 0.0053 0.08 0.4138 pinene 0.21 0.1346 0.12 0.4477 0.24 0.0175 0.25 0.0724 0.05 0.7644 0.11 0.2914 0.11 0.4086 0.29 0.0675 0.25 0.0170 0.11 0.4355 0.26 0.1105 0.16 0.1287 Myrcene 0.26 0.0542 0.11 0.4802 0.23 0.0115 0.27 0.0519 0.13 0.4111 0.10 0.3534 0.19 0.1642 0.01 0.9353 0.16 0.1153 0.26 0.0586 0.03 0.8428 0.23 0.0274

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390 Table 7 8 Comparison of simple regression ( r and P value) of grapefruit, orange and combined data in regards to physical and chemical characteristics (with volatile components) at 70oF for 12 weeks ( P Bold numbers are for significant relationships, Underlined variables are potential harvest indicators, Y; significant in MSR and N; not significant in MSR. Variable Weight loss (%) Decay (%) TSS: acid ratio Grapefruit Orange Combined G rapefruit Orange Combined Grapefruit Orange Combined Bloom 0.12 0.3897 0.10 0.5471 0.10 0.3336 0.41 0.0023 0.48 0.0016 0.43 <.0001 0.90 <.0001 0.78 <.0001 0.58 <.0001 FDF 0.56 Y <.0001 0.57 Y 0.0001 0.53 Y <.0001 0.40 0.0024 0 .23 0.1596 0.06 0.5623 0.03 0.8361 0.22 0.1746 0.29 0.0052 Firmness 0.50 0.0001 0.22 0.1698 0.02 0.8377 0.17 0.2159 0.08 0.6357 0.11 0.3070 0.09 0.5249 0.21 0.1820 0.27 0.0080 Color 0.09 0.4964 0.10 0.5469 0.04 0.7052 0.36 0.0068 0.66 Y <.000 1 0.33 0.0013 0.43 0.0010 0.56 0.0002 0.58 <.0001 TSS: acid 0.14 0.3049 0.44 0.0040 0.23 0.0251 0.47 0.0003 0.69 Y <.0001 0.42 <.0001 1.00 1.00 1.00 Flavedo sucrose 0.52 Y <.0001 0.30 0.0589 0.43 <.0001 0.16 0.2505 0.295 0.0736 0.21 0.0405 0. 42 0.0014 0.32 0.0451 0.30 0.0028 Flavedo red. sugars 0.55 Y <.0001 0.02 0.9104 0.37 0.0002 0.38 0.0047 0.26 0.1023 0.16 0.1223 0.37 0.0064 0.64 <.0001 0.36 0.0003 Albedo sucrose 0.43 0.0013 0.30 0.0598 0.24 0.0175 0.31 0.0243 0.69 Y <.00 01 0.02 0.8764 0.31 0.0201 0.68 <.0001 0.21 0.0449 Albedo red. sugars 0.38 0.0045 0.06 0.7074 0.25 0.0163 0.29 0.0316 0.18 0.2768 0.09 0.3988 0.53 <.0001 0.52 0.0006 0.44 <.0001 Geranyl 0.01 0.9272 0.27 0.0936 0.01 0.8889 0.31 0.0232 0.34 0.0330 0.19 0.0698 0.41 0.0020 0.76 <.0001 0.21 0.0451 Linalool 0.06 0.6662 0.34 0.0332 0.11 0.2888 0.11 0.4471 0.36 0.0223 0.14 0.1617 0.40 0.0025 0.43 0.0053 0.08 0.4138 pinene 0.19 0.1610 0.24 0.1404 0.08 0.4356 0.16 0.2353 0.13 0.4070 0.11 0.2704 0.11 0.4355 0.26 0.1105 0.16 0.1287 Myrcene 0.13 0.3537 0.22 0.1678 0.03 0.7757 0.23 0.0994 0.08 0.6157 0.16 0.1215 0.26 0.0586 0.03 0.8428 0.23 0.0274

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391 Table 7 9 Summary of physical and chemical characteristics that showed significant relationship with post storage variables in MSR & SR and may be used to determine harvest date of Marsh grapefruit and Valencia orange for storage at 40oF and 70oF. Harvest index MSR SR Species Grapefruit Orange Grapefruit Orange 40F 70F 40F 70F 40F 70F 40F 70F Bloom Both TSS: acid Both FDF Both Firmness Both Color Orange Flav. Red. Sug Grapefruit Flav. Sucrose Grapefruit Alb. Sucrose Both Linalool Both Geranyl acetate Orange

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392 A PC-1 (34%)PC-1 (34%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44PC-2 (14%)P C 2 ( 1 4 % ) -4-4 -3-3 -2-2 -1-1 00 11 22 33 Scoes4SC4SC4SC4SC4SC4SC4OC4OC4OC4OC4OC4OC4NC4NC4NC4NC4NC4NC4DC4DC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5MyC5MyC B PC-1 (34%)PC-1 (34%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (14%)P C 2 ( 1 4 % ) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 gBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA Figure 7 1. Principal component analysis of Marsh grapefruit control fruit harvested in 2004/2005 season and stored for 12 weeks at 40OF including all variables except volatile components. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control (n = 27). (B) Loadings plot of PC 1 and PC 2 using 17 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -galactosidase, ABA = abscisic acid) and fruit age calculated from full bl oom to harvest time (Bloom). PCs total variance = 48%.

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393 A PC-1 (25%)PC-1 (25%) -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33PC-2 (20%)P C 2 ( 2 0 % ) -3-3 -2-2 -1-1 00 11 22 33 Scoes5SC5SC5SC5SC5SC5SC5NC5NC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlC B PC-1 (25%)PC-1 (25%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5PC-2 (20%)P C 2 ( 2 0 % ) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 gBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA Figure 7 2. Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 40OF including all variables except volatile components. (A) Scores pl ot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gro wth regulators, WG = water stress growth regulators (n = 54). (B) Loadings plot of PC 1 and PC 2 using 17 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = abscisic acid) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 45%.

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394 A PC-1 (25%)PC-1 (25%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33PC-2 (16%)P C 2 ( 1 6 % ) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 4SC4SC4SC4SC4SC4SC4OC4OC4OC4OC4OC4OC4NC4NC4NC4NC4NC4NC4DC4DC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5MyC5MyC5SC5SC5SC5SC5SC5SC5NC5NC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlC B PC-1 (25%)PC-1 (25%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5PC-2 (16%)P C 2 ( 1 6 % ) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 BloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA Figure 7 3. Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 40OF including all variables except volatile comp onents. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = con trol, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 81). (B) Loadings plot of PC 1 and PC 2 using 17 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = de ca yed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = abscisic acid) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 41%. B

PAGE 395

395 A PC-1 (35%)PC-1 (35%) -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44PC-2 (17%)P C 2 ( 1 7 % ) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 Scoes4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyC B PC-1 (35%)PC-1 (35%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (17%)P C 2 ( 1 7 % ) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 gBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene Figure 7 4. Principal component analysis of Marsh grapefruit control fruit harvested in 2004/2005 season and stored for 12 weeks at 40OF including all variables. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul. C = control (n = 18). (B) Loadings plot of PC 1 and PC 2 using 22 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachm ent force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = abscisic acid, Nootkatone, Geranyl acetate, Linalool, A pinene, Myrcene = volatile components) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 52%.

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396 A PC-1 (25%)PC-1 (25%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55PC-2 (18%)P C 2 ( 1 8 % ) -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55 66 77 Scoes5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC B PC-1 (25%)PC-1 (25%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (18%)P C 2 ( 1 8 % ) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 gBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene Figure 7 5. Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 40OF incl uding all variables. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 36). (B) Loadings plot of PC 1 and PC 2 using 22 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = de cayed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -galactosidase, ABA = abscisic acid, Nootkatone, Geranyl acetate, Linalool, A pinene, Myrcene = volatile components) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 53%.

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397 A PC-1 (24%)PC-1 (24%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55PC-2 (17%)P C 2 ( 1 7 % ) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55 4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC B PC-1 (24%)PC-1 (24%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (17%)P C 2 ( 1 7 % ) -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 BloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene Figure 7 6. Principal component analysis of Marsh gr apefruit harvested over 2 seasons and stored for 12 weeks at 40OF including all variables. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 54). (B) Loadings plot of PC 1 and PC 2 using 22 physical and chemical characterist ics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -galactosidase, ABA = abscisic acid, Nootkatone, Geranyl acetate, Linalool, A pinene, Myrcene = volatile components) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 41%.

PAGE 398

398 A PC-1 (37%)PC-1 (37%) -3-3 -2-2 -1-1 00 11 22 33 44 55 66PC-2 (13%)P C 2 ( 1 3 % ) -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 Scoes4SC4SC4SC4SC4SC4SC4OC4OC4OC4OC4OC4OC4NC4NC4NC4NC4NC4NC4DC4DC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5MyC5MyC B PC-1 (37%)PC-1 (37%) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3PC-2 (13%)P C 2 ( 1 3 % ) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 gBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA Figure 7 7. Principal component analysis of Marsh grapefruit control fruit harvested in 2004/2005 season and stored for 12 weeks at 70OF including all variables except volatile components. (A) Scores plot of PC 1 and PC 2 usi ng harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control (n = 27). (B) Loadings plot of PC 1 and PC 2 usin g 16 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -galactosidase, ABA = abscisic acid) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 50%.

PAGE 399

399 A PC-1 (26%)PC-1 (26%) -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55 66 77PC-2 (19%)P C 2 ( 1 9 % ) -4-4 -3-3 -2-2 -1-1 00 11 22 33 Scoes5SC5SC5SC5SC5SC5SC5NC5NC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlC B PC-1 (26%)PC-1 (26%) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (19%)P C 2 ( 1 9 % ) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 gBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA Figure 7 8. Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 70OF including all variables except volatile components. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 54). (B) Loadings plot of PC 1 and PC 2 using 16 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = s ucrose, Red = reduc ing sugars, A -mannosidase, B -galactosidase, ABA = abscisic acid) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 45%.

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400 A PC-1 (27%)PC-1 (27%) -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55 66PC-2 (14%)P C 2 ( 1 4 % ) -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 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 B PC-1 (27%)PC-1 (27%) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (14%)P C 2 ( 1 4 % ) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 BloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA Figure 7 9. Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 70OF including all variables except volatile components. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct ., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 81). (B) Loadings plot of PC 1 and PC 2 using 16 physical and chemical cha racteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing suga rs, A -mannosidase, B -galactosidase, ABA = abscisic acid) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 41%.

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401 A PC-1 (37%)PC-1 (37%) -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44PC-2 (16%)P C 2 ( 1 6 % ) -3-3 -2-2 -1-1 00 11 22 33 44 55 Scoes4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyC B PC-1 (37%)PC-1 (37%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (16%)P C 2 ( 1 6 % ) -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 gBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene Figure 7 10. Principal component analysis of Marsh grapefruit control fruit harve sted in 2004/2005 season and stored for 12 weeks at 70OF including all variables. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control (n = 18). (B) Loadings plot of PC 1 and PC 2 using 21 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FD F = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A mannosidase, B -galactosidase, ABA = abscisic acid, Nootkatone, Geranyl acetate, Linaloo l, A -pinene, Myrcene = volatile components) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 53%.

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402 A PC-1 (24%)PC-1 (24%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55 66PC-2 (20%)P C 2 ( 2 0 % ) -7-7 -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 Scoes5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC B PC-1 (24%)PC-1 (24%) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (20%)P C 2 ( 2 0 % ) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 gBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene Figure 7 11. Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 70OF including all variables. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 36). (B) Loadings plot of PC 1 and PC 2 using 21 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, Bgalactosidase, ABA = abscisic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 44%.

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403 A PC-1 (25%)PC-1 (25%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44PC-2 (16%)P C 2 ( 1 6 % ) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44 55 4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC B PC-1 (25%)PC-1 (25%) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (16%)P C 2 ( 1 6 % ) -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 BloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene Figure 7 12. Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 70OF including all variables. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 54). (B) Loadings plot of PC 1 and PC 2 using 21 physical and chemical characteri stics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -m -mannosidase, B -galactosidase, ABA = abscisic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 41%. B

PAGE 404

404 A PC-1 (40%)PC-1 (40%) -3-3 -2-2 -1-1 00 11 22 33 44 55PC-2 (27%)P C 2 ( 2 7 % ) -9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSW6JlSW6JlSW B PC-1 (40%)PC-1 (40%) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5PC-2 (27%)P C 2 ( 2 7 % ) -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 LoadingsLoadingsBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Figure 7 13. Principal component analysis of Valencia orange harvested in 2005/2006 season and stored for 12 weeks at 40OF including all variables except volatile components. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 6 = 2006, Ja = Jan., Mr = Mar., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 60). (B) Loadings plot of PC 1 and PC 2 using 11 physical and chemical characteristi cs (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 67%.

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405 A PC-1 (35%)PC-1 (35%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44PC-2 (22%)P C 2 ( 2 2 % ) -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSW B PC-1 (35%)PC-1 (35%) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (22%)P C 2 ( 2 2 % ) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 BloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. AldehydesAldehydesGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrceneValenceneValencene Figure 7 14. Principal component analysis of Valencia orange harvested in 2005/2006 season and stored for 12 weeks at 40OF including all variables. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 6 = 2006, Ja = Jan., Mr = Mar., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regulators (n = 40). (B) Loadings plot of PC 1 and PC 2 using 17 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injured fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, Aldehydes, Geranyl acetate, Linalool, A -pinene, Myrcene, Valencene = volatile components) and fruit age calc ulated from full bloom to harvest time (Bloom). PCs total variance = 57%.

PAGE 406

406 A PC-1 (39%)PC-1 (39%) -4-4 -3-3 -2-2 -1-1 00 11 22 33 44PC-2 (28%)P C 2 ( 2 8 % ) -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSW6JlSW6JlSW B PC-1 (39%)PC-1 (39%) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (28%)P C 2 ( 2 8 % ) -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 BloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Figure 7 15. Principal component analysis of Valencia orange harvested in 2005/2006 season and stored for 12 weeks at 70OF including all variables except volatile comp onents. (A) Scores plot of PC 1 and PC 2 using harvest date (blue = early, red = mid, green = late) and treatments. 6 = 2006, Ja = Jan., Mr = Mar., My = May, Jl = Jul., C = control, W = water stress, G = growth regulators, WG = water stress growth regula tors (n = 60). (B) Loadings plot of PC 1 and PC 2 using 10 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = fruit detachment force, Firmness = peel turgidity, Color = p eel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars) and fruit age calculated from full bloom to harvest time (Bloom). PCs total variance = 67%.

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407 A PC-1 (34%)PC-1 (34%) -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 44PC-2 (24%)P C 2 ( 2 4 % ) -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00 11 22 33 6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSW B PC-1 (34%)PC-1 (34%) -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4PC-2 (24%)P C 2 ( 2 4 % ) -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 BloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. AldehydesAldehydesGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrceneValenceneValencene Figure 7 16. Principal component analysis of valencia orange harvested in 2005/2006 season and stored for 12 weeks at 70OF including all variables. (a) Scores plot of pc -1 and pc 2 using harvest date (blue = early, red = mid, green = late) and treatments. 6 = 2006, ja = jan., mr = mar., my = may, jl = jul., c = control, w = water stress, g = growth regulators, wg = water stress growth regulators (n = 40). (b) Lssoadings plot of pc 1 and pc 2 using 16 physical and chemical characteristics (TSS: acid = juice TSS: acid ratio, % weight l= fruit weight loss, % decay = decayed fruits, fdf = fruit detachment force, firmness = peel turgidity, color = peel color, flav. = flavedo, alb. = albedo, sucr/sucro = sucrose, red = reducing sugars, aldehydes, geranyl acetate, linalool, a pinene, myrcene, valencene = volatile component s) and fruit age calculated from full bloom to harvest time (bloom). Pcs total variance = 58%.

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408 CHAPTER 8 CONCLUSIONS Florida is the leading US producer of citrus fruits. Citrus is harvested based on internal quality (edibility), but most handling and sto rage problems are related to peel condition. Peel changes have not been adequately related to best harvest time and this research evaluated physical and chemical measurements that might reflect peel maturation and senescence. A combination of water stress and growth regulators that advance or delay maturity, respectively, can be used to assist in determin ing the safe harvest period may be helpful in this regard. Changes in TSS: acid ratio, peel color, peel turgidity, percent fruit drop, and fruit character istics during storage (weight loss, decay and chilling injury) over different harvest date s were studied, and combined with results water stress and growth regulators Some promising results were achieved from this combination; partly infer red from the e ffect s of WS and GR in advancing or delaying the physiological changes over time. Growth regulators show ed more pronounced effects than water stress on maturity Based on these physiological changes of the peel during storage (weight loss, decay and chilli ng injury) suggested safe harvest windows were the period from January to March for Marsh grapefruit and the period from March to May for Val e ncia orange. Sugar level in the peel of Marsh grapefruit and Valencia orange were increasing as fruit ma turation progressed, reaching a peak in mid -season, then decreas ing again toward the end of the season. This increase in mid -season period was associated with noticeable reduction in chilling injury, and the decrease in sugar content by late season was ass ociated with increase in chilling injury incidence again. More significant results occurred with March grapefruit than Valencia orange mainly because grapefruit is more susceptible to chilling injury than orange.

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409 Fruit can be harvested within this mid-season period, with good juice quality and less senescent peel Th erefore levels of peel sugar might be used in a peel senescence index. galactosidase increased gradually during the first stages of fruit development then decreased with the p rogress of fruit maturation with little increase again late season in senescent peel. -mannosidase fluctuated early in the harvest season with a general trend to increase in senescent peel The increase in both enzymes activities was accompani ed by a decrease in fruit firmness and increase in juice quality, suggesting that the y might be indicators of peel senescence. Based on glycosidases activity, the suggested harvest window of Marsh grapefruit is the February March period. Application of w ater stress and growth regulators to affect glycosidases activity and adjust the time of harvest needs more investigation, but from the few significant results, it can be noticed that growth regulators delay the activity of albedo mannosidase until May, associated with acceptable fruit peel turgidity These results also show that GR had more effect than WS on glycosidases in terms of changing their level s and possible relationship with peel maturation rate. Harvest window can be e xtended to be from February to April, and m onitoring peel soft en ing might be a more practical means of determin in g senescent peel. Abscisic acid level increase d toward the end of the season, and it increased more after the water stress treatment, associate d with the natural maturation processes, such as decrease in peel turgidity and fruit detachment force. Suggested harvest date is February March period. Growth regulators alone or combined with water stress suppress the activity of ABA compared to the co ntrol and water stress treatment and w ere associated with reduction in color index and acceptable fruit turgidity C hanges in physical measurements were probably not a result of ABA

PAGE 410

410 level, but were related to peel maturation rate, so it is possible to rela te changes in ABA level to changes in peel maturation rate, and use ABA as indicator of peel senescence index. Volatile component results showed differences among all components in response to harvest date, water stress and growth regulators. For all stud ied components, except nootkatone, these differences in the level and the trend with fruit maturation showed too many variations that c ould not be used to define peel senescence index accurately. More research is required on the qualitative and quantitativ e changes in oil composition with harvest date to evaluate changes with physiological age of the peel. Combining the effects of harvest date, water stress and growth regulators and physical characteristics of the peel with their effects on chemical chara cteristics in one matrix may give us a broader view of peel maturity and how to identify a peel senescence index accurately based on any changes in physical and chemical charcteristics associated with changes in postharvest parameters (weight loss, decay and chilling injury) This can be done based on how chemical constituents may reflect the stage of peel maturation, and indicate how water stress and growth regulators can or cannot be used as tools to adjust the internal level of the chemical constituents of the peel These changes in chemical constituent associated with changes in physical characteristics can be used to determine the safe harvest window and/or the cut off points of harvest period to avoid periods of immature peel or senescent peel to reduce fruit susceptibility to postharvest problems such as, decay and physiological disorders, while still h aving acceptable internal quality. These results from the research point of view can be translated into a practical way to help citrus growers. F or exa mple, parameters such as, peel turgidity or detachment force can be determined at the field using a hand -held peel turgidity device or detachment force device. So, the grower can easily measure these parameters at weekly basis, may be two weeks

PAGE 411

411 or one mont h, before the suggested harvest window to harvest the fruit at a proper time of peel maturity. Our results show that s uggested harvest window of Marsh grapefruit is January March, and for Valencia orange is March May H owever, to identify a peel se nescence index more accurately, further research on more seasons, as well as further studies on chemical and molecular levels are needed How changes in peel characteristics can be altered by adjusting maturation and senescence of the peel with the application of water stress and growth regulators may also still have value in further studies to find consistent changes in physical and chemical characteristics with peel maturation. These data gave an idea about peel maturation throughout the season with indic ation to the best harvest time based on changes in peel health after harvest expressed as weight loss, decay and chilling injury, which were associated with other physical and chemical measurements. However, in this study p rincipal component analysis (PCA) and multiple stepwise regression (MSR) analysis were used to obtain a broader picture about the window of peel maturity that is related to common postharvest problems, weight loss, decay and chilling injuries. The simple regression (SR) analysis was done after MSR to see if other variables that did not get used in MSR might relate to post storage variables. Both MSR and SR came up with some promising results from the interval between bloom and harvest date, peel color, detachment force, flavedo reducing su gars, albedo sucrose and juice TSS: acid ratio, which is mainly an index of pulp maturity, but still seems to indicate similar changes in rate of peel and pulp maturation. Results suggested that mid-season harvest period is the best for both grapefruit and orange as the best period for harvest for storage, because most peel problems happened early and late in the harvest season; two periods that probably represent the immature and senescence stages of the peel, respectively,

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412 Suggested harvest window of M arsh grapefruit harvested for 40oF storage starts in December (263267 days from bloom date) at FDF = 8.7 Kg and end in March (352377 days from bloom date) or early April (382 384 days from bloom date) at FDF = 6.4 Kg, while fruit harvested for 70oF stor age should be harvested in Late November (254 258 days from bloom date) or early December (263 267 days from bloom date) and the cut -off point should be by March (352 357 days from bloom date). Valencia orange should be harvested by March (355360 days f rom bloom date) for both 40oF and 70oF storage at peel turgidity = 9.71 Kg and the harvest window end by May (440445 days from bloom date) or early June (445450 days from bloom date) for 40oF storage and by late April (440445 days from bloom date) for 70oF storage at peel turgidity = 7.85 Kg Hoever, with these findings, it should be considered that with limited seasons, days from bloom date may change, but with more seasons different measurements cn be compared to see if they adjust together as clmate change with different weather. Days from bloom date, FDF, Firmness, TSS: acid ratio, albedo sucrose and linalool can be used as indicators for both grapefruit and orange but color and geranyl acetate are only for orange and flavedo sugars are only for gra pefruit. Calendar date (days from bloom date), TSS: acid FDF, firmness and color are good and quick candidates to predict optimal harvest time that indicate level of peel maturation to reduce postharvest loss Using these variables as peel maturity indice s would require more research over more than two seasons to collects chemical and physical peel characteristics together in one data matrix for a recommended harvest period.

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413 APPENDIX A TABLES

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414 Table A 1. Effect of water stress and growth regulators on chemical and physical characteristics of Marsh grapefruit during 2005/06 and 2006/07 seasons Cont; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. TSS:acid ratio Color index Puncture resist. (Kg) FD F (Kg) Month Treat. 2005/06 2006/07 2005/06 2006/07 2005/06 2006/07 2005/06 2006/07 Jan. Cont 8.560.01 8.580.14 0.860.12 NA 4.670.28 6.140.23 7.710.72 9.290.29 WS 8.500.08 8.350.61 0.930.06 NA 5.300.42 5.590.18 7.68054 10.680.09 GR 8. 550.19 8.330.15 1.260.21 NA 5.170.03 5.880.20 7.900.33 11.420.29 WS*GR 8.430.06 7.290.59 1.170.03 NA 5.030.57 5.710.15 7.990.29 11.320.66 Mar. Cont 9.070.12 8.680.12 0.660.10 NA 5.840.09 5.360.11 3.890.66 6.920.80 WS 8.910.09 8.330.31 0.680.10 NA 5.010.04 5.430.08 5.860.51 7.930.22 GR 9.050.10 8.580.36 1.180.08 NA 5.930.15 5.750.09 4.840.53 7.620.39 WS*GR 9.090.09 7.900.42 0.880.14 NA 5.910.15 6.030.06 5.270.64 8.250.54 May Cont 10.150.29 9.890.46 1.030.05 0.590.20 5.340.15 5.640.14 9.490.45 8.560.43 WS 10.260.03 9.660.12 1.180.02 0.660.25 5.570.07 5.480.07 8.430.41 8.200.17 GR 10.230.20 9.590.50 1.420.08 1.190.37 5.960.23 5.960.29 8.700.28 8.320.40 WS*GR 10.280.16 9.690.34 1.360.08 0.780.07 5.580.08 5.830.27 8.460.24 8.860.54 Jul. Cont 13.310.80 NA 0.940.13 NA 4.890.26 NA 8.860.52 NA WS 13.080.04 NA 1.140.10 NA 4.980.19 NA 10.210.63 NA GR 13.910.34 NA 0.970.13 NA 4.850.12 NA 8.300.58 NA WS*GR 13.58046 NA 1.010.26 NA 4.680.13 NA 8.570.41 NA Table A 1. Continued %WL 2005/06 %Decay 2005/06 % CI 2005/06 Month Treat. 12 weeks @ 40 o F 12 weeks @ 70 o F 12 weeks @ 40 o F 12 weeks @ 70 o F 12 weeks @ 40 o F Jan. Cont 2.560.29 5.53 0.55 0.550.30 47.226.76 29.446.26 WS 2.470.28 5.550.60 1.670.48 57.4110.37 38.337.72 GR 2.580.32 5.160.53 0.180.18 33.895.15 38.707.23 WS*GR 2.540.27 5.240.50 0.370.25 43.896.43 38.707.41 Mar. Cont 2.960.43 2.830.29 3.881.18 39. 816.41 26.675.13 WS 2.240.27 3.450.42 0.740.34 44.266.14 18.524.99 GR 2.510.33 2.800.27 2.220.89 41.485.24 23.155.32 WS*GR 2.230.26 3.570.41 0.550.40 47.045.61 16.303.90 May Cont 3.610.42 3.420.35 3.521.22 22.783.46 46.676.67 WS 3.310.27 4.450.47 0.740.43 36.484.49 40.186.60 GR 3.290.44 3.750.38 1.850.94 32.414.36 50.006.97 WS*GR 3.510.44 4.030.42 2.591.22 31.673.76 50.936.81 Jul. Cont 4.470.54 5.160.61 5.551.40 59.075.82 52.417.31 WS 3.710.45 5.91 0.66 2.960.65 64.445.87 53.898.18 GR 3.960.46 5.570.60 2.961.04 60.185.18 45.747.00 WS*GR 3.660.43 6.110.61 8.701.38 58.525.23 46.307.62 Values are means SE NA is not available value

PAGE 415

415 Table A 2 Effect of water stress and growth regul ators on chemical and physical characteristics of Valencia orange during 2005/06 and 2006/07 seasons. Cont; control, WS; water stress, GR; growth regulators, and WS*GR; water stress & growth regulators. TSS:acid ratio Color index Puncture resist. (Kg ) FDF (Kg) Month Treat. 2005/06 2006/07 2005/06 2006/07 2005/06 2006/07 2005/06 2006/07 Jan. Cont 10.490.63 11.501.18 1.430.28 NA 9.040.24 6.560.30 11.500.56 16.290.14 WS 9.730.51 12.221.17 1.690.23 NA 8.380.36 6.090.07 11.100.53 15.020.2 6 GR 10.350.70 11.261.33 0.480.19 NA 8.930.47 6.030.11 11.470.34 16.310.45 WS*GR 10.290.58 11.321.46 0.650.47 NA 9.650.51 6.460.14 11.350.52 13.050.22 Mar. Cont 15.771.49 17.202.40 3.220.11 NA 9.710.25 5.920.18 8.420.42 14.200.20 WS 14.181.48 16.442.43 3.430.18 NA 9.550.35 6.080.07 8.570.86 13.470.36 GR 17.112.10 18.352.92 2.910.11 NA 10.460.17 6.170.07 9.130.27 13.640.10 WS*GR 14.200.98 14.371.18 2.290.15 NA 9.560.16 6.250.11 8.360.62 14.170.54 May Cont 25.893.80 25.534.58 4.010.65 3.230.27 7.850.27 5.470.04 10.560.57 12.330.76 WS 21.862.18 23.563.49 3.970.21 3.060.38 7.770.24 5.320.14 11.030.23 12.610.43 GR 25.202.90 22.663.18 3.640.12 3.050.32 7.680.22 5.570.23 11.100.21 12.5 80.18 WS*GR 23.912.56 25.374.40 3.510.28 3.260.17 7.920.09 5.48012 11.570.50 11.470.27 Jul. Cont 49.288.49 32.936.97 3.670.40 2.040.36 7.630.23 4.710.15 10.010.16 15.140.84 WS 41.885.16 37.274.26 3.860.36 2.150.23 6.580.30 4.570 .18 10.150.67 14.940.40 GR 45.927.43 31.067.43 3.760.14 0.820.76 6.950.19 4.770.10 10.420.26 13.590.75 WS*GR 44.494.29 35.811.96 3.620.45 2.500.38 6.710.14 4.790.06 10.670.55 15.250.58

PAGE 416

416 Table A 2. Continued %WL 2005/06 %Decay 2005/06 % CI 2005/06 Month Treat. 12 weeks @ 40 o F 12 weeks @ 70 o F 12 weeks @ 40 o F 12 weeks @ 70 o F 12 weeks @ 40 o F Jan. Cont 3.650.38 8.060.73 0.740.34 26.114.32 6.112.02 WS 3.390.39 6.990.67 1.670.77 19.633.14 0.620.43 GR 3.540.38 7.43 0.65 1.300.77 23.523.69 1.850.82 WS*GR 3.230.34 7.520.74 2.041.18 18.153.44 1.110.54 Mar. Cont 3.640.44 4.440.50 3.151.06 24.445.24 9.072.35 WS 3.550.44 4.580.49 0.370.25 39.264.98 6.302.03 GR 3.520.43 4.580.46 2.410.93 37.964. 95 6.851.56 WS*GR 3.130.36 5.320.55 1.670.48 40.005.97 10.371.29 May Cont 3.770.36 5.350.48 5.182.02 46.306.61 5.372.53 WS 3.870.38 5.330.45 4.632.02 49.264.74 2.590.57 GR 3.880.38 5.370.45 6.112.71 47.595.76 2.781.02 WS*GR 3. 760.35 5.610.54 0.930.59 36.855.32 7.781.66 Jul. Cont 5.030.57 7.080.81 9.812.20 52.224.45 17.592.96 WS 5.030.58 6.620.76 10.742.51 55.553.66 21.672.78 GR 4.750.52 6.600.69 3.520.74 68.153.62 32.412.80 WS*GR 4.630.50 7.830.84 1 0.372.01 66.854.55 19.813.40 Values are means SE NA is not available value

PAGE 417

417 Table A 3 Effect of soil coverage with Tyvek on chemical and physical characteristics of Valencia orange during 2005/2006 and 2006/2007 seasons. Cont; control, WS; w ater stress, GR; growth regulators, and WS*GR; water stress & growth regulators. TSS:acid ratio Color index Puncture resist. (Kg) FDF (Kg) Month Treat. 2005/06 2006/07 2005/06 2006/07 2005/06 2006/07 2005/06 2006/07 Jan. Cont 10.490.63 8.920.44 1.4 30.28 NA 9.040.24 6.190.15 11.500.56 16.141.18 Tyvek 8.380.12 9.280.46 1.050.36 NA 8.540.21 6.610.32 9.720.39 14.850.28 Mar. Cont 15.771.49 11.330.47 3.220.11 NA 9.710.25 6.090.18 8.420.42 13.091.07 Tyvek 11.190.21 11.421.34 4.24 0.10 NA 10.170.13 6.690.02 7.890.73 12.810.48 May Cont 25.893.80 15.580.35 4.010.65 3.100.52 7.850.27 5.760.11 10.560.57 10.940.29 Tyvek 14.070.78 15.120.18 4.720.06 2.990.33 7.630.20 5.830.10 8.580.34 11.650.83 Jul. Cont 49.288.49 23.120.60 3.670.40 1.691.09 7.630.23 5.760.13 10.010.16 14.760.16 Tyvek 27.22 24.553.25 4.13 1.830.25 NA 6.060.10 NA 13.290.91 Table A 3. Continued %WL 2005/06 %Decay 2005/06 % CI 2005/06 Month Treat. 12 weeks @ 40 o F 12 weeks @ 70 o F 12 weeks @ 40 o F 12 weeks @ 70 o F 12 weeks @ 40 o F Jan. Cont 3.650.38 8.060.73 0.740.34 26.114.32 6.112.02 Tyvek 3.910.43 6.870.59 0.550.30 16.302.89 6.302.41 Mar. Cont 3.640.44 4.440.50 3.151.06 24.445.24 9.072.35 Tyvek 3.710.48 3.83 0.37 1.300.48 20.553.23 4.070.83 May Cont 3.770.36 5.350.48 5.182.02 46.306.61 5.372.53 Tyvek 3.790.47 4.730.48 0.000.00 32.225.70 3.050.96 Jul. Cont 5.030.57 7.080.81 9.812.20 52.224.45 17.592.96 Tyvek 4.820.93 5.370.98 5.553.1 8 48.895.55 14.993.07 Values are means SE NA is not available value

PAGE 418

418 T able A 4 Day s between bloom date and harvest dates of Marsh grapefruit and Valencia orange in three seasons. Bloom dates of Marsh grapefruit and Valencia orange in thr ee seasons from Albrigo (2004; 2005; 2006) 2004/2005 season 2005/2006 season 2006/2007 season Days from bloom Days from bloom Days from bloom Harvest date Grapfruit bloom = Mar 7 th Orange bloom = Mar 5 th Harvest date Grapfruit bloom = Mar 10 th Orange bloom = Mar 7 th Harvest date Grapfruit bloom = Mar 8 th Orange bloom = Mar 4 th Sept 20 th 193 Sept 8 th 178 Sept 3 rd 175 Oct 18 th 221 Nov 15 th 248 Nov 4 th 234 Nov 9 th 241 Dec 13 th 276 Jan 10 th 303 Jan 3 rd 293 296 Jan 4 th 296 300 Feb 7 th 330 Mar 4 th 357 Mar 2 nd 352 355 Mar 1 st 353 357 Apr 1 st 384 May 1 st 41 4 May 1 st 411 414 May 1 st 413 417 Jun 1 st 444 Jul 5 th 474 477 Jul 1 st 473 477

PAGE 419

419 APPENDIX B FIGURES

PAGE 420

420 0 10 20 30 40 50 60 70 80 90 Sep-03 Oct-03 Nov-03 Dec-03 Jan-04 Feb-04 Mar-04 Apr-04 May-04 Jun-04 Jul-04 Aug-04 Sep-04 Oct-04 Nov-04 Dec-04 Jan-05 Feb-05 Mar-05 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 Jul-06 Aug-06 Sep-06 Oct-06 Nov-06 Dec-06 Jan-07 Feb-07 Mar-07 Apr-07 May-07 Jun-07 Jul-07 Aug-07 Sep-07 Month Temperature (F) Figure B 1. Monthly Temperatures (F) of winter haven area from Sept 2003 to Sept 2007 (NOAA, 20 03; NOAA, 2004; NOAA, 2005; NOAA, 2006; NOAA, 2007a; NOAA, 2007b) 0 2 4 6 8 10 12 14 Sep-03 Oct-03 Nov-03 Dec-03 Jan-04 Feb-04 Mar-04 Apr-04 May-04 Jun-04 Jul-04 Aug-04 Sep-04 Oct-04 Nov-04 Dec-04 Jan-05 Feb-05 Mar-05 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 Jul-06 Aug-06 Sep-06 Oct-06 Nov-06 Dec-06 Jan-07 Feb-07 Mar-07 Apr-07 May-07 Jun-07 Jul-07 Aug-07 Sep-07 Month Percipitation (Inches) Figure B 2. Monthly Precipitation (inches) of Winter Haven area from Sept 2003 to Sept 2007 (NOAA, 2003; NOAA, 2004; NOAA, 2005; NOAA, 2006; NOAA, 2007a; NOAA, 2007b)

PAGE 421

421 0 50 100 150 200 250 300 350 400 Control Jan at harvest Mar May July Control Jan @40F Mar May July Control Jan @70F Mar May July WS Jan at harvest Mar May July WS Jan @40F Mar May July WS Jan @70F Mar May July GR Jan at harvest Mar May July GR Jan @40F Mar May July GR Jan @70F Mar May July WS & GR Jan at harvest Mar May July WS & GR Jan @40F Mar May July WS & GR Jan @70F Mar May July Traatment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure B 3. Changes in peel sugar content of water stress and growth regulators treated Marsh grapefruit at harvest and after 12 weeks storage at 40oF and 70oF during the 2005/2006 season. Values are mean SE.

PAGE 422

422 0 50 100 150 200 Control Jan at harvest Mar May July Control Jan @40F Mar May July Control Jan @70F Mar May July WS Jan at harvest Mar May July WS Jan @40F Mar May July WS Jan @70F Mar May July GR Jan at harvest Mar May July GR Jan @40F Mar May July GR Jan @70F Mar May July WS & GR Jan at harvest Mar May July WS & GR Jan @40F Mar May July WS & GR Jan @70F Mar May July Treatment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure B 4. Changes in peel sugar content of water stress and growth regulators treated Valencia orange at harvest and after 12 weeks storage at 40oF and 70oF during the 2005/2006 season. Values are mean SE.

PAGE 423

423 0 50 100 150 200 Control Jan at harvest Mar May July Control Jan @40F Mar May July Control Jan @70F Mar May July Tyvek Jan at harvest Mar May July Tyvek Jan @40F Mar May July Tyvek Jan @70F Mar May July Traetment & Harvest date Sugar content (ug/mg D.W.) Flav. Sucrose Flav. Red. Sug. Alb. Sucrose Alb. Red. Sug. Figure B 5. Effect of soil coverage with Tyvek on sugar content of Valencia orange peel at harvest and after 12 weeks storage at 40oF and 70oF during the 2005/2006 season. Values are mean SE.

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424 0 2 4 6 8 10 Control Jan Mar May July WS Jan Mar May July GR Jan Mar May July WS & GR Jan Mar May July Harvest date ABA conc. (mg/g DW) Flav. ABA 05/06 Alb. ABA 05/06 Flav. ABA 06/07 Alb. ABA 06/07 Figure B 6. Effect of water stress and growth regulators on ABA level of Marsh grapefruit peel durin g 2005/2006 and 2006/2007 seasons. Values are mean SE.

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425 Figure B 7 Principal component analysis of Mar sh grapefruit harvested in 2004/2005 season and stored for 12 weeks at 40OF using 17 physical and chemical charcterist cs (without volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 27). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling i njuried fruits FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -gal = -galactosidase, ABA = a bscicic acid, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 48%. PC-1 (34%)PC-1 (34%) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8PC-2 (14%)P C 2 ( 1 4 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 p4SC4SC4SC4SC4SC4SC4OC4OC4OC4OC4OC4OC4NC4NC4NC4NC4NC4NC4DC4DC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5MyC5MyCBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA

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426 Figure B 8 Principal component analysis of Marsh grape fruit harvested in 2005/2006 season and stored for 12 weeks at 40OF using 17 physical and chemical charcteristcs (without volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 54). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injuried fruits FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -gal = -galactosidase, ABA = a bscicic acid, Bloom = fruit age calculated from full bloom to harvest time, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 45%. PC-1 (25%)PC-1 (25%) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6PC-2 (20%)P C 2 ( 2 0 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 p5SC5SC5SC5SC5SC5SC5NC5NC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlCBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA

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427 Figure B 9 Principal component analysis of Mar sh grapefruit harvested over 2 season s and stored for 12 weeks at 40OF using 17 physical and chemical charcteristcs (without volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 8 1 ). TSS: acid = juice TSS: acid ratio, % We ight L = fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injuried fruits FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -m annosidase, B -gal = -galactosidase, ABA = a bscicic acid, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 41%. PC-1 (25%)PC-1 (25%) -1-1 -0.9-0.9 -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8PC-2 (16%)P C 2 ( 1 6 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 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% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA

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428 Figure B 10. Principal component analysis of Marsh grapefruit harvested in 2004/2005 season and stored for 12 weeks at 40OF using 22 physical and chemical charcteristcs (with volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 18). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injuried fruits FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -gal = -galactosidase, ABA = a bscicic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 52 %. PC-1 (35%)PC-1 (35%) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8PC-2 (17%)P C 2 ( 1 7 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 p4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyCBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene

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429 Figure B 11. Principal component analysis of Mars h grapefruit harvested in 2005/2006 season and stored for 12 weeks at 40OF using 22 physical and chemical charcteristcs (with volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 36). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injuried fruits FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = a lbedo, Sucr/Sucro = suc rose, Red = reducing sugars, A -mannosidase, B -gal = -galactosidase, ABA = a bscicic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 43 %. PC-1 (25%)PC-1 (25%) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11PC-2 (18%)P C 2 ( 1 8 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 p5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlCBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatonNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene

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430 Figure B 12. Principal component analysis of M arsh grapefruit harvested over 2 seasons and stored for 12 weeks at 40OF using 22 physical and chemical charcteristcs (with volatile components) and fruit age (red) with indication of harvest time an d treatments (blue) in the PC 1/PC 2 biplot (n = 54). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injuried fruits FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B -gal = -galactosidase, ABA = a bscicic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 41 % PC-1 (24%)PC-1 (24%) -1-1 -0.9-0.9 -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8 0.90.9 11PC-2 (17%)P C 2 ( 1 7 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlCBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatonNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene

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431 Figure B 13. Principal component analysis of Ma rsh grapefruit harvested in 2004/2005 season and stored for 12 weeks at 70OF using 16 physical and chemical charcteristcs (without volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 27). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Fl av. = f lavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = a bscicic acid, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 50 %. PC-1 (37%)PC-1 (37%) -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11PC-2 (13%)P C 2 ( 1 3 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 p4SC4SC4SC4SC4SC4SC4OC4OC4OC4OC4OC4OC4NC4NC4NC4NC4NC4NC4DC4DC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5MyC5MyCBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA

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432 Figure B 14. Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 70OF using 16 physical and chemical charcteristcs (without volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 54). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = f lavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase B galactosidase, ABA = a bscicic acid, Bloom = fruit age calculated from full bloom to harvest time, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 45 %. PC-1 (26%)PC-1 (26%) -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11PC-2 (19%)P C 2 ( 1 9 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 p5SC5SC5SC5SC5SC5SC5NC5NC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlCBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA

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433 Figure B 15. Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 7 0OF using 16 physical and chemical charcteristcs (without volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 8 1 ). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decaye d fruits, FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = f lavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = a bscicic acid, Bloom = fruit age calcul ated from full bloom to harvest time, 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regu lators. PCs total variance = 41 %. PC-1 (27%)PC-1 (27%) -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8 0.90.9 11PC-2 (14%)P C 2 ( 1 4 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 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% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABA

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434 Figure B 16. Principal component analysis o f Marsh grapefruit harvested in 2004/2005 season and stored for 12 weeks at 70OF using 21 physical and chemical charcteristcs (with volatile compo nents) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 18). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = f ruit detachment force, Firmness = peel tur gidity, Color = peel color, Flav. = f lavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = a bscicic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 53%. PC-1 (37%)PC-1 (37%) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8PC-2 (16%)P C 2 ( 1 6 % ) -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 p4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyCBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene

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435 Figure B 17. Principal component analysis of Marsh grapefruit harvested in 2005/2006 season and stored for 12 weeks at 70OF using 21 physical and chemical charcteristcs (with volatile components) and frui t age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 36). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = f laved o, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = a bscicic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components, Bloom = fruit age calculated from full bloom to harvest time, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 44%. PC-1 (24%)PC-1 (24%) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11PC-2 (20%)P C 2 ( 2 0 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlCBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatonNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene

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436 Figure B 18. Principal component analysis of Marsh grapefruit harvested over 2 seasons and stored for 12 weeks at 70OF using 21 physical and chemical charcteristcs (with volatile components) and fruit age (red) with indication of ha rvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 54). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = f lavedo, Alb = a lbedo, Sucr/Sucro = su crose, Red = reducing sugars, A -mannosidase, B galactosidase, ABA = a bscicic acid, Nootkatone, Geranyl acetate, Linalool, A -pinene, Myrcene = volatile components, Bloom = fruit age calculated from full bloom to harvest time, 4 = 2004, 5 = 2005, 6 = 2006, S = Sept., O = Oct., N = Nov., D = Dec., Ja = Jan., F = Feb, Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 41%. PC-1 (25%)PC-1 (25%) -1-1 -0.9-0.9 -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8PC-2 (16%)P C 2 ( 1 6 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 11 4SC4SC4SC4SC4OC4OC4OC4OC4NC4NC4NC4NC4DC4DC4DC4DC5JaC5JaC5JaC5JaC5FC5FC5FC5FC5MrC5MrC5MrC5MrC5AC5AC5AC5AC5MyC5MyC5MyC5MyC5SC5SC5SC5SC5NC5NC5NC5NC6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlCBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. Flav. B-gaFlav. B-gaFlav. A-maFlav. A-maAlb. B-galAlb. B-galAlb. A-manAlb. A-manFlav. ABAFlav. ABAAlb. ABAAlb. ABANootkatoneNootkatoneGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrcene

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437 Figure B 19. Principal component analysis of Valencia orange harvested over in 2005/ 2006 season and stored for 12 weeks at 40OF using 11 physical and chemical charcteristcs (without volatile components) and fruit age (red) with indication of har vest time and treatments (blue) in the PC 1/PC 2 biplot (n = 60). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, % Chill. I = chilling injuried fruits FDF = fruit detachment force, Firmness = peel turgidity, Co lor = peel color, Flav. = flavedo, Alb. = albedo, Sucr/Sucro = sucrose, Red = reducing sugars, Bloom = fruit age calculated from full bloom to harvest time, 6 = 2006, Ja = Jan., Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = g rowth regulators, WG = water stress growth regulators. PCs total variance = 67%. PC-1 (40%)PC-1 (40%) -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8 0.90.9 11PC-2 (27%)P C 2 ( 2 7 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 p6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSW6JlSW6JlSWBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red.

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438 Figure B 20. Principal component analysis of Valencia orange harvested over in 2005/ 2006 season and stored for 12 weeks at 40OF using 1 7 phys ical and chemical charcteristcs (with volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 4 0 ). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits % Chill. I = chilling injuried fruits FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = flavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, Aldehydes, Geranyl acetate, Linalool, A pinene, Myrcene, Vale ncene = Volatile components, Bloom = fruit age calculated from full bloom to harvest time, 6 = 2006, Ja = Jan., Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 5 7 %. PC-1 (35%)PC-1 (35%) -1-1 -0.9-0.9 -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8 0.90.9 11PC-2 (22%)P C 2 ( 2 2 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 p6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSWBloomBloom% weight l% weight l% decay% decay% chill. I% chill. IFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. AldehydesAldehydesGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrceneValenceneValencene

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439 Figure B 21. Principal component analysis of Valencia orange harvested over in 2005/ 2006 season and stored for 12 weeks at 70OF using 10 physical and chemical charcteristcs (without volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 60). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruits, FDF = f ruit detachment force, Firmness = peel turgidity, Col or = peel color, Flav. = f lavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, Bloom = fruit age calculated from full bloom to harvest time, 6 = 2006, Ja = Jan., Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 67 %. PC-1 (39%)PC-1 (39%) -1-1 -0.9-0.9 -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8PC-2 (28%)P C 2 ( 2 8 % ) -1-1 -0.9-0.9 -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 6JaWG6JaWG6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSW6JlSW6JlSWBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. ReFlav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red.

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440 Figure B 22. Principal component analysis of Valencia orange harvested over in 2005/ 2006 season and stored for 12 weeks at 70OF using 16 phys ical and chemical charcteristcs (with volatile components) and fruit age (red) with indication of harvest time and treatments (blue) in the PC 1/PC 2 biplot (n = 40). TSS: acid = juice TSS: acid ratio, % Weight L= fruit weight loss, % Decay = decayed fruit s, FDF = f ruit detachment force, Firmness = peel turgidity, Color = peel color, Flav. = f lavedo, Alb. = a lbedo, Sucr/Sucro = sucrose, Red = reducing sugars, Aldehydes, Geranyl acetate, Linalool, A -pinene, Myrcene, Valencene = Volatile components, Bloom = f ruit age calculated from full bloom to harvest time, 6 = 2006, Ja = Jan., Mr = Mar., A = Apr., My = May, Jl = Jul., C = control, W = water stress, G = gr o wth regulators, WG = water stress growth regulators. PCs total variance = 58%. PC-1 (34%)PC-1 (34%) -1-1 -0.9-0.9 -0.8-0.8 -0.7-0.7 -0.6-0.6 -0.5-0.5 -0.4-0.4 -0.3-0.3 -0.2-0.2 -0.1-0.1 00 0.10.1 0.20.2 0.30.3 0.40.4 0.50.5 0.60.6 0.70.7 0.80.8 0.90.9 11PC-2 (24%)P C 2 ( 2 4 % ) -1-1 -0.8-0.8 -0.6-0.6 -0.4-0.4 -0.2-0.2 00 0.20.2 0.40.4 0.60.6 0.80.8 p6JaWG6JaWG6JaWG6JaWG6JaW6JaW6JaW6JaW6JaG6JaG6JaG6JaG6JaC6JaC6JaC6JaC6JaSW6JaSW6JaSW6JaSW6MrWG6MrWG6MrWG6MrWG6MrW6MrW6MrW6MrW6MrG6MrG6MrG6MrG6MrC6MrC6MrC6MrC6MrSW6MrSW6MrSW6MrSW6MyWG6MyWG6MyWG6MyWG6MyW6MyW6MyW6MyW6MyG6MyG6MyG6MyG6MyC6MyC6MyC6MyC6MySW6MySW6MySW6MySW6JlWG6JlWG6JlWG6JlWG6JlW6JlW6JlW6JlW6JlG6JlG6JlG6JlG6JlC6JlC6JlC6JlC6JlSW6JlSW6JlSW6JlSWBloomBloom% weight l% weight l% decay% decayFDFFDFFirmnessFirmnessColorColorTSS:acidTSS:acidFlav. SucrFlav. SucrFlav. Red.Flav. Red.Alb. SucroAlb. SucroAlb. Red. Alb. Red. AldehydesAldehydesGeranylGeranylLinaloolLinaloolA-pineneA-pineneMyrceneMyrceneValenceneValencene

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472 BIOGRAPHICAL S KETCH Shamel Mohamed Alam Eldein was born on 1974 in Menoufia, Egypt. In June 1994, he obtained his bachelor degree in Agricultural Sciences from Menoufia University, Egypt. In May 1999, he obtained his master degree in Horticultural Sciences from Tanta Un iversity, Egypt. In 2003 he was granted an Egyptian governmental mission to continue his study in USA, and in June 2004, he was admitted as a Ph.D. student in the Ph.D. program in the Horticultural Sciences Department at the University of Florida. After c ompleting his Ph.D. program, Shamel plans to return to Egypt to fulfill an appointed position as an assistant professor in the D epartment of Horticultural Sciences at Tanta University. He will teach and conduct research hoping to promote the excitement and enthusiasm to his students in Egypt, which he has experienced from his professors in USA.