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Evaluation of Aroma Volatiles and Their Odor Activity in a Population of Tangerine Hybrids

Permanent Link: http://ufdc.ufl.edu/UFE0024825/00001

Material Information

Title: Evaluation of Aroma Volatiles and Their Odor Activity in a Population of Tangerine Hybrids
Physical Description: 1 online resource (99 p.)
Language: english
Creator: Miyazaki, Takayuki
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Tangerine (Citrus reticulata Blanco) is well known for its pleasant aroma, flavor and ease of consumption. With the desirable combination of sugars and acids, volatile compounds contribute to the essential organoleptic attributes for consumer acceptance. While aroma volatiles in orange juice have been well studied, little information is available on those found in fresh tangerine. There is a growing need to evaluate tangerine aroma components and their odor characteristics for improvement in fruit quality. Twenty tangerine hybrids were harvested from November 2007 to March 2008, and five commercial cultivars were used as reference. Aroma volatiles were sampled from hand-squeezed juice by the headspace solid phase microextraction (SPME) method, and analyzed by gas chromatography-mass spectrometry (GC-MS). In total, more than 200 volatiles were identified in all samples. Two principal component analyses (PCA), based on relative peak areas (content) of all volatiles and 11 chemical classes, clearly separated the samples with richness in volatiles, such as sesquiterpenes and esters, and genetic background from sweet orange. Furthermore, a cluster analysis grouped these hybrids using qualitative (presence and absence) volatile composition. While ?Murcott? contained high levels of carotenoid derived volatiles, those compounds were absent in its progenies issued from the cross with 8-9. 9-4 times Blood4x and two unknown samples were grouped in the same cluster due to their peculiar terpene profiles. In addition, aroma active compounds in five selected samples were evaluated by three panelists using gas chromatography-olfactometry (GC-O) and time intensity (Osme) method. Forty nine odorants were found in a consensus and comprised of monoterpenes, aldehydes, esters, alcohols, ketones, phenol and ether. Hexanal, ethyl 2-methylbutanoate, unknown peak (No. 9), 1-octen-3-one, ?-myrcene, 1,8-cineole, linalool, (E,E)-2,4-nonadienal with descriptors of green/grassy, fruity, metallic, mushroom, metallic, green, floral and fatty, respectively, were intense aroma compounds in all five samples. Differences between sample aroma profiles were found for compounds having descriptors of fruity, green/metallic/fatty, terpeney and green/grassy notes as the top notes, and five minor aroma categories. Perceived aroma intensity determined the specific aroma character of each sample. This is the first time that the aroma volatile profile and sensory quality of fruit from fresh tangerine hybrids with various genetic origins were investigated.
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 Takayuki Miyazaki.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Gmitter, Frederick G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024825:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024825/00001

Material Information

Title: Evaluation of Aroma Volatiles and Their Odor Activity in a Population of Tangerine Hybrids
Physical Description: 1 online resource (99 p.)
Language: english
Creator: Miyazaki, Takayuki
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Tangerine (Citrus reticulata Blanco) is well known for its pleasant aroma, flavor and ease of consumption. With the desirable combination of sugars and acids, volatile compounds contribute to the essential organoleptic attributes for consumer acceptance. While aroma volatiles in orange juice have been well studied, little information is available on those found in fresh tangerine. There is a growing need to evaluate tangerine aroma components and their odor characteristics for improvement in fruit quality. Twenty tangerine hybrids were harvested from November 2007 to March 2008, and five commercial cultivars were used as reference. Aroma volatiles were sampled from hand-squeezed juice by the headspace solid phase microextraction (SPME) method, and analyzed by gas chromatography-mass spectrometry (GC-MS). In total, more than 200 volatiles were identified in all samples. Two principal component analyses (PCA), based on relative peak areas (content) of all volatiles and 11 chemical classes, clearly separated the samples with richness in volatiles, such as sesquiterpenes and esters, and genetic background from sweet orange. Furthermore, a cluster analysis grouped these hybrids using qualitative (presence and absence) volatile composition. While ?Murcott? contained high levels of carotenoid derived volatiles, those compounds were absent in its progenies issued from the cross with 8-9. 9-4 times Blood4x and two unknown samples were grouped in the same cluster due to their peculiar terpene profiles. In addition, aroma active compounds in five selected samples were evaluated by three panelists using gas chromatography-olfactometry (GC-O) and time intensity (Osme) method. Forty nine odorants were found in a consensus and comprised of monoterpenes, aldehydes, esters, alcohols, ketones, phenol and ether. Hexanal, ethyl 2-methylbutanoate, unknown peak (No. 9), 1-octen-3-one, ?-myrcene, 1,8-cineole, linalool, (E,E)-2,4-nonadienal with descriptors of green/grassy, fruity, metallic, mushroom, metallic, green, floral and fatty, respectively, were intense aroma compounds in all five samples. Differences between sample aroma profiles were found for compounds having descriptors of fruity, green/metallic/fatty, terpeney and green/grassy notes as the top notes, and five minor aroma categories. Perceived aroma intensity determined the specific aroma character of each sample. This is the first time that the aroma volatile profile and sensory quality of fruit from fresh tangerine hybrids with various genetic origins were investigated.
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 Takayuki Miyazaki.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Gmitter, Frederick G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024825:00001


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1 EVALUATION OF AROMA VOLATIES AN D THEIR ODOR ACTIVITY IN A POPULATION OF TANGERINE HYBRIDS By TAKAYUKI MIYAZAKI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Takayuki Miyazaki

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3 To my family, friends, girlfriend

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4 ACKNOWLEDGMENTS I grea tly thank my advisor Dr. Frederick G. Gmitter, Jr. for his financial and academic support to pursue my degree and Dr. Anne Plotto at USDA/ARS, Citrus & Subtropical Products Laboratory, for her thoughtful advi ce and guidance during the course of this study. I also would like to extend my gratitude to Dr. Jude Grosser for serving on the supervisory committee. Appreciation is extended to the members of Dr. Gmitters lab, Drs. Chunxian Chen, Xiuli Shen, Qibin Yu, Madhugiri Rao, Jaya Soneji, and Fan Jing for their useful advice and friendship. I thank Margie Wendell and Misty Holt for their technical assistance. Also, I appreciate many people helping me at the faculty of the Departme nt of Horticultural Sciences and the Citrus Research and Education Center, University of Florida. I am very grateful to Dr. Elizabeth Baldwin for allowing me to use the gas chromatography and mass spectrometry, gas chromatography-olfactom etry and all the othe r lab equipments in conducting this research project. I thank Mr. Timothy Tillman for hi s support and guidance to set up the equipments, Keith Williamson and Dr. Jinh e Bai for their help and care in the USDA laboratory. I also thank Dr. Jose Reyes for providing me the computer software to carry out the experiment using gas chromatography-olfactometry. I am truly thankful for my family for unc onditional encouragement and support. Thanks also go to many friends and professors in Japan for supporting me to go abroad and pursue the interest in my major. I thank the Rotary Foundation for providing me one year of scholarship and supporting my stay in the U.S. Finally, I would like to deeply thank Tomoko Shinoda for giving constant love and waiting for me to complete my MS degree.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................... ...............9 CHAP TER 1 INTRODUCTION .................................................................................................................. 11 2 REVIEW OF LITERATURE .................................................................................................16 Origin and Taxonomy of Citrus .............................................................................................. 16 Analysis of Aroma Volatiles .................................................................................................. 19 Gas Chromatography-Olf actom etry (GC-O) ...................................................................19 GC-O Methods ................................................................................................................20 SPME Method .................................................................................................................22 Citrus Aroma and Flavor ........................................................................................................23 Tangerine/Mandarin Aroma ............................................................................................ 24 Orange Aroma .................................................................................................................25 Grapefruit and Pummelo Aroma ..................................................................................... 27 Aroma Volatile Biosynthetic Pathways .................................................................................. 28 Terpenoid Aroma Volatiles ............................................................................................. 29 Fatty Acid Derived Aroma Volatiles ............................................................................... 30 Amino Acid Derived Aroma Volatiles ............................................................................ 31 Improvement of Citrus Aroma and Flavor ...................................................................... 32 3 DISTRIBUTION OF AROMA VOLATI LE C OMPOUNDS IN TANGERINE HYBRIDS AND PROPOSED INHERITANCE .................................................................... 35 Introduction .................................................................................................................. ...........35 Materials and Methods ...........................................................................................................37 Plant Materials .................................................................................................................37 Sample Preparation .......................................................................................................... 38 Optimization of Volatile Sampling ................................................................................. 38 Headspace Sampling and GC-MS analysis ..................................................................... 39 Volatile Compound Identification ...................................................................................40 Statistical Analyses .......................................................................................................... 40 Results and Discussion ........................................................................................................ ...40 Principal Component Analysis (PCA) ............................................................................. 42 Cluster Analysis (CA) Based on Qualitative Volatile Composition ............................... 43

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6 Cluster 1 (C1) ........................................................................................................... 43 Cluster 2 (C2) ........................................................................................................... 44 Cluster 3 (C3) ........................................................................................................... 45 Cluster 4 (C4) ........................................................................................................... 46 Cluster 5 (C5) ........................................................................................................... 47 4 CHARACTERIZATION OF AROMA VOLAT ILES IN T ANGERINE HYBRIDS BY GAS CHROMATOGRAPHY-OLFACTOMETRY ..............................................................62 Introduction .................................................................................................................. ...........62 Materials and Methods ...........................................................................................................64 Sample Preparation .......................................................................................................... 64 Gas Chromatography-Olfactometry ................................................................................ 64 Osme Analysis .................................................................................................................65 Statistical Analysis .......................................................................................................... 66 Results and Discussion ........................................................................................................ ...67 Consensus of Tangerine Arom a Active Compounds ...................................................... 67 Terpenes ...................................................................................................................68 Aldehydes ................................................................................................................. 68 Esters ........................................................................................................................ 70 Alcohols ................................................................................................................... 70 Ketones ..................................................................................................................... 71 Phenol ....................................................................................................................... 72 Ether ......................................................................................................................... 72 Unknown Compounds ..............................................................................................73 Tangerine Aroma Profiles ............................................................................................... 74 5 SUMMARY AND CONCLUSIONS .....................................................................................82 APPENDIX A OPTIMIZATION OF SPME FOR GC -MS AND GC-O ANALYSIS .................................. 85 LIST OF REFERENCES ...............................................................................................................86 BIOGRAPHICAL SKETCH .........................................................................................................99

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7 LIST OF TABLES Table page 1-1 Total production of tanger ine and mandarin fruits in 2007 ............................................... 15 3-1 List of samples and corresponding selection nam es or parentage, hybrid numbers, harvest dates and sample codes .......................................................................................... 49 3-2 List of aroma volatiles detected by GC-MS am ong samples and identified by linear retention index (LRI) on DB-5, DB-wax column and confirmed with chemical standards. Volatiles are listed according to their frequency of appearance, 48% to 100%, in samples ...............................................................................................................51 3-3 List of aroma volatiles detected by GC-MS am ong samples and identified by linear retention index (LRI) on DB-5, DB-wax column and confirmed with chemical standards. Volatiles are listed according to their frequency of appearance, 1% to 47%, in samples .................................................................................................................53 3-4 Tangerine aroma volatiles am ong 11 chemical classes ..................................................... 55 3-5 Amount (relative peak area) of aroma volatiles arranged by 11 chem ical classes in 25 samples ....................................................................................................................... ........57 3-6 Samples in five clusters formed by clus ter analysis based on presence and absence of volatiles, and their num ber of vol atiles in classified compounds ...................................... 61 4-1 Consensus of aroma active compounds in five tangerine hybrids determ ined by GCMS and GC-O using Osme analysis .................................................................................. 77 4-2 Tangerine aroma active com pounds in 8 chem ical classes ................................................ 79 4-3 List of tangerine aroma activ e com pounds in 8 aroma categories ..................................... 80

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8 LIST OF FIGURES Figure page 2-1 Terpenoid aroma volatiles................................................................................................ ..33 2-2 Fatty acid derived aroma volatiles ..................................................................................... 34 3-1 Pedigree of tangerine hybrids. ...........................................................................................50 3-2 Principal component analysis by using volatile relative peak areas am ong samples ........ 58 3-3 Principal component analysis by usi ng volatile peak areas of classified compound categories among sa mples .................................................................................................. 59 3-4 Cluster analysis by using volatile presence and absence am ong samples.. ....................... 60 4-1 Aroma profiles of five tangerine hybrids. .......................................................................... 81

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF AROMA VOLATIES AN D THEIR ODOR ACTIVITY IN A POPULATION OF TANGERINE HYBRIDS By Takayuki Miyazaki December 2009 Chair: Frederick G. Gmitter, Jr. Major: Horticultural Science Tangerine ( Citrus reticulata Blanco) is well known for its pleasant aroma, flavor and ease of consumption. With the desirable combina tion of sugars and acids, volatile compounds contribute to the essential organoleptic attribut es for consumer acceptance. While aroma volatiles in orange juice have been we ll studied, little information is available on those found in fresh tangerine. There is a growing need to evalua te tangerine aroma com ponents and their odor characteristics for improvement in fruit quality. Twenty tangerine hybrids were harvested from November 2007 to March 2008, and five commercial cultivars were used as reference. Aroma volatiles were sampled from hand-squeezed juice by the headspace solid phase microextra ction (SPME) method, and analyzed by gas chromatography-mass spectrometry (GC-MS). In to tal, more than 200 volatiles were identified in all samples. Two principal component analyses (PCA), based on relative peak areas (content) of all volatiles and 11 chemical classes, clearly separated the samples with richness in volatiles, such as sesquiterpenes and esters, and geneti c background from sweet orange. Furthermore, a cluster analysis grouped these hybrids using qualitative (presence and absence) volatile composition. While Murcott contained high levels of carotenoid deri ved volatiles, those

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10 compounds were absent in its progenies issued from the cross with 8-9. 9-4 Blood4x and two unknown samples were grouped in the same cluste r due to their peculi ar terpene profiles. In addition, aroma active compounds in five selected samples were evaluated by three panelists using gas chromatography-olfactomet ry (GC-O) and time intensity (Osme) method. Forty nine odorants were found in a consensus and comprised of monoterpenes, aldehydes, esters, alcohols, ketones, phenol and ether. Hexanal, ethyl 2-methylbutanoate, unknown peak (No. 9), 1octen-3-one, -myrcene, 1,8-cineole, linalool, ( E E )-2,4-nonadienal with descriptors of green/grassy, fruity, metallic, mushroom, metallic, green, floral and fatty, respectively, were intense aroma compounds in all five samples. Differences between sample aroma profiles were found for compounds having descriptors of fr uity, green/metallic/ fatty, terpeney and green/grassy notes as the top not es, and five minor aroma categor ies. Perceived aroma intensity determined the specific aroma character of each sample. This is the first time that the aroma volatile profile and sensory qual ity of fruit from fresh tanger ine hybrids with various genetic origins were investigated.

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11 CHAPTER 1 INTRODUCTION Citrus is one of the m ost economically impor tant and widely grown fruit crops in the world. Citrus is believed to have originated in tropical and subt ropical regions of Southeast Asia, where it was domesticated at least 4,000 years B.C. and then spread to other continents (Webber, 1967). Citrus production has exceeded other fruit crops such as grapes, bananas and apples (FAO, 2009). Mandarin and tangerine ( Citrus reticulata Blanco) production is the second largest market of citrus fruit following oranges ( C. sinensis L. Osb.) in the world. China is the largest tangerine producing country, followed by Spain and Brazil (Table 1-1). Spain has been significantly successful in the trade of seedless Clementine varieties, accounting for over 50 % of worlds exports of fresh mandarin/tangerine fruits. The tangerine fruit is well known for its pleas ant aroma and flavor, desirable combination of sugar and acid and ease of consumption. For hu man benefits, citrus fruits are a major source of vitamin C. Carotenoids known as antioxidants are more abundant in tangerine than orange (Mayne, 1996; U.S. Department of Agriculture -Nutrition Coordinating Center, 1998; Goodner et al., 2001). Other antioxidants such as flavonoids and phenolics, having a role in disease prevention, could also benefit consumer healt h. In addition, most tangerines are much more tolerant of citrus canker than are grapefruit and could be an alternative or complement to grapefruit for fresh market productio n in Florida (Gottwald et al., 2002). Food flavors are commonly characterized by a combination of aroma, basic tastes (e.g., sweetness, sourness, bitterness, saltiness and um ami) and mouth sensation such as astringency. Volatile compounds are responsible for aroma and flavor, and are analyzed by isolation, identification and quantification. Citrus aroma vola tiles have usually been extracted by solvent, distillation, sorptive and headspace methods. Solid phase microextraction (SPME) is a

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12 solventless headspace sampling method (Arthur and Pawliszyn, 1990). It is a rapid and relatively inexpensive technique, thereby ha ving been applied to many studies on aroma volatiles in citrus juice (Steffen and Pawliszyn, 1996; Rega et al ., 2003). The development of gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) has enabled flavor researchers to identify and quantify volatiles in food and bevera ges. To date, volatiles in citrus juice and peel oil have been widely investigated. Most ar oma compounds arise as a result of degradation reactions from carbohydrates, fatty acids and am ino acids (Schwab et al., 2008). Above all, esters play an important role in overall aroma an d flavor of climacteric horticultural crops such as apples (Defilippi et al., 2005) and bananas (Shiota, 1993 ). In non-climacteric citrus, monoterpenes (C10H16) and sesquiterpenes (C15H24) are the major volatile compounds produced, and citrus aroma is the result of a combination of volatile components such as terpenes, esters, aldehydes, ketones, and alcohols (N isperos-Carriedo et al., 1990). Gas chromatography-olfactometry (GC-O) is the most appropriate method to detect and evaluate odor active components from a complex mixture of volatiles, using a human nose as a detector (van Ruth, 2001). The various GC-O techniques, measuring odor activity, can be classified into three categories: time intensity, detection frequency and dilution to threshold methods. A time-intensity method, Osme (Greek wo rd for smell), takes into consideration a psychophysical law, estimating the magnitude of an odorant (McDaniel et al ., 1990). So far, the sensory properties of volatiles in orange juice have been well investig ated (Hinterholzer and Schieberle, 1998; Tnder et al., 1998; Buettner et al., 2001b; Rouseff a nd Prez-Cacho, 2007). Some major aroma active compounds were known to impart odor notes such as citrus, fruity, floral and green to orange juice aroma. While there have been several reports on aroma volatiles

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13 in tangerine peel and essence oil (Moshona s and Shaw, 1974; Shaw, 1979, Wilson and Shaw, 1981), very little is known regarding those in fresh tangerine fruits. Citrus fruits with desirable flavor associated with aroma volatiles, as well as sugars and acids, could increase consumption and market value, thereby resulting in greater economic returns to growers, shippers a nd packers. Improvements in fruit quality and characteristics, as well as improvements in pest and disease resist ance, tolerance to various environmental stress factors, horticultural pe rformance, and productivity are the primary goals of citrus breeding (Gmitter et al., 2007). However, the conventiona l citrus breeding based on hybridization and selection has been hampered by the characteristics of long juvenility, high heterozygosity, gametophytic selfand cross-incompatibility and apomixis. Most of the grown citrus scion and rootstock cultivars at present arose as chance seedlings or limb and bud sport mutations, rather than from organized breeding programs (Hodgson, 1967). Fruit aroma and fl avor are affected by a complex system of genes, and environmental (temperature, irrigation, soil condition, illumination, etc.) and postharvest (storage, processing, etc.) factors. Understanding plant genetics and genomics, as well as food science, will enhance the efficiency to improve fruit quality in breeding programs. The fundamental data of fruit aroma and flavor components will provide the basis for developing methods for marker-assisted sele ction (MAS), to efficiently select at an early stage the superior individuals with high fruit quality from tangerine breeding programs. The main objectives of this study were: 1. To identify the aroma volatile compounds in juice samples of tangerine hybrids and commercial cultivars by GC-MS, 2. To analyze intervarietal relationships from volatile profiles by using multivariate statistics, principal component analysis (P CA) and cluster analysis (CA),

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14 3. To characterize aroma active compounds of some selected hybrids by GC-O and investigate the contribution of each vol atile to overall fresh fruit aroma.

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15 Table 1-1. Total production of tangerin e and mandarin fruits in 2007 (FAO, 2009) Country Production (tons) China 14,152,000 Spain 2,080,700 Brazil 1,271,000 Japan 853,000 Turkey 738,786 Italy 702,732 Iran 702,000 Thailand 670,000 Egypt 660,000 Pakistan 640,000 Others* 4,043,768 Total (in the world) 26,513,986 Others include 328,000 tons of United States of America.

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16 CHAPTER 2 REVIEW OF LITERATURE Origin and Taxonomy of Citrus The center of origin and diversity of Citrus and its rela ted genera is still unclear, but most researchers have considered that Citrus is native to Southeast Asia, especially to east India, north Burma and southwest China, extending to Malay ar chipelago and the East Indies, northeastern Asia, and Japan (Scora, 1975; Gmitter and Hu, 1990). The genus Citrus belongs to the Rutaceae family, subfamily Aurantioideae. The true citrus fruit trees include six closely related genera: Fortunella (kumquat), Eremocitrus (Australian desert lime), Poncirus (trifoliate orange), Clymenia Microcitrus (Australian wild lime) and Citrus The taxonomic classification of Citrus species is very complicated due to nucellar embryony, self-compatibility and incompatibility, many natural and artificial hybridization, wide dispersion and long history of cultivation (Cameron and Frost, 1968; Moore, 2001; Cornlio et al., 2003). In the past, studies on taxonomy and phylogeny were concluded based on morphological and geographical information. According to Swingle and Reece (1967) and Tanaka (1977), the genus Citrus consists of 16 and 162 species, respectively; indeed, these two taxon omic system differ widely. Swingle and Reece (1967) recognized three mandarin species, C reticulata C tachibana (a wild species from Japan) and C indica (a wild species from India), wh ereas Tanaka (1977) separated the mandarins into 36 species. Hodgson (1967) recognized 36 Citrus species and classified the mandarins into five classe s: the satsuma mandarins ( C unshiu), the King mandarins ( C noblis ), the Mediterranean mandarins ( C deliciosa ), the common mandarins ( C reticulata ) and the small-fruited mandarins. Tangerine is grouped into the mandarin species C reticulata and usually characterized by deep orange and red color, sweet flavor and easiness of peelin g. Swingle and Reece (1967) noted

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17 that mandarins include monoembryonic and polyembryonic cultivars, as well as self-fertile and self-incompatible types. Mandarins are the most phenotypically heterogeneous group in Citrus (Hodgson, 1967; Moore, 2001). Although mandarins ha d been cultivated in China and Japan from ancient times, mandarins were introduced to the West (England) from China for the first time in 1805 by Sir Abraham Hume, and they only subsequently spread to the Mediterranean region (Webber, 1967; Soost and Roose, 1996). Biochemical studies focusing on various types of compounds such as limonoids (Dreyer et al., 1972) and flavonoids (Albach and Redman, 1969) have been conducted to assess taxonomic relationship of Citrus Esen and Scora (1975) widely analy zed the enzymatic browning capacity of young shoot homogenates in 428 accessions of th e Aurantioideae including 10 citrus species, and confirmed its species-specificity. Barrett and Rhodes (1976) performed a comprehensive phylogenetic study of 146 morphological and biochemical tree, leaf, flower and fruit characteristics. These two st udies suggested that citron ( C medica ), mandarin ( C reticulata ) and pummelo ( C grandis ) are the primary species of Citrus DNA-based molecular markers and DNA sequences have been applied in many research fields such as phylogeny, species identification, associ ation studies and breeding during the last few decades. A molecular marker may be found at a particular position of genome, and then associated with a gene or trait of interest. Molecular marker techniques are more accurate, reproducible and time-saving than conventional survey only ba sed on phenotypic characteristics of samples, to elucidate genetic relationship or to select interesting genotypes. So far, phylogenetic relationships in Citrus have been studied by seve ral marker techniques: e.g., random amplified polymorphic DNA (RAPDs) marker (Coletta Filho et al., 1998; Federici et al., 1998), inter-simple sequence repeat markers (ISSRs) (Fang and Roose, 1997) and sequence-

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18 characterized amplified regions (SCARS) (Nicolosi et al., 2000). These molecular marker analyses support the pro position that there are three primary species in Citrus (Federici et al., 1988; Nicolosi et al., 2000). More recently, Bayer et al. (2009) showed the pummelo and all it derivatives (grapefruit, sweet orange, sour ora nge, lemon, Tahitian lime) were grouped in the same clade, using maternally inherited chlo roplast DNA sequences. The three primary species, with other genera of tribe Citre ae, were clearly separated into di fferent phylogenetic groups. It is generally accepted that the wide variety of current citrus speci es were originated from the hybridization between and among these true species, as well as fr om their crossing with closely related genera and perhaps extin ct ancestral species. For instan ce, sweet oranges are thought to be predominantly of mandarin germplasm introgressed with pummelo (Barrett and Rhode, 1976; Nicolosi et al., 2000). Although some studies re vealed a large molecular heterogeneity in mandarin accessions, there were nonetheless high genetic similarities among the samples. Thus, it has been concluded that manda rin is a single species, consis ting of a large number of hybrids (Coletta Filho et al., 1998). In relation to the present study, variety cl assification based on volatile compounds has been conducted in lemon essential oil components (Scora and Malik, 1970), mandarin essential oils (Merle et al., 2004), tangerine ju ice (Kerbiriou et al., 2007) and yuzu ( C junos ) peel oil (Lan-Phi et al., 2009). Since many volatiles have been found in citrus fruits, the multivariate statistical analyses such as principal component analysis (P CA) and cluster analysis (CA) have been valuable tools to differ entiate individual varieties. Comparisons of citrus hybrids with their parents have been c onducted based on aroma volatile composition in juice of interspecific hyb rids between orange and pummelo and between tangelo (i.e., a cross between ta ngerine and grapefruit) and grapef ruit (Shaw et al., 2001), leaves

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19 and peels of a somatic tetraploid hybrid betw een Mexican lime and Star Ruby grapefruit (Gancel et al., 2002), leaves of 13 interpecific and intergeneric soma tic tetraploid hybrids (Gancel et al., 2005). Shaw et al. (2001) quantita tively compared juice volatile compounds of Shamouti orange, Nakon pummelo and their hybrid. The same 39 volatile components were quantified among them, and 13 volatile s of the hybrid showed inte rmediate levels between the parents. The other compounds were about equally divided between values higher than those present in either parent, and thos e equal to or lower than those present in either parent. Although allotetraploids contain the diploid sets of ch romosomes from each parent, their qualitative (presence/absence) and quantitative (content) ar oma composition were widely different from that of parents (Gancel et al., 2002, 2005). The somatic tetraploids from mand arin and non-mandarin (lime, lemon, sweet orange, kumquat or Poncirus ) parents significantly lowered the production of most aroma volatiles of the latter parent (Gan cel et al., 2005). The same author also reported the quantitative and qualita tive levels of proteomes in the tetraploids (mandarin + lime, mandarin + kumquat) were closer to mandarin parents, suggesting that the manda rin parent was highly dominant (Gancel et al., 2006). The study on volatil e composition in the hybrids and its parents can lead perhaps to the basic knowledge of inhe ritance mechanisms of genes involved in aroma volatile synthesis. Analysis of Aroma Volatiles Gas Chromatography-Olfactometry (GC-O) Food flavor is comm only divided into the subs ets of smell and taste, which are perceived in the nose and mouth, re spectively. Odor perception can be considered the response to aroma active volatiles that enter through the nostrils (orthona sal), and aroma perception is caused by volatiles entering from the mouth and respirat ory system (retoronasal) (van Ruth, 2001).

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20 Over the last decades, the rapid progress in flavor research has been made due to improvement in instruments and analytical ch emistry. Since modern gas chromatography was invented in 1952 (Bartle and Myer s, 2002), it has allowed research ers to separate, identify and quantify volatile compounds. The application of GC to evaluate sensory quality of food aroma was first published by Fuller and coworkers in 1964 (Zellner et al., 2008). Later, Dravnieks and O'Donnella (1971) reported more sophisticated GC-O system with humidified air to reduce nasal dehydration. Until now, GC-olfactometry is the most appropriate analytical solution that uses human assessors to detect odor active co mpounds eluting from a GC separation. An individual volatile com pound has essentially three prope rties related to its odor potential or activity for humans: absolute thresh old, intensity as a function of concentration and quality (Delahunty, 2006). It is es sential to estimate the sensoria l contribution of each component to overall food aroma by using it s odor detection, threshold, perceived odor intensity and duration of odor activity as well as odor qualit y. Therefore, various GC-O methods have been developed in the past two decades: detection fr equency method, dilution to threshold analysis (AEDA) and CharmAnalysisTM, and direct intensity (Osme) and posterior intensity methods. GC-O Methods The trad itional threshold analysis of food flavor is measured in water or in air (Plotto et al., 2004). The ratio of concentration to its odor threshold is calculated to estimate th e importance of each aroma active compound, and this value is called aroma value, odor unit, odor value, flavor unit or odor active value (Delahunty et al., 2006). In dilution analysis, an aroma extract is diluted, usually as a series of 1:2 or 1:3 dilu tions, and each dilution is analyzed by GC-O until no odor is perceived by panelists (Acr ee et al., 1984). Panelists record individual response (yes or no) to a dilution and usually record an odor de scription. In AEDA first proposed by Ulrich and Grosh (1987), the panelists evaluate samples in increasing dilution order. The maximum dilution

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21 at which odor can still be perceived is expressed as dilution factor (FD) value. Another type of method, CharmAnalysisTM (Acree et al., 1984), presents the dilutions in randomized order to avoid bias introduced by knowledge of the samples. Dilution analysis is the most widely applied method (Zellner et al., 2008), however, this met hod does not consider the actual odor intensity of aroma volatiles in intact samples. Moreover, dilution procedures are time-consuming and therefore the number of a ssessors is very limited. The detection frequency method proposed by Linsse n et al. (1993) requi res a panel of 6 to 12 assessors that smell the GC effluents once, whereas dilution analyses require only1 to 3 panelists that perform the olfactory test multip le times. Pollien et al. (1997) developed this method for more consistent resu lts and no training for the panelists. The number of assessors detecting an odorant at a particul ar retention time is used as an estimate of the odor intensity. Thus, this method also does not present actu al intensities of odor in food samples. As concentration increases, odor intensity may also contribute to increa se; however, detection frequency can not increase. This method is time-s aving and easy to handle, and therefore it has been used to determine aroma impact components in several food products: guava fruit puree (Jordn et al., 2003), hand-squeezed juices of four orange varieties (Arena et al., 2006), and wine (Falco et al., 2008). The direct intensity or Osme method was deve loped by McDaniel et al. (1990) to directly measure the perceived odor intensity using tr ained panelists. Therefore, this method is differentiated from the others due to its consideration of psyc hophysical law. The odor intensity, duration of odor activity and verb al descriptor of each compound are recorded with variable scales and computer-based device s. The generated figure of reten tion time (or index) vs. average maximum odor intensity is called an Osmegram, representing the significance of each compound

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22 in food aroma. Although this method is time-saving and requires a small nu mber of panelists, it has not been applied to many studies: apple (Plo tto et al., 2000), cooked mussels (Guen et al., 2000), cashew apple (Garruti et al., 2003) and grapefruit oil (Lin and Rouseff, 2001). The comparison of global analysis based on detectio n frequency, Osme and AEDA showed that their results were well correlated, especially for the most potent odorants in cooked mussels (Guen et al., 2000). Also, van Ruth (2004) compared the de tection frequency and time intensity method for 6 volatile compounds, and concluded higher re peatability of the former method and higher discrimination of the latter method. Thus, me thod selection depends on the study objective, performance of panelists and time availability. SPME Method Arom a volatiles tend to reside in the headspace above solid or liquid, being less soluble in the presence of water. The concentration of volatiles above food products ranges from about 10-4 to 10-10 g/L (Reineccius, 2005), and hen ce extraction of aroma volatiles is a critical factor for the following analyses using GC-MS, GC-O, etc. Vo latile compounds in citrus fruits can be extracted by several different methods: distilla tion (Kirchner et al., 1953 ; Radford et al., 1974), static headspace (Nisperos-Carriedo and Sh aw., 1990), solvent extraction (Buettner and Schiertle, 1999; Chisholm et al., 2003), and a pur ge and trap method (Cadwallader and Xu, 1994; Shaw et al., 2000). However, disadvantages of these methods are artifact production, the limited number of detected volatiles, and compositional changes as we ll as time-consuming extraction procedures. The SPME method, first developed by Pawliszy ns group (Arthur and Pawliszyn, 1990), is a solvent-free, rapid, simple and relatively inexpensive technique that can reduce or eliminate the above problems. SPME involves exposing a fused s ilica with a polymeric coating to a sample or its headspace. The absorbed volat iles are thermally desorbed in the injector of a GC, GC-MS or

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23 GC-O. So far, this method has been widely app lied to the studies on citrus juice volatiles. Rega et al. (2003) reported the optimized SPME conditi ons for orange juice flavor by sniffing the overall SPME extracts and eval uating their odor repr esentativeness. This study provided the valuable information regarding to fiber selection and sample equilibrium time for SPME of citrus aroma volatiles. Citrus Aroma and Flavor Am ong 6 closely related genera as described earl ier, most flavors of commercial value are found in the genus Citrus and subgenus Eucitrus (Rouseff and Prez-Cach o, 2007). Citrus flavor is the result of complex combina tions of aroma volatiles and soluble solids. An odor is usually elicited by a combination of vol atile compounds each of which imparts its own smell. Soluble solids are mainly comprised of sugars (e.g., sucr ose, fructose and gluc ose), organic acids (e.g., citric acid and malic acid), and the flavonoi d subgroup. These components are responsible for three basic tastes, sweetness, sourness, and bitterness, respectively. Most of our knowledge about citrus volatiles has been gained from studies of processed juices and the peel essential oils essence oils, and aqueous essences used to flavor juice products (Shaw, 1991). Conversely, studies on aroma volatiles in fresh citrus fruit have not much been reported. Most citrus aroma volatiles are mainly classified into terpene hydrocarbons, aldehydes, esters, alcohols and ketones, and many are commonl y found within citrus species. Some citrus species, such as lemon and grapefruit, contai n one or two major aroma impact compounds. For instance, a terpene aldehyde citral is considered to be one ma jor aroma impact volatile in lemon. However, no single volatile in tangerine/mandari n and orange can be considered a single characteristic impact compound (Shaw, 1991). Aroma and flavor of commercially important citrus fruits, tangerine/manda rin, orange and grapefruit/pummelo, are described below.

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24 Tangerine/Mandarin Aroma Com pared to orange fruit, very little information about tangerine/mandarin aroma and flavor is available, perhaps because tangerine is mostly consumed as fresh fruit and hence its juice and peel is less used for the beverage a nd fragrance industry. Furt her, tangerine/mandarin juice products have been more difficult to ma rket because of undesirable off-flavors during processing and storage (Moshonas and Shaw, 1997). The aroma volatiles in mandarin (tangerine) peel oil have been relatively well studied. The major volatiles in tangerine essence and peel oil are d-limonene, myrcene and -pinene, together accounting for more than 90% of all detected compounds (Moshonas and Shaw, 1974; Coleman and Shaw, 1972). However, these volatiles may not be major aroma active compounds due to their high thresholds (Plotto et al ., 2004). The characteristic smell of mandarin peel oil has been believed to be caused by methyl N-methylanth ranilate and thymol. Wilson and Shaw (1981) reported that it was necessary to add -pinene and -terpinene with methyl N-methylanthranilate and thymol, in order to give the tangerine peel oil an aroma similar to that of Sicilian mandarin peel oil. Chisholm et al. (2003) found almost 50 odor active compounds in Clementine peel oil using CharmAnalysisTM, and approximately 80% of its aroma was due to the aldehydes. Buettner et al. (2003) also analyzed arom a volatiles in Clementine peel oil by AEDA. Of 42 odor active compounds detected, linalool, ( E ,E )-2,4-decadienal, wine lactone, -pinene, myrcene and octanal showed high FD factors. Interestingly, many aroma active compounds in these studies impart non-citrus odor notes, described as green, metallic, floral etc. Sawamura et al. (2004) identified 39 volatile compounds in cold-pressed oil of Ponkan ( C reticulata ) and characterized their odors by AEDA technique. Coupled with sensory evaluation, it was concluded that two aldehydes, octa nal and decanal, played an im portant role as Ponkan-like odor along with (R)-(+)-limonene as a backgr ound component for the overall aroma.

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25 The studies on tangerine/mandarin juice aroma s eem to have been conducted mostly in the main producing countries such as Japan and Spain. Yajima et al. (1979) identified 68 and 72 aroma volatiles in aroma concentrate from ju ice and in peel oil of Satsuma mandarin ( C unshiu ), respectively. Although most of the same hydrocar bons were identified in both samples, they consisted of 60.5 and 98.0% of total content, respectively. Therefor e, the qualitative (presence/absence) and quantitative (cont ent) composition of the oxygenated compounds differed widely. More recently, Prez-Lpez and collaborators (2006a ) quantifie d volatile compounds in two Spanish mandarin (Fortuna a nd Clemenules) juices by GC-MS. This study focused on how pasteurization and storage of ju ice affected on the vol atile compounds, and only five compounds were quantified. Orange Aroma Orange juice is the m ost widely consumed fru it juice in the world. Many aroma volatiles of freshly squeezed and processing orange juices ha ve been reported. Up to now, more than 300 volatiles have been reported in fresh orange juice. However, less than 25 appear to have significant odor activity (Prez -Cacho and Rouseff, 2008). These compounds mainly consist of terpene hydrocarbons, aldehydes, esters and alcohols. Terpenes are one of the most abundant groups of volatiles in freshly squeezed orange juice (Nisperos-Carriedo and Shaw, 1990; Bylaite and Meyer, 2006). Although limonene usually accounts for 90% of all terpenes in orange, the ro le of limonene for overall aroma has not been clearly proven (Shaw, 1991; Bazemore et al., 1999) It might be possible that limonene functions as a lifting agent for other vol atiles in a similar way as etha nol does in wine (Prez-Cacho and Rouseff, 2008). The odor qualities of major terpenes are usually characterized as citrus-like and minty (limonene), ethereal and piney ( -pinene), wet soil and mossy (myrcene) and terpene-like and pungent ( -pinene). A sesquiterpene valencen e is also present in orange

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26 juice in high quantity (Nisperos-Carriedo and Shaw, 1990; Bylaite and Meyer, 2006), however, its contribution to orange juice aroma has not ye t been verified in GC-O studies (Hinterholzer and Schieberle, 1998; Buettn er and Schiebertle, 2001b). Aroma active aldehydes are f ound in several different fo rms: saturated aliphatic, unsaturated aliphatic, terpenic and phenolic aldehydes (Prez-C acho and Rouseff, 2008). Most aldehydes are major contributors to the fresh, pungent odor quality of orange juice (Buettner and Schieberle, 2001b). Shaw (1991) re ported that the three homologous straight-chain aldehydes, octanal, nonanal, and decanal were implicated as important contribu tors to orange juice aroma. Hinterholzer and Schieberle (1998) characterized their odor qualitie s as green, citrus-like and soapy, respectively, and found ( Z )-hex-3-enal (green and leaf-lik e odor) had the highest FD factor among all the detected aldehydes. Esters are well known to impart a fruity note to orange juice. Of the identified esters, ethyl butanoate is present in orange juice at 500 to 10,000 times its flavor threshold value in water (Shaw, 1991) and it has been therefore been receiving attention. Indeed, dilution analysis (Hinterholzer and Schieberle, 1998; Buttner and Schieberle, 2001b) and detection frequency methods (Rega et al., 2003) showed that this compound was one of the most potent odorants in orange juice. Ethyl acetate is found at relative ly high concentration in orange juice (Moshonas and Shaw, 1987; Nisperos-Carriedo and Shaw, 1990; Moshonas and Shaw, 1994). However, the orthonasal threshold of ethyl a cetate (6,038 g/L) in reconstituted pump-out (deodorized orange juice concentrate) is significantly higher than th ose of ethyl butanoate (1.71 g/L) (Plotto et al., 2008). Therefore, ethyl acetate does not seem to ma ke a direct contribution to orange flavor (Shaw, 1991). It is clear that este rs are important to fresh orange flavor, but further research on

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27 their balance and interaction is needed to unders tand the precise role in orange flavor (Shaw, 1991). Alcohol is also major aroma component in oran ge juice. The quantity of ethanol is usually the highest among all alcohols in fresh orange ju ice. Although a large nu mber of alcohols are isolated from orange juice, only a few are consid ered to contribute to or ange juice aroma (Shaw, 1991). Three aliphatic alcohols, 1-hexanol, ( Z )-3-Hexen-1-ol and 1-octa nol, were reported as aroma active compounds in orange ju ice (Rega et al., 2003), as well as terpene alcohols such as linalool and geraniol (Mahattanata wee et al., 2005). Linalool is th e most potent alcohol in orange juice, characterized as flora l and sweety (Shaw, 1991; Hi nterholzer and Schieberle, 1998; Buttner and Schieberle, 2001b) Also, a terpene ketone -ionone is characterized as strong floral odor. Mahattanatawee et al (2005) reported that linalool and -ionone each contribute 22% of the floral note in orange juice aroma. Grapefruit and Pummelo Aroma Grapefruit is thought to be derived from a backcross between pumm elo and sweet orange (Nicolosi et al., 2000). Grapefruit flavor is us ually distinguished from mandarin/tangerine and sweet orange due to its characteristic sourne ss and bitterness. The b itterness is caused by the accumulation of flavanone-glycosides, mostly flavanone neohesperidosides such as naringin (Frydman et al, 2004). On the other hand, non-bitt er citrus fruits contain mostly tasteless flavanone rutinosides. The major pummelo varie ties lack the bitterness characteristic of grapefruit, but bitter pummelos do exist (Hodgson, 1967). The volatile composition in grapefruit is simila r to that of mandarin/tangerine and orange (Cadwallader and Xu, 1994; Shaw et al., 2000). However, nootkatone (MacLeod and Buigues, 1964) and 1-p-menthene-8-thiol (Demole et al., 1982) have been considered as the primary flavor-impact compounds in grapefruit. Nootkatone is used commercially in artificially flavored

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28 grapefruit beverages and perfumes (Wilson a nd Shaw, 1978). The high level of nootkatone is generally associated with pummelo and some pummelo hybrids including grapefruit. For instance, the high content was detected in cold-pressed oils from 8 pummelo varieties (Sawamura et al., 1991) and peels of 10 grapefruit varieties (Ort uo et al., 1995). However, the threshold of nootkatone is high as previously determined, about 1 ppm in water (Berry et al., 1967; Haring et al., 1972). Its contribution to overall grapefruit aroma is still unclear. Since nootkatone levels of grapefruit increases duri ng fruit maturation (del Ro et al., 1992) and postharvest treatment (Biolatto et al., 2002), it is proposed as an indicator of fruit quality. 1-p-Menthene-8-thiol has been proposed as a character impact compound due to its low odor threshold and high content in grapefruit juice. Demole et al. (1982) determined the threshold of 1.0 10-7 ppm in water and found 200-fold higher concentration in the juice. Using the AEDA method, Buettner and Schieberle (1999) have reported that 1-p-menthene-8-thiol significantly contributed to grap efruit aroma, along with severa l other compounds such as ethyl butanoate, ( Z )-3-hexenal and 1-hepten-3-one. Aroma Volatile Biosynthetic Pathways A large number of arom a volatiles have already been found in plants but most of the enzymes and genes involved in volatile produc tion are still unknown (Schwab et al., 2008). Various aroma volatile compounds with saturated, unsaturated, single-chain, branched-chain and cyclic structures are derived from carbohydrate, fatty acid and amino acid pools. The characteristic flavor of fruits, su ch as bananas, peaches, pears (a ll climacteric fruits) and cherries (non-climacteric fruit), develops entirely during a rather brief ripening period (Reineccius, 2005). Several studies on apple aroma synt hesis showed that ethylene played an important role in fruit ripening and also expression of genes related to volatile synthesis (Schaf fer et al., 2007). Since esters contribute greatly to aroma of many fruits (e.g., apple, melon and banana), most of the

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29 studies on aroma synthesis have focused on the last step of ester fo rmation with alcohol acyltransferase (AAT). As mentioned earlier, it seems that several terp enes, esters, aldehydes, alcohols, etc, contribute to citr us aroma. These compounds are mainly derived from terpenoids, fatty acids and amino acids. Terpenoid Aroma Volatiles Terpenoids are the largest and m ost diverse family of natural products, consisting over 40,000 individual compounds (Aharoni et al., 2005). Th e terpenoids play di verse functional roles in plants as hormones (gibberell ins, abcisic acid), photosynthetic pigments (phytol, carotenoids) and electron carriers (ubiquinone, plastoquinone ) (McGarvey and Croteau, 1995). In addition, monoterpenoid (C10H16) and sesquiterpenoids (C15H24) commonly found in citrus species serve as attractants for seed-dispersing animals, compon ents of desirable aroma/flavor, and flavorings and fragrances for human beings. In Citrus terpenes are produced especially in le aves, fruit epidermis (flavedo) and juice (Dornelas and Mazzafera, 2007). Terpenoids are derived from common terpene building units isopentenyl diphosphate (IPP) a nd its isomer dimethylallyl diphospate (DMAPP). These two compounds are de novo synthesized from acetyl CoA via the acetate-mevalonate pathway in cytoplasm, and pyruvate with gyceralde hydes-3-phosphate via the non-mevalonate (methylerythritol phosphate, MEP) pathway in plastids (Tholl, 2006) (Figure 2-1). The cytosolic pathway is responsible for the synthesis of sesquiterpenes, whereas monoterpenes and carotenoids are produced in pl astids. Following the isomerization of IPP to DMAPP by isopentenyl diphosphate (IPP) isomerase, both compounds are condensed to form geranyl diphosphate (GPP; C10). Subsequently, farnes yl diphosphate (FPP; C15) and geranylgeranyl diphosphate (GGPP; C20) are formed by sequential addition of IPP. GPP, FPP and GGPP are the direct precursors of terpenoids, which are s ynthesized to monoterpen es, sesquiterpenes and

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30 diterpenes, respectively. Moreover, several aroma volatiles such as -ionone and -damacenone are derived from carotenoids which precu rsor is GGPP (Baldwin et al., 2000). Recently, several studies on terpene synthases have been reported in Citrus. Maruyama et al. (2001) reported the cloning and functional expression of the ( E )-farnesene (sesquiterpene) synthase gene from young leaves of Yuzu ( C junos ). This is the first report of the cloning of a terpene synthase from a Rutaceous plant. Lck er et al. (2002) isolat ed four monoterpene synthase cDNAs by random sequencing of fl avedo-derived cDNA library of lemon ( C limon). These cDNAs were functionally expressed in Escherichia coli and three different major products, (+)-limonene (two cDNAs), -pinene and -terpinene were identified by GC-MS using the cDNA-encoded enzyme and GPP. Sharon-Asa et al. (2003) and Shimada et al. (2004) also conducted similar studies with valencene synthase of Valencia or ange and monoterpene synthases of Satsuma mandarin, respectively. The former study showed valencene accumulation and the gene expression was responsive to ethylene accumulated toward fruit maturation, suggesting its importance for aroma production even in non-climacteric citrus fruit. Fatty Acid Derived Aroma Volatiles The m ost common fatty acids in citrus juices are palmitic, palmitoleic, oleic, linoleic and linolenic acids (Nordby and Nagy, 1969). Galactolip ids and phospholipids are rich in lipoprotein membranes of fruit cells and sub-cellular or ganelles such as mitochondria and lamellae of chloroplast (Goldschmidt, 1977). During the fruit ripening stage, membrane degradation occurs, resulting in the release of free fatty acids. Many aliphatic straight-chain alcohols, aldehydes, ketones, acids and esters are formed from fatty acids via three processes: -oxidation, -oxidation and the lipoxygenase pathway. The oxidation of unsaturated fatty aci ds forms aldehydes and their subs equent reduction to alcohols. Rowan et al. (1999) demonstrated th at straight chain ester volatile s were derived from fatty acids

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31 via the above three processe s in apples, and several C6 aldehydes and alcohols were intermediate products from linolenic and linoleic acids to esters (F igure 2-2). For instance, ( Z )-hex-3-enal (C6H10O), derived from linolenic acid, is an odoractive volatile in handsqueezed juices of Valencia and Navel oranges (B uettner and Schieberle, 2001b). Amino Acid Derived Aroma Volatiles Branched-chain and arom atic volatiles (alcohol s, aldehydes, acids, esters, sulfur-containing volatiles) are derived from amino acid (Sch wab et al., 2008). Alt hough their biosynthetic pathways are still unclear, synthesis of branched-c hain esters originated from leucine, isoleucine and valine have been relatively well studied, probab ly due to their characteristic fruity odors in banana (Tressel and Drawert, 1973), strawberry (Prez et al., 2002) and apple (Matich and Rowan, 2007). Branched-chain esters as well as straight-chain es ters derived from fatty acids, are formed from the same reaction; alcohol ac yltransferase (AAT) catal yzes alcohol and acyl CoA, following the reduction of aldehyde to alcohol by alcohol dehydrogenase (ADH). In fresh orange juice, many esters have already been found by several researchers; however, only a few of these are known as aroma activ e volatiles: methyl butanoate, ethyl acetate, ethyl butanoate, ethyl-2-methylpropanoate, ethyl-2-methylbutanoate, ethyl hexanoate and et hyl octanoate (PrezCacho and Rouseff, 2008). Sulfur-containing volatiles derive d from methionine and cysteine contribute to the odor of garlic and onions (Jones et al., 2004). These volatil es are also important to some fruits such as durian (Weenen et al., 1996), passion fruit (T ominaga and Dubourdien, 2000) and pineapple (Umano et al., 1992). Several hydrogen sulfides we re found in the headspace above fresh orange, grapefruit, tangerine, lemon, lime, tangelo a nd tangor juices (Shaw and Wilson, 1982). In processed mandarin (Araki and Sakakiba ra, 1991) and orange juice (Prez-Cacho et al., 2007),

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32 dimethyl sulfide was found and its odor quality was characterized as s ulfur, giving undesirable smell to juice aroma. Improvement of Citrus Aroma and Flavor In conventional citrus breeding, breeders put a lot of efforts and tim e toward their breeding programs, in order to identify supe rior individual plants with desi rable traits. Citrus breeding has been impeded by several reproductiv e characteristics such as long juvenility, gametophytic selfand cross-incompatibility, and nucellar embryony (Talon and Gmitter, 2008). The juvenile periods of citrus seedlings range from one to tw enty years, though typically most will flower and fruit within 3 to 7 years (Talon and Gmitter, 2008). The sources of citrus genetic variation can currently arise not only from seed/bud introduction, natural mutants and sexual hybrids, but from irradiated budlines, somaclones, somatic hybrids/c ybrids and molecular genetics (Gmitter et al., 2007). However, genetic improvement of aroma a nd flavor of mandarin fruits still highly depends on the traditional breedi ng strategy from sexual hybridizati on (pollination) to selection. Therefore, the development of molecular mark ers that can associate the genotype with the phenotype of interest can greatly accelerate citrus breeding.

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33 Figure 2-1. Terpenoid aroma volatiles (Reproduced with permission from Elsevier. Tholl, D. 2006. Terpene synthases and the regulation, di versity and biological roles of terpene metabolism. Curr. Opin. Plant Biol. 9: 297-304.)

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34 Figure 2-2. Fatty acid derive d aroma volatiles (Reproduced w ith permission from American Chemical Society. Rowan, D. D., A. J. M., S. Fielder, and M. B. Hunt. 1999. Biosynthesis of straight-chain ester volatile s in red delicious and granny smith apples using deuterium-labeled precusors J. Agr. Food Chem. 47: 2553-2562.)

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35 CHAPTER 3 DISTRIBUTION OF AROMA VOLATILE C OMPOUNDS IN TANGERINE HYBRIDS AND PROPOSED INHERITANCE Introduction Florida fresh tangerines (Citrus r eticulata Blanco) as well as oranges ( C. sinensis L. Osb.) and grapefruits ( C. paradisi Macf.) are one of the largest agricultural commodities in the U.S. citrus market. While grapefruits are very sensit ive to endemic citrus canker disease, tangerines are fairly tolerant and could pr ovide an alternative to grapefru it production for the future fresh fruit market (Gottwald et al., 2002). The fresh ta ngerine fruit is widely consumed due to its desirable fruit quality of aroma/flavor and eas e of peeling. In addition, the high nutritional content from vitamin C and carotenoids in ta ngerines can benefit human health. From 2007 to 2008, five and a half million 95-lb boxes of all Flor ida tangerines (early tangerines, Fallglo and Sunburst; late tangerine, Murcott) were produced, with the valu e of production of $37.8 million (U.S. Department of Agriculture, 2009). A pproximately 60 % of Florida tangerines were utilized for the fresh fruit market. Improvement in fruit quality is one of the primary goals of fresh tangerine breeding programs in Florida. The commercial tangerine fru its are currently graded by inspectors based on only fruit appearance such as color, size and damage (U.S. De partment of Agriculture, 1997). Therefore, new quality standards that describe fla vor attributes should be created to be used in the selection of high quality fruits. The conventional citrus breeding based on crossing and selection requires a large amount of space, a nd considerable time and labor during the long juvenile period of citrus trees; su ch efforts are rather costly to conduct. In the last decades, the advancement of plant genetics and genomics has e nhanced the efficiency to improve fruit quality in breeding programs. The appl ication of molecular markers to citrus breeding may allow breeders to select efficiently superior recombinants improved for multiple traits by conventional

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36 breeding efforts, using marker-assisted sel ection (MAS) (Gmitter et al., 2007). Molecular markers associated with citrus fruit quality can be a valuable tool for genetic improvement leading to the faster release of new superior scions a nd rootstocks. High-value fruits would result in greater economic impact on the Florida citrus industry. Aroma, as well as taste, color and texture, is one of the most important quality attributes of citrus fruits. So far, the aroma volatiles of th e major processing orange and grapefruit cultivars have been well investigated. Over 300 aroma vol atiles have been already reported from gas chromatography (GC) and gas chromatography-m ass spectrometry (GC-MS) analyses on fresh orange juices (Prez-Cacho and Rouseff, 2008). It is well known that citrus aroma components are a mixture of monoterpenes, se squiterpenes, alcohols, aldehydes, acids, esters, ketones, etc. Although there have been several reports on arom a volatiles in tangerine essence and peel oil (Coleman and Shaw, 1972; Moshonas and Shaw, 1974; Buettner et al., 2003, Chisholm et al., 2003; Sawamura et al., 2004), very little information is availa ble regarding those in fresh tangerine fruits (Elmaci and Altug, 2005, Pr ez-Lpez and Carbonell-Barranchina, 2006a; Kerbiriou et al., 2007; Ba rboni et al., 2009). The information on volatile quality and quantity can be useful to evaluate tangerine fruit quality. In addition, peel a nd juice volatile composition have been analyzed for classification of different citrus fruits: yuzu ( C. junos Sieb.) cultivars (Lan-Phi et al., 2009), lemon ( C. limon Burm.) cultivars (Allegrone et al., 2006), grapefruit hybrids (Shaw et al., 2001) and tangerine/mandarin hybrids (Ker biriou et al., 2007; Barb oni et al., 2009). The differentiation of tangerine hybrids based on their aroma profiles may lead to better understanding of genetic c ontrol of aroma production. The main objectives of this study were to investigate aroma volatile compounds in a population of tangerine hybrids, and analyze inter-varietal relati onships from volatile profiles by

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37 using multivariate statistics, principal component analysis (PCA) and cluster analysis (CA). It was hypothesized that if similari ties were present, they c ould be due to common genetic background (Kerbiriou et al., 2007 ). The study on aroma volatiles present among tangerine hybrids of diverse origins would provide fundament al information on fruit quality, maturity and development of early DNA-based MAS of interesting individuals. Materials and Methods Plant Materials All tangerine hybrids were originated from the University of Florida Citrus Research and Education Center (UF-CREC) breeding program The trees were grown under the same environmental conditions of soil, irrigation, a nd illumination at the CREC groves. Fruit were harvested from November 2007 until March 2008 (Table 3-1). Twelve of the 56 tangerine hybrids evaluated in 2006 to 2007 (Kerbiriou et al., 2007) were selected for re-evaluation: sample a, c and d (three seedlings issued from a 8-9 Murcott cross); sample b and g (two seedlings of a Robinson Fairchild cross); samp le i (Fallglo Fairchild); sample l (8-9); sample m (8-10); sample n (8-9 Orlando); sample p and r (two seedlings from unknown parentage); and sample y (8-9 Val4x). Eight new hybrids and 5 named commercial cultivars as references were also included for the present study. These hybrids have various genetic backgrounds from tangerines, oranges and grapef ruits (Figure 3-1) (H odgson, 1967; Wutscher et al., 1973; Futch and Jackson, 2003a, 2003b; Jackson and Futch, 2003a, 2003b, 2003c, 2003d, 2003e). Each sample was a juice composite from approximately 50 to 60 fruits harvested from one tree. A total of 25 samples were prepared at the USDA/ARS Citrus and Subtropical Products Laboratory.

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38 Sample Preparation Fruit were soaked in warm water with 200 mL detergent (DECCO 241 Fruit and Vegetable Kleen, Monrovia, CA, USA) in a 16 L bucket, wash ed for about 30 s, and rinsed. Fruits were then sanitized in 10 L water at 30 to 35 C us ing a 100 ppm peroxyacetic acid solution (Biosafe Systems, East Hartford, CT, USA) for 3 min prior to processing in the lab. Individual fruits were cut in half on sterile foil, and juiced manually w ith an electric juicer (Model 3183; Oster, Rye, NY, USA). The fruit were juiced carefully for 3 s, avoiding any scrapi ng of the albedo or squeezing of the flavedo to prevent potential pe el components from contaminating the juice. Most of the seeds were removed, and aliquots (2.5 mL) of tangerine juice were placed in 20 mL glass vials (Gerstel, Inc., Baltimore, MD, USA) along with saturated sodium chloride solution (2.5 mL) to help drive volatiles into the headspace and stabilize any potentia l enzymatic activity. 3-Hexanone (1 ppm) was also added into the vials as an internal standard. The vials were capped with magnetic crimp caps containing Teflon-coated septa and stored at -20 C until analyzed. Optimization of Volatile Sampling Direct gas chrom atography-olfactometry (D-G C-O) is a technique to evaluate odor from headspace solid phase microextraction (SPME) extr acts (Lecanu et al., 2002). It was used to determine the optimum extraction conditions so th at the odor of the headspace extract was the most representative of that of the reference tangerine juice samples. A divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/P DMS) SPME fiber (Supelco, Bellefonte, PA, USA) was used due to its superior extraction perf ormance for orange juice (Rega et al., 2003). After a sample e quilibration time of 30 min at 40 C in a water-bath (Baxter Scientific Products, Cincinnati, OH, USA), the fiber was exposed to the juice headspace for 3, 30 or 60 min at 40 C. The fiber was then introduced into the injector of the gas chromatography (GC) (Perkin Elmer, Autosystem XL, Waltham, MA USA) for 3 min at 240 C for desorption of

PAGE 39

39 volatiles. The GC was equipped with a sniffing port (Olfactory Detector Port ODP 2, Gerstel), and a 15-cm deactivated silica column (0.32-mm in ternal diameter) to assess global odor without chromatographic separation (Jouquand et al., 2008). The flow rate of the carrier gas (He) was 18 mL min-1, and the oven temperature was kept at 70 C. A similarity test was performed in triplicate for the different extracts (3, 30 and 60 mi n of fiber exposure times) with three panelists. Each panelist smelled the odor of the extract from the GC sniff port and then rated their similarity to the sample using a 10-cm scale ranging from 0 (far from the sample) to 10 (close to the sample). As a result, th e condition of 60 min fiber expos ure time generated the most representative tangerine odor and therefore was applied to further volatile analyses (Table A-1). Headspace Sampling a nd GC-MS analysis The extraction of arom a volatiles was perf ormed by a SPME method with an MPS-2 autosampler (Gerstel). The vials were incubated at 40 C for 30 min and then a 2-cm SPME fiber (50/30 m DVB/CAR/PDMS) was inserted into the h eadspace of the sample vial and exposed for 60 min. The analytes were thermally desorbed in the GC injector (splitless mode) port for 3 min at 250 C. The separation of volatile com pounds was accomplished using an Agilent 6890 GC (Agilent Technologies, Santa Clara, CA, US A) equipped with DB-5 (60 m length, 0.25 mm i.d., 1.00 m film thickness; J&W Scientific, Fols om, CA, USA) and DB-Wax (60 m length, 0.25 mm i.d., 0.50 m film thickness; Agilent Technologies Santa Clara, CA, USA) columns, coupled with a 5973N MS detector (Agilent Technologies). The column oven was programmed to increase at 4 C min-1 from the initial 40 C to 230 C, then ramped at 100 C min-1 to 260 C and held for 11.70 min for a total ru n time of 60 min. Helium was used as carrier gas at flow rate of 1.5 mL min-1. Inlet, ionizing source, and transfer line were kept at 250, 230, and 280 C, respectively. Mass units were monitored from 40 to 250 m/z and ionized at 70 eV. Data were

PAGE 40

40 collected using the ChemStation G1701 AA data system (Hewlett-Packard, Palo Alto, CA, USA). Samples were run in triplicate on a DB-5 column, with a blank run between each hybrid to assure fiber cleanness between samples. A mixture of C-5 to C-15 n-alkanes was run at the end of each day to calculate retention indices (RIs) (Goodner, 2008). Samples were also analyzed (one run per sample) on a DB-Wax column to identify potential co-eluting compounds on the DB-5 column. Volatile Compound Identification Volatile compounds we re identif ied by the comparison of their retention indices (RI) and mass spectra with library entr ies (NIST/EPA/NIH Mass Spectral Library, version 2.0d; National Institute of Standards and Technology, Gaithers burg, MA, USA). Chemical authentic standards, when available, were run on both columns and their RIs and spectra confirmed compound identities. Statistical Analyses Volatile compounds we re semi-quantified by calculating each peak area relative to the peak area of the internal st andard. A principal component an alysis (PCA) was performed to differentiate individual samples based on thei r volatile composition using XLSTAT software (Addinsoft, Paris, France). With the data transformed into presence/absen ce (1/0) of volatiles, a cluster analysis was performed to find correla tions between volatile composition and sample origin (Kerbiriou et al., 2007). Clusters were formed using th e unweighted pair-group average method with arithmetic mean (UPGMA) and the Ku lczynski coefficient, to measure similarities between samples (XLSTAT, Addinsoft). Results and Discussion A total of 203 com pounds were identified by GC-MS in the 25 samples (Table 3-2 and 33). The average number of volatiles per sample was 77, ranging from 52 to 118, and more than

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41 77 compounds were detected in 12 samples. The volatiles were widely distributed among samples, however, only a small number of volatiles (5 to 26 volatiles) cont ributed to more than 90% of the total content. Many volatile compounds in Table 3-2 and 3-3 have already been reported in tangerine essence, peel oi l and juice (Moshonas and Shaw; 1972, 1974, 1997; Coleman and Shaw, 1972; Minh Tu et al., 2002; Elmaci and Altug, 2005; Njoroge et al., 2005; Prez-Lpez et al., 2006a, 2006b; Dharmawan et al ., 2007; Kerbiriou et al ., 2007; Barboni et al., 2009). All samples contained 15 volatiles in common: ethanol, hexanal, -pinene, -myrcene, octanal, -terpinene, p-cymene, d-limonene, terpinolene, dehydro-p-cymene, linalool, nonanal, decanal, -terpineol and d-carvone (Table 3-2). Although the samples have various genetic backgrounds from mandarin/tangeri ne, orange and grapefruit (F igure 3-1), the same seven monoterpenes (Table 3-2) were detected in a ll samples. d-Limonene was the most abundant compound, representing 40.6 to 82.5% of the tota l aroma volatiles. Among 22 identified monoterpene hydrocarbons, four monoterpenes were also present in relatively high amount in most samples: -myrcene (0.5.9% of relative p eak area), p-cymene (0.3.0%), phellandrene (0.5%) and dehydro-p-cymene ( 0.5.5%). The other major compounds present in large amount in samples were valencen e (0.9%), hexanal (0.1.1%) and linalool (0.5 9.5%). Conversely, fifty five volat iles were present in only one or two samples (1%, Table 33) and therefore might be more cultivar specific. Most volatiles were classified into 6 chemi cal classes: monoterpenes, sesquiterpenes, aldehydes, esters, alcohols and ketones (Table 3-4 and 3-5). The number of volatiles in these classes alone accounts for 76.8% of the total (203 volatiles). In addition, terpene-derived aroma volatiles were grouped into several classes: e g., neral and geranial in aldehyde, linalool and citronellol acetates in ester, linalool and citr onellol in alcohol and and -ionones in ketone

PAGE 42

42 (Table 3-4). Most volatiles are or iginated from enzymatic synthesis in terpenoid and fatty acid metabolisms, thereby playing an important role in aroma production in samples. Only a few volatiles were detected in other chemical classes: phenols, ethers acids and epoxides (Table 3-4 and 3-5). Principal Component Analysis (PCA) Princip al component analysis is a multiv ariate statistical method to reduce the dimensionality of the data sets into fewer compon ents when there are large amounts of variables, and identify patterns of similarities and dissimila rities in data (Iezzoni and Pritts, 1991; Ringnr, 2008). The relative peak areas ( volatile relative content) were semi-quantified by dividing the chromatographic peak areas of compounds with th at of the internal st andard. These variables were used in the Pearsons correla tions, the basis of this analysis. The first and second principal components (F1 and F2) represente d 39.80% of the total variance (Figure 3-2A). The third component e xplained an additional 11.69% of the variance (Figure 3-2B). The plot of scor es in the PCA analysis illustra tes that samples q (9-4 Blood orange4x), t (Temple) and u (Sangu inelli orange) were clearly diffe rent from the others due to their volatile profile. These samples were much rich er in volatiles than th e average (77 volatiles), since they contained 118, 109 a nd 111 volatiles, respectively. According to the first two components biplot data of this PCA analysis (Figure 3-2A), many vectors representing sesquiterpenes were in the dire ction of the first quadrant (uppe r right), along with some on the fourth quadrants (lower right), and most vectors representing este rs were located on the fourth quadrant (lower right) (data not shown). Therefore, sample q (9-4 Blood4x) may be distinguished by its high number and amount of sesquiterpenes, and samples t (Temple) and u (Sanguinelli) by their a bundance in sesquiterpenes and esters as can be seen in Table 3-5.

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43 Another type of PCA analysis was performed using relative peak areas of total volatiles in each 11 chemical classes (Figure 3-3). Ketones a nd alcohols were highly correlated with each other, as well as correlated with aldehydes. Samples u (Sanguinelli) and t (Temple) had high scores on PC1 (esters, other, ketones and alcohols ), sample y (8-9 Val4x) had a high score on PC2, and samples s (Murcott) and v (Fortune Murcott) had relativel y high negative scores on PC2. In general, this graph shows that the samples on the right side, q (9-4 Blood4x), s (Murcott), t (Temple), u (Sa nguinelli), v (Fortune Murco tt), w (Ortani que) and y (8-9 Val4x) showed high total content of volatiles except of ethers and phenols. Indeed, sample t (Temple) produced 3.1, 6.9 and 5.3 times more se squiterpenes, esters and ketones than the averages of all samples, respectively (Table 3-5). Sample u also contained high amounts of sesquiterpenes, esters and alc ohols. On the left side of Fi gure 3-3, the samples with more mandarin/tangerine genetic com ponents had less volatiles. Cluster Analysis (CA) Based on Qualitative Volatile Composition The m ain goal of cluster analysis is to assi gn individual samples into groups. This analysis is based on distance measures between groups of variables (clusters), being often applied with molecular marker techniques to assess ge netic relationship among samples based on presence/absence of certain genes or alleles (Coletta F ilho et al., 1998; Chao et al., 2004). In the present study, a dendrogram provides informa tion on volatile qualitati ve differences among samples as well as relationships of their vola tile composition and gene tic background (Figure 34). Cluster 1 (C1) Cluster 1 grouped sam ples a (8-9 Murcott ) and b (Robinson Fairchild) that contain less volatiles than the average of all sa mples. These samples are characterized by having less aldehydes (except sample b), esters and ke tones (Table 3-6). The aliphatic aldehydes,

PAGE 44

44 pentanal, ( E )-2-hexenal, heptanal and (E )-2-heptenal were not detected in sample a (8-9 Murcott), but were in all other samples. Sa mple b (Robinson Fairchild) was the only sample without any esters; esters usually impart a fruity note to orange juice (Plotto et al., 2008). On the other hand, these samples were the riches t in monoterpenes among the 5 clusters both in quality (Table 3-6) and amount (Table 35). Above all, the highest amounts of -myrcene, dlimonene and -terpinene contribute to the distinctive volatile profile of sample a (8-9 Murcott). Since samples a (8-9 Murcott) and b (Robins on Fairchild) are grouped in the same cluster as determined by their volatile profile, it is hypothesized that they have similar genetic background. Indeed, they both have Clementine as a grandparent: 8-9 is a cross between Clementine and Minneola. Robinson and F airchild are both hybrids from Clementine and Orlando (Figure 3-1). Cluster 2 (C2) Cluster 2 grouped sam ples c (8-9 Murcott) an d l (8-9), and had le ss volatiles than the average samples, especially less sesquitepene s and aldehydes, like cluster 1 (Table 3-6). According to the relationship between volatile composition and parentage, sample c (8-9 Murcott) as well as sample a (8-9 Murcott) in C1 was more similar to sample l (8-9) than s (Murcott) in terms of qu alitative volatile composition, indicati ng that the seed parent might be a dominant parent for its aroma volatile production. However, it is not a rule since sample d (89 Murcott) is in the same cluste r 3 as its parent Murcott (sampl e s). It has been observed that some volatiles in parents are not synthesized in progenies, and conversely some new volatiles are synthesized in progenies (Ker biriou et al., 2007; Barboni et al., 2009). In the current study, 1octen-3-one was found in both pare nts (sample l, 8-9; sample s, Murcott), whereas it was absent in the progenies, samples c, a and d (all are 8-9 Murco tt). This volatile is the most

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45 intense odor active aliphatic ket one, imparting a mushroom-like odor to orange juice (PrezCacho and Rouseff, 2008). The content of 1-octe n-3-one in sample s (Murcott) was the highest and more than twice the average of all samples. Thus, its produc tion was likely to be suppressed in the progenies. Moreover, samples c, a and d (all are 8-9 Murcott) do not contain aroma volatiles from carotenoids, incl uding neral and gera nial, which are also synthesized from geraniol (Lewinsohn et al., 2005). Their seed parent (sample l, 8-9) contains 6-methyl-5-hepten2-one and geranyl acetone, degraded from lycopene and -carotene, respectively. In addition, cyclocitral, -ionone and dihydroactinidiolide, degradation products from -carotene, were only present in the pollen parent (sam ple s, Murcott). For example, -ionone is well known as an aroma active compound, and its odor is usually described as floral in orange juice (Hinterholzer and Schieberle, 1998; Mahattanatawee et al., 2005). This suggests that the carotenoid degradations to aroma volatiles might be regulated by that spec ific hybridization. Some sesquiterpenes ( -cubebene, -caryophyllene, -muurolene, calamenene) were detected in the progenies, but not in their parents (sample l, 89; sample s, Murcott). These compounds might be inherited from the pare nts with additive genetic effects. Cluster 3 (C3) Cluster 3 grouped 12 samples equally divided into ones that contain m ore or less volatiles than the average of all samples (77 volatiles) Most samples share common genetic background with others in this cluster. Samples d (8-9 Murcott), v (Fortune Murcott) and x (8-8 Murcott) have the common pollen parent Murcott. Also, sample s j and k are siblings of the same parents, Fallglo and Fairchild. Samples e, g and h are siblings originated from the same cross between Robinson and Fairchild. Sample e as well as b in C1 (both are Robinson and Fairchild) were harvested on November 16th, 2007, which had 61 and 66 volatiles, respectively. Samples g and h (both are Robi nson and Fairchild), harvested on December

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46 14th, 2007, contained 84 and 82 volatiles, respec tively. These samples are originated from different trees (siblings) of the same parents, though their volatile composition of early and late harvests was quite different, highlighting the re lationship between aroma volatile production and fruit maturity. Cluster 4 (C4) Cluster 4 contained samples with higher than av erage number of volatiles except sample n (8-9 Orlando). Samples in this cluster contai n a distinctly higher numb er of volatiles in the sesquiterpenes and esters categor ies (Table 3-6). Samples t (Tem ple) and u (Sanguinelli), the outliers in the PCA analysis, ar e grouped in this cluster due to their distinctive volatile composition, as previously mentioned. Sample n (8 -9 Orlando) is also characterized by its richness in esters, and samples i (Fallglo Fairchid), w (Ortanique) and y (8-9 Val4x) are characterized by their high content of sesquiterp enes and esters. With regard to samples u (Sanguinelli), w (Ortanique) and y (8-9 Val4x), the sesquite rpene valencene was the second most abundant volatile, accounting for 12.5, 24.8 a nd 11.9% of relative pe ak area, respectively (data not shown). Nootkatone, a pu tative derivative from valencen e, was also detected in the three samples and sample t (Temple). This compound is a major aroma impact compound that contributes to characteristic aroma and flavor of grapef ruit and pummelo (MacLeod and Buigues, 1964; Sawamura and Kuriyama, 1988). These samples are grouped in the same cluster, probably because they have some orange genetic background that brings th e production of esters. Kerbiriou et al. (2007) had f ound a similar clustering in 20062007. Temple and Ortanique are believed to be tangors, or iginated in Jamaica (Hodgson; 1967; Blazquez, 1967; Jackson and Futch, 2003e). Sanguinelli is a blood orange. Val4x is a tetraploid Valencia orange. Sample i (Fallglo Fairch ild) does not have a direct orange parent, however, its orange background may be originated from grandpa rent Temple (Figure 3-1).

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47 Cluster 5 (C5) Cluster 5 g rouped samples that are outliers based on their volatile composition. Samples p and r are themselves in a sub-cluster (Figure 3-4). The parentage of samples p and r are unknown, though they may be originated from the same parents due to their very similar volatile composition. The marked characteristics of thes e samples are significan tly lower amounts of total volatiles and an absence of sesquiterpenes In addition, aldehydes are rich in samples p and r, accounting for 31.0% and 33.9% of relative pe ak area, respectively. 2-Methyl-2-hexenal, ( E ,Z )-2,4-heptadienal and ( E ,E )-2,4-heptadienal were detected onl y in the three samples in this cluster. Most aldehydes present in samples ha ve already been found in many citrus fruits (Sawamura and Kuriyama, 1988; Shaw, 1991; Sh aw et al., 2000, Allegrone et al., 2006; Kerbiriou et al., 2007; Prez-C acho and Rouseff, 2008), and they are well known to impart green and fatty notes. Therefore, the hi gh level of aldehyde cont ent might negatively influence the overall aroma of samples p and r as well as q (9-4 Blood4x). As shown in Figure 3-2, Table 3-5 and Table 3-6, sample q (9-4 Blood4x) is also clearly characterized by a high number and quantity of sesquiterpenes Sample q (9-4 Blood4x) contained 33 compounds of the 39 different sesqu iterpenes detected in the 25 samples. This sample and sample y (8-9 Val4x) in C4 have two-thirds chromosome complement from Blood4x (tetraploid Ruby blood orange) or Val4x, respectively. While cluster 4 grouped samples that have some orange in their backgrou nd, this sample is differentiated from them due to its lower amount of esters. Interestingly, although sample q (9-4 Blood4x) contained the highest number of volatiles (118 volatiles) (Table 3-6), their amount was only slightly higher than the average of the 25 samples and similar to those of the siblings of 9-4, or 8-9 and 8-10 (all are ClementineMinneola) (T able 3-5). Sample y (8-9 Va l4x) in C4 contained higher

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48 number of volatiles (93) than its parent -9 (65) (Table 3-6), wh ereas the total content is similar to that of the parent (Table 3-5). From these results, it may be deduced that sa mples q (9-4 Blood4x) and y (8-9 Val4x) can synthesize aroma volatiles from the pollen pa rents, though the total volatile content is regulated by the seed parents. So far, inheritanc e of genes involved in vola tile synthesis and their expression in sexual citrus progenies are still unk nown. Gancel et al. (2005) showed that leaf volatile composition of a somatic hybrid (2n = 4x = 36) between W illow leaf mandarin ( C deliciosa Ten.) with sweet orange was similar to that of the mandarin parent. Since the parents 94 and 8-9 have background from mandarin, its gene tic components might play an important role in the control of aroma production. The volatile difference among hybrids provide s fundamental information for future improvement in tangerine aroma and flavor. Fu rther research is needed to understand the relationship between aroma volatiles and sample aroma/flavor, as described in Chapter 4.

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49 Table 3-1. List of samples and corresponding se lection names or parentage, hybrid numbers, harvest dates and sample codes Selection name or parentage Hybrid number Harvest date Sample code 8-9 Murcott 1 16 Nov. 2007 a 8-9 Murcott 2 16 Nov. 2007 c 8-9 Murcott 3 16 Nov. 2007 d Robinson Fairchild 1 16 Nov. 2007 b Robinson Fairchild 2 16 Nov. 2007 e Robinson Fairchild 3 14 Dec. 2007 g Robinson Fairchild 4 14 Dec. 2007 h Fallglo 16 Nov. 2007 f Fallglo Fairchild 1 14 Dec. 2007 i Fallglo Fairchild 2 14 Dec. 2007 j Fallglo Fairchild 3 14 Dec. 2007 k 8-9 1 14 Dec. 2007 l 8-10 1 14 Dec. 2007 m Unknown 11 Jan. 2008 o Unknown 11 Jan. 2008 p Unknown 11 Jan. 2008 r 8-9 Orlando 1 11 Jan. 2008 n 9-4 Blood4x 1 11 Jan. 2008 q Murcott 14 Feb. 2008 s Temple 14 Feb. 2008 t Sanguinelli 14 Feb. 2008 u Fortune Murcott 1 14 Feb. 2008 v Ortanique 25 Mar. 2008 w 8-8 Murcott 1 25 Mar. 2008 x 8-9 VAL4x 1 25 Mar. 2008 y Each sample is a juice composite from fruits ha rvested from one tree of individual hybrid or commercial cultivar.

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50 Figure 3-1. Pedigree of tangerine hybrids.

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51Table 3-2. List of aroma volat iles detected by GC-MS among samples and identifi ed by linear retention i ndex (LRI) on DB-5, DBwax column and confirmed with chemical standards. Volatiles are listed according to their frequency of appearance, 48% to 100%, in samples 48% to 71%a 72% to 95%b 96% to 100%c LRI LRI LRI DB-5 DB-wax DB-5 DB-wax DB-5 DB-wax ( E)-2-Pentenald 755 1167 Acetaldehyde 469 659 Ethanold 489 929 Benzaldehyded 985 1604 ( E)-2-Decenal 1262 1868 Hexanald 805 1123 Allo-ocimene 1132 1384 -Phellandrened 1025 1195 -Pinened 956 1061 -Selinene 1545 2228 -Terpinened 1073 1253 -Myrcened 999 1189 -Cubebened 1363 1496 Perillaldehyded 1293 2447 Octanald 1013 1283 -Caryophyllened 1499 2013 Cadinene 1557 2309 -Terpinened 1035 1239 -Selinene 1530 Propanald 511 747 p-Cymened 1042 1272 (-)-Panasinsen 1577 2393 Terpinene-4-old 1196 1765 d-Limonened 1049 1223 -Cyclocitral 1234 1854 p-Menth-1-en-9-al 1231 1839 Terpinolened 1100 1282 2-Undecenald 1366 2255 Copaened 1401 1551 Dehydro-p-cymened 1105 1446 ( Z )-p-Mentha-2,8-dien-1-ol 1150 1933 Caryophyllened 1458 1801 Linaloold 1105 1600 -Terpineold 1162 1796 1-Octen-3-one 988 Nonanald 1109 1384 Octyl acetated 1202 1491 6-Meth yl-5-hepten2-one 994 1316 Decanald 1205 1538 -Elemene 1409 1754 p-Menth-1-en-9-al isomer 1234 -Terpineold 1208 2026 Calamenened 1566 2631 1-Penten-3-oned 676 1057 d-Carvoned 1257 2225 -Calacorened 1593 3117 ( E)-2-Nonenald 1163 1615 Acetone 509 775 1 Unknown monoterpenee Geranyl acetoned 1454 2680 Ethyl acetated 599 870 2 Unknown sesquiterpenesf Valencened 1539 2191 Pentanald 690 1005 4 Unknownsg -Ionone 1506 3187 ( E)-2-Hexenald 864 1233 2 Unknown monoterpenese Heptanald 913 1208 2 Unknownsg ( E)-2-Heptenald 970 1315 -Phellandrened 1052 1229 ( E)-2-Octenald 1067 1433 1,3,8-p-Menthatriene 1127 1392

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52Table 3-2. Continued 48% to 71%a 72% to 95%b 96% to 100%c LRI LRI LRI DB-5 DB-wax DB-5 DB-wax DB-5 DB-wax (+/-)-4-Acetyl-1 methylcyclohexene 1144 1670 Dihydrocarvoned 1212 1819 a Volatiles detected in 12 to 17 out of 25 samples b Volatiles detected in 18 to 23 out of 25 samples c Volatiles detected in 24 to 25 samples d Volatiles confirmed with chemical standards e Unknown monoterpenes unidentified by GC-MS f Unknown sesquiterpenes unidentified by GC-MS g Unknown volatiles unidentified by GC-MS except monoterpenes and sesquiterpenes

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53Table 3-3. List of aroma volat iles detected by GC-MS among samples and identifi ed by linear retention i ndex (LRI) on DB-5, DBwax column and confirmed with chemical standards. Volatiles ar e listed according to their fr equency of appearance, 1 % to 47%, in samples 1% to 11%a 12% to 23%b 24% to 47%c LRI LRI LRI DB-5 DB-wax DB-5 DB-wax DB-5 DB-wax 2-Methyl-2-propanol 530 Ethyl propanoated 699 974 Butanald 583 861 2-Butanone 583 894 Ethyl butanoated 798 1075 Nerald 1241 2004 ( E)-2-Butenal 639 1084 Methyl hexanoated 929 1208 Geraniald 1268 2005 ( Z )-3-Hexenal 799 Ethyl hexanoated 1001 1240 Methyl acetate 531 784 1-Octen-3-ol 987 1436 Ethyl 3-hydroxyhexanoated 1128 1967 3-Pentanone 685 1002 p-Mentha-3,8-diene 1084 1271 Ethyl octanoated 1188 1427 4-Heptanone 878 1162 Octanoic Acid 1152 -Muurolene 1534 2160 4-Methyl-hexanal 889 Camphord 1171 2076 Caryophyllene oxided 1658 3584 Styrened 911 1262 Bornyl acetate 1294 Nootkatoned 1881 7433 -Thujened 942 1063 ( E, E )-2,4-Decadienald 1320 Dimethyl sulfide 529 1,8-Cineoled 1054 1228 ( Z )-Carvyl acetated 1332 Methyl butanoated 712 1004 Dodecanal 1411 2076 -Elemened 1351 Ethyl 2-methylbutanoated 856 1054 -Ionone epoxide 1511 3500 (S)-Perillyl acetate 1442 Sabinened 991 1191 Undecanald 1305 1782 -Guaiened 1459 2212 Hexyl ethanoate 1018 1266 Citronellol acetated 1341 1913 -Farnesene 1518 2200 3-Carened 1031 1187 -Pinened 1003 1149 ( E, E )-2,4-Hexadienald 923 1397 ( E)-Ocimene 1056 1241 Thymold 1289 4594 Ethyl tiglate 944 1245 -Cubebene 1463 1 788 Neryl acetated 1350 2126 1-Methylbutyl butanoate 1025 1-Penten-3-old 671 1150 Geranyl acetated 1370 2261 Methyl octanoated 1120 1373 2-Pentanone 674 1005 Selina-3,7(11)-diene 1600 2137 Hexyl butanoated 1188 2,3-Pentanedioned 683 Juniper camphor 1741 5244 ( Z )-Carveold 1229 2536 3-Methyl-butanold 729 1164 2 Unknown sesquiterpenesf Citronellold 1236 2186 Ethyl 2-butenoate 848 1194 7 Unknownsg ( E)-Carveold 1243 2664 2-Methyl-2-hexenal 884 Nonanoic acid 1248 Hexanoic acidd 965

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54Table 3-3. Continued 1% to 11%a 12% to 23%b 24% to 47%c LRI LRI LRI DB-5 DB-wax DB-5 DB-wax DB-5 DB-wax Nonyl acetated 1300 1689 2,3-Octanedione 991 Methyl geranate 1315 (E, Z )-2,4-Heptadienal 1007 1517 exo-2-Hydroxycineole acetate 1343 ( E, E )-2,4-Heptadienald 1022 1532 -Terpinyl acetated 1350 2063 Thymol methyl ether 1231 1766 Ylangene 1396 Linalool acetated 1242 1638 Decyl acetated 1403 1973 -Ionone 1439 2735 -Farnesene 1460 1918 -Elemene 1455 1890 1 Unknown monoterpenee 2,6-Bis(1,1-dimethylethyl)-phenol 1523 5439 11 Unknown sesquitepenesf Nerolidold 1585 3646 12 Unknownsg Dihydroactinidiolide 1592 3 Unknown sesquitepenesf 10 Unknownsg a Volatiles detected in 1 to 2 out of 25 samples b Volatiles detected in 3 to 5 out of 25 samples c Volatiles detected in 6 to 11 out of 25 samples d Volatiles confirmed with chemical standards e Unknown monoterpene unidentified by GC-MS f Unknown sesquiterpenes unidentified by GC-MS g Unknown volatiles unidentified by GC-MS except monoterpene and sesquiterpenes

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55Table 3-4. Tangerine aroma volat iles among 11 chemical classes Monoterpenes Sesquiterpenes Aldehydes Esters Alcohols -Thujene -Elemene Acetaldehyde Methyl acetate Ethanol -Pinene -Cubebene Propanal Ethyl acetate 2-Methyl-2-propanol Sabinene Ylangene Butanal Ethyl propanoate 1-Penten-3-ol -Myrcene Copaene ( E)-2-Butenal Methyl butanoate 3-Methylbutanol -Pinene -Elemene Pentanal Ethyl butanoate 1-Octen-3-ol -Phellandrene -Elemene ( E)-2-Pentenal Ethyl 2-butenoate Linalool 3-Carene Caryophyllene ( Z )-3-Hexenal Ethyl 2-methylbutanoate cis-p-Mentha-2,8-dien-1-ol -Terpinene -Farnesene Hexanal Methyl hexanoate -Terpineol p-Cymene -Guaiene ( E)-2-Hexenal Ethyl tiglate Terpinene-4-ol d-Limonene -Cubebene 2-Methyl-2-hex enal Ethyl hexanoate -Terpineol -Phellandrene -Caryophyllene 4-Methyl-hexanal Hexyl ethanoate ( Z )-Carveol ( E)-Ocimene -Farnesene Heptanal 1-Methyl butyl butanoate Citronellol -Terpinene -Selinene ( E, E )-2,4-Hexadienal Methyl octanoate ( E)-Carveol p-Mentha-3,8-diene -Muurolene ( E)-2-Heptenal Ethyl 3-hydroxyhexanoate Nerolidol Terpinolene Valencene Benzaldehyde Hexyl butanoate Juniper camphor Dehydro-p-cymene -Selinene ( E, Z )-2,4-Heptadienal Ethyl octanoate 1,3,8-p-Menthatriene Cadine ne Octanal Octyl acetate Allo-ocimene Calamenene ( E, E )-2,4-Heptadienal Linalool acetate 4 Unknown monoterpenes (-)-Panasinsen ( E)-2-Octenal Bornyl acetate -Calacorene Nonanal Nonyl acetate Selina-3,7(11)-diene ( E)-2-Nonenal Methyl geranate 18 Unknown sesquiterpenes Decanal ( Z )-Carvyl acetate p-Menth-1-en-9-al Citronellol acetate p-Menth-1-en-9-al isomer Exo-2-hydroxycineole acetate -Cyclocitral Neryl acetate Neral -Terpinyl acetate ( E)-2-Decenal Geranyl acetate Geranial Decyl acetate Perillaldehyde (S )-Perillyl acetate Undecanal ( E, E )-2,4-Decadienal 2-Undecenal Dodecanal

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56Table 3-4. Continued Ketones Phenols Ethers Acids Epoxides Others Acetone Thymol 1,8-Cineole Hexanoic acid -Ionone epoxide Dimethyl sulfide 2-Butanone 2,6-Bis(1,1-dimethylethyl)-phenol Thymol methyl ether Octanoic Acid Caryophyllene oxide Styrene 1-Penten-3-one Nonanoic acid (+/-)-4-Acetyl-1-methylcyclohexene 2-Pentanone 35 Unknowns 2,3-Pentanedione 3-Pentanone 4-Heptanone 1-Octen-3-one 2,3-Octanedione 6-Methyl-5-hepten-2-one Camphor Dihydrocarvone d-Carvone -Ionone Geranyl acetone -Ionone Dihydroactinidiolide Nootkatone

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57Table 3-5. Amount (relative peak area) of aroma volatiles arranged by 11 chemical classes in 25 samples. M=Murcott; R=Robinson; FC=Fairchild; FG=Fallg lo; F=Fortune; O=O rlando; T= Temple; SANG= Sanguinelli; ORT=Ortanique hybrid sample code Monoterpenes Sesquiterpenes Aldehydes Esters Alcohols Ketones Phenols Ethers Acids Epoxides Others Total 8-9 M a 27.58 0.09 0.61 0.01 0.80 0.02 0.002 0 0 0 0.01 29.13 R FC b 16.05 0.18 0.42 0 0.87 0.20 0 0 0 0 0.03 17.76 8-9 M c 9.04 0.08 0.41 0.01 0.48 0.12 0.015 0.198 0 0 0.02 10.39 8-9 M d 13.74 0.05 0.50 0.03 0.37 0.18 0 0.130 0 0 0.09 15.09 R FC e 10.36 0.03 0.83 0.06 0.36 0.25 0 0.114 0 0 0.11 12.12 FG f 3.69 0.46 0.78 0.03 0.27 0.13 0 0 0 0. 005 0.06 5.43 R FC g 6.22 0.66 0.80 0.03 0.47 0.16 0 0.08 0 0.004 0.06 8.49 R FC h 10.61 0.05 0.96 0.05 0.74 0.36 0 0.17 0 0.003 0.13 13.08 FG FC i 17.01 1.73 0.46 0.24 0.73 0.19 0 0 0 0 0.11 20.46 FG FC j 7.76 0.04 0.59 0.00 0.66 0.19 0 0 0 0.005 0.05 9.30 FG FC k 4.88 0.67 0.71 0.01 0.46 0.14 0 0.105 0 0.006 0.05 7.03 8-9 l 10.03 0.11 0.32 0.05 0.63 0.04 0.064 0.003 0 0 0.15 11.40 8-10 m 5.88 2.45 0.72 0.02 0.76 0.07 0.043 0.024 0 0 0.10 10.05 8-9 O n 8.55 0.32 0.43 0.23 0.42 0.09 0 0.157 0 0 0.03 10.22 Unknown o 5.64 0.13 0.98 0.01 0.31 0.22 0 0.068 0.004 0 0.06 7.43 Unknown p 1.75 0 0.93 0.03 0.10 0.14 0 0 0 0.003 0.03 2.98 9-4 Blood4x q 7.62 3.99 1.14 0.02 0.49 0.35 0 0 0.003 0 0.34 13.94 Unknown r 1.80 0 1.11 0.03 0.11 0.18 0 0 0 0.005 0.04 3.27 M s 11.78 0.03 1.57 0.07 0.65 0.48 0 0 0 0.003 0.12 14.69 T t 19.44 3.30 2.03 1.32 3.43 0.48 0 0 0 0 0.71 30.70 SANG u 19.05 5.56 0.99 1.11 1.35 0.63 0 0 0 0 0.35 29.03 F M v 17.63 0.03 1.07 0.35 0.72 0.35 0 0 0.003 0 0.11 20.26 ORT w 8.35 2.13 0.65 0.44 0.27 0.37 0 0 0 0 0.15 12.36 8-8 M x 4.95 0.03 0.73 0.04 0.18 0.10 0.030 0 0.003 0.002 0.05 6.10 8-9 Val4x y 6.12 4.31 0.58 0.59 0.47 0.24 0.022 0.044 0.051 0 0.33 12.76

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58 Figure 3-2. Principal component analysis by using volatile relati ve peak areas among samples. Each percent of variance explained by the three factors is shown in parenthesis. Letters refer to sample codes (Table 3-1).

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59 Figure 3-3. Principal component analysis by using volatile peak areas of classified compound categories among samples. Letters refe r to sample codes (Table 3-1).

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60 Figure 3-4. Cluster analysis by using volat ile presence and absence among samples. Lette rs indicate sample codes (Table 3-1).

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61Table 3-6. Samples in five clusters form ed by cluster analysis based on presence a nd absence of volatiles, and their number of volatiles in classified compounds. M=Murco tt; R=Robinson; FC=Fair child; FG=Fallglo; F=Fortune; O=Orlando; T= Temple; SANG= Sanguinelli; ORT=Ortanique Cluster no. Sample code Sample name Monoterpenes Sesquiterpenes Aldehydes Esters Alcohols Ketones Phenols Ethers Acids Epoxides Others Total C1 a 8-9 M 18 10 9 2 5 4 1 0 0 0 3 52 b R FC 19 11 17 0 9 5 0 0 0 0 5 66 C2 c 8-9 M 17 7 14 2 6 6 2 2 0 0 6 62 l 8-9 16 8 14 2 6 8 2 1 0 0 8 65 C3 f FG 13 8 16 2 3 8 0 0 0 1 7 58 m 8-10 18 17 21 3 6 7 1 1 0 0 15 89 d 8-9 M 13 8 16 2 4 4 0 1 0 0 10 58 e R FC 14 5 17 2 5 8 0 1 0 0 9 61 k FG FC 14 11 21 1 5 8 0 1 0 2 9 72 g R FC 14 14 23 5 6 9 0 1 0 1 12 85 o Unknown 14 12 24 2 5 9 0 1 1 0 12 80 h R FC 15 8 26 3 6 8 0 1 0 1 14 82 j FG FC 16 8 22 1 5 9 0 0 0 1 8 70 x 8-8 M 14 6 20 2 5 11 2 0 1 1 6 68 s M 15 4 26 5 7 12 0 0 0 1 12 82 v F M 15 5 24 9 7 10 0 0 1 0 9 80 C4 n 8-9 O 14 13 16 10 4 6 0 1 0 0 5 69 i FG FC 16 20 19 11 5 8 0 0 0 0 11 90 y 8-9 Val 4x 13 19 20 9 6 11 1 1 2 1 10 93 t T 14 19 24 17 7 12 0 0 0 1 15 109 u SANG 17 23 18 20 8 11 0 0 0 1 13 111 w ORT 14 20 19 10 6 12 0 0 0 1 13 95 C5 q 9-4 Blood 4x 14 33 28 4 7 9 0 0 1 0 22 118 p Unknown 10 0 26 2 5 9 0 0 0 1 8 61 r Unknown 8 0 25 2 4 9 0 0 0 1 7 56

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62 CHAPTER 4 CHARACTERIZATION OF AROMA VOLATI LES IN T ANGERINE HYBRIDS BY GAS CHROMATOGRAPHY-OLFACTOMETRY Introduction Tangerine is one of the most popular citrus c onsum ed as fresh fruit due to its ease of peeling, delicate and pleasant flavor. Aroma volatil es, as well as sugars an d acids, are critical factors to evaluate fruit quality a nd contribute to the or ganoleptic quality of fresh tangerine fruit. Since high quality citrus fruit can lead to greater economic return to the industry, improvement in fruit aroma and flavor has been one of the primar y goals of fresh fruit breeding programs. Citrus breeders have selected superior genotypes from th e segregating populations and released some of them as commercial cultivars, such as Fallglo and Sunburst tangeri nes (Jackson and Futch, 2003b; Futch and Jackson, 2003c). However, there are no available fruit quality criteria that address tangerine aroma and flavor in Florida-grown fruit that enter commercial channels (U.S. Department of Agriculture, 1997), so the succ ess of breeding programs highly depends on the ability and long experience of the breeders. The evaluation of tange rine aroma profile as well as their chemical composition has become an importa nt research objective in the breeding program, facilitating the process of screening high quality fruit and selection of in teresting hybrids with desirable aroma and flavor. The sensation of smell is triggered by aroma volatiles entering the nostrils, mouths and respiratory system (van Ruth, 2001). The improvem ent of analytical inst ruments and techniques has led to a large number of volatiles reported in fruits and vegetables. So far, over 7,000 aroma volatiles have been identified in food (Zellner et al., 2008). However, it is indicated that only a small fraction of many volatiles present in food make a direct contribution to the odor. Gas chromatography-olfactometry (GC-O) is a valuab le tool to determine aroma activity using the human nose as a detector. Aroma volatiles extr acted from samples ar e separated by the GC

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63 column and eluted from the sniffing port, from which the panelists can smell and characterize aroma active compounds. Combining this instrumental and sensor y analysis, several techniques have been proposed: aroma extraction diluti on analysis (AEDA) (Ulrich and Grosh, 1987), CharmAnalysis (Acree et al., 1984) and detection fr equency method (Linssen et al., 1993). McDaniel et al. (1990) devel oped the time intensity (Osme) method to directly measure perceived odor intensity by trained panelists. While AEDA and CharmAnalysis are based on olfactory thresholds, Osme is the only method that considers Stevens law of psychophysics which states that the response to an odor stimulus with incr easing concentration follows a sigmoidal curve (Stevens, 1957). This method has been applied to several aroma studies, among which studies on apples (Plotto et al., 2000), cashew apples (Garruti et al., 2003), grapefruit oil (Lin and Rouseff, 2001), and unpasteurized and ex cessively heated orange juice (Bazemore et al., 1999). Tangerine aroma is due to a complex combination of several volatile compounds in the proper proportions such as hydr ocarbons, aldehydes, esters, al cohols and ketones (Shaw, 1991). Among more than 300 volatile compounds reported from GC-MS studies in fresh orange juice, less than 25 appear to have si gnificant odor activity at levels found (Prez-Cacho and Rouseff, 2008). Unfortunately, there is very little information available on aroma volatiles and their odor activity in fresh tangerines. So far, GC-O has been applied to characterize aroma volatiles in only a few tangerine peel oils: Ponkan ( C reticulata ) (Sawamura et al., 2004) and Clementine peel oil (Buettner et al., 2003; Chisholm et al., 2003). Elmaci and Altug (2005) detected 26 volatile compounds in three mandarin cultivars (Satsuma, Bodrum, Clementine) using dynamic headspace analysis and a sensory descriptive panel. Based on the volatile quantitative data, they concluded the key aroma impact compounds were limonene, -terpinene, p-cymene, myrcene, -

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64 pinene, -pinene, and -terpinolene in all samples. Howe ver, their results do not give information on important aroma active compounds without consideration of odor threshold. With volatile identification and quantification by GC-MS, the GC-O technique can play an important role for assessing key aroma com pounds and later developing molecular markers associated with their synthe sis and sensory attributes. In the previous chapter, vola tiles were analyzed in 25 ta ngerine hybrids or commercial cultivars by GC-MS. This chapter describes th e characterization of ar oma volatiles in five selected tangerine hybrids using GC-O. Materials and Methods Sample Preparation Based on GC-MS analysis (Figure 3-4) and a trained taste panel (data not shown), five sam ples were selected for GC-O analysis due to their unique and diverse aromas: sample l (8-9), m (8-10), p (Unknown), v (Fortune Murcott) and y (8-9 Va l4x). Fruit received from the field were juiced on the same day and aliquots were frozen at -20 C. Juicing was done to carefully avoid incorporating any pe el oil into the juice. Aliquots of frozen juice were thawed in water, and a volume of 2.5 mL juice was introduced into 20 mL glass vials (Gerstel). Saturated aqueous sodium chloride (2.5 mL) was also adde d to help drive volatiles into the headspace and stabilize any potential enzymatic activity. The vi als were capped with magnetic crimp caps containing Teflon-coated septa and stored at -20 C until analyzed, within 3 weeks. Gas Chromatography-Olfactometry Each sam ple was equilibrated in a water-ba th (Baxter Scientific Products) for 30 min at 40C. A 2-cm SPME fiber (50/30 m DVB/Carboxen/PDMS; Supelco) was then exposed to the headspace for 60 min at 40 C. After exposure, the SPM E fiber was inserted into the injector of a gas chromatography (Perkin Elmer) to desorb th e extract for 5 min at 250 C. The GC was

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65 configured with a flame ionization detector (FID) and an olfactor y detection port (Gerstel). An HP-5 capillary column ( 30 m length, 0.32 mm i.d., 0.25 m film thickness; Agilent Technologies) was installed to separate each volatile compound. The oven temperature was 40 C for 2 min (0 to 2 min run tim e), increased to 180 C at 6 C min-1 (2 to 25.33 min), then to 250 C at 10 C min-1 (25.33 to 32.33 min) and then held for 7 min (32.33 to 39.33 min). The flow rate of the carri er gas (He) was 1.75 mL min-1. The column effluent was split (3:1) between the sniffing port and the FID, a nd the sniffing port was connected to a humidified air make-up (8.9 mL min-1). Linear retention indice s (RI) of volatile compounds were calculated using a series of n-alkanes (C-5 to C-15, C-17 and C-18) that was run under the same chromatographic conditions. Osme Analysis Training ses sions were conducted to familiarize the panelists (1 male and 2 females) with the optimum positioning, time intensity device and verbal descriptors. Test samples were analyzed after panelists demonstrated reliable consistency and reproducibili ty. Panelists adjusted their seating positions to evaluate comfortably the GC effluents during 30 min sessions. To simultaneously record a chromatogram and an ol factogram, a data acquisition system model NI USB-6210 and a computer program written in LabV IEW 8.5 (National Instruments, Austin, TX, USA) were used to interface the GC-O to th e computer and the panelist. Every 200 ms, the program simultaneously collects gr aphs and saves voltage data from the GC and aroma intensity. Panelists actuate a large slide bar on the front panel (computer screen) with a mouse to conveniently report aroma intens ity. The 10-point intensity scal e (0 = no aroma perceived, 10 = extremely strong) was used. Aroma descript ors were manually written on a notebook. Each

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66 sample was evaluated three times per panelist, ea ch replicate representi ng a different GC run. The samples were run in a random order of presenta tion to avoid introducing bias into the results. The criteria to develop a consen sus was: 1) first identify th e aroma active peaks that each panelist detected in two out of three replications, 2) and then compare the peaks of 3 panelists that met in the criteria, 3) select the peaks that two out of three panelists detected and 4) calculate the average retention times, RI s and maximum odor intensities ( Imax) of three panelists (Bazemore et al., 1999). Volatile compounds were identified by their RIs from FID and GC-MS analysis. Their aroma descriptors were also comp ared with published information to confirm the compound identities. Statistical Analysis Data were analyzed by using the following linear m odel: Xijkl = + i + j + ij + j( k) + ijkl where Xijkl is the l th perceived intensity of a compound of the j th hybrid evaluated by the i th panelist in the k th replicate, is the overall mean intensity, i is the effect of the i th panelist, j is the effect of the j th hybrid, ij is the effect of the interaction between the i th panelist and the j th hybrid, j( k) is the effect of the k th replicate nested within the j th hybrid, and ijk is the random residuals (Lea et al., 1997). Paneli st and hybrid are considered as fixed effects, and all other terms are considered as random effects. Replicates were nested within hybrids, because panelists evaluated a different vial from the same jui ce in each replicate. Unlike most sensory data analysis, each panelist is considered as the fixed effect in this study. The reason is that panelists are calibrated like an instrument and do not represent a random section of the population. Statistical analysis was performed by using PR OC GLM in SAS statistical software version 9.1 (SAS Institute Inc., Cary, NC). Separation of means was performed with the Fishers least significant difference (LSD) test with <0.05.

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67 Results and Discussion Consensus of Tangerine Aroma Active Compounds When analyzed by GC-MS, the number of volatiles in 8-9 (sam ple l), 8-10 (sample m), Unknown (sample p), Fortune Murcott (sam ple v) and 8-9 Val4x (sample y) was 65, 89, 61, 80 and 93, respectively, with a total of 150 diffe rent volatiles (Table 3-6). When the same samples were analyzed by GC-O, 119 aroma act ive peaks were detected, though many generated peaks had low intensity and inconsistent detect ion among three panelists and replications per sample. Therefore, a consensus among all three re plications and three panelists was built, with 49 aroma active compounds that were detected at l east in two replications out of three for each panelist, and by at least two of th e three panelists (Table 4-1). Acco rding to this consensus, eight volatiles showed a strong aroma activity in most samples: hexanal, ethyl 2-methylbutanoate, unknown (No. 9), 1-octen-3-one, -myrcene, 1,8-cineole, linalool and ( E ,E )-2,4-nonadienal (Table 4-1). Their average intensity among the fi ve samples was 5.0 to 7.4 on a 10 point scale, so they may largely contribute to the overall ar oma. Moreover, 12 compounds had relatively high aroma intensity greater than 5 in a few samp les: ethyl butanoate, heptanal, octanal, phellandrene, -terpinene, unknown (No. 27 and 30), camphor, d-carvone, ( E )-2-decenal, 2undecenal and -damascenone. As shown in Table 4-2, vola tile compounds were classified into monoterpenes (7 volatiles), aldehydes (12), esters (3), alcohols (4), ketones (7), phenol (1), ether (1) and unknowns (14). It is to be noted that ev en though sesquiterpenes we re identified in these samples by GC-MS (chapter 3), none had odor activ ity in the amount present in the samples. Odor-active peaks were also grouped based on the si milarity of their aroma descriptors: fruity, green/metallic/fatty, terpeney (minty/piney), gr een/grassy, citrus, mushroom, floral and other (Table 4-3).

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68 Terpenes Am ong 61 terpene hydrocarbons found by GC-MS, seven monoterpenes exhibited aroma activity. d-Limonene was the most abundant in all samples, accounting for 40.6 to 76.4% of the total content by GC-MS. However, because it has a high odor threshold (13,700 g/L in a deodorized orange juice matrix (DOJ) (Plotto et al., 2004), its citrus-lik e odor was not detected by GC-O. -Myrcene, one of the richest volatiles had the highest intensity among seven terpenes, with the threshold of 773 g/L in DOJ (Plotto et al., 2004). This compound possessed a strong green and metallic odor, whic h may negatively affect the ar oma and flavor characteristics. However, its intensity was equally perceived be tween samples (Table 4-1). Rega et al. (2003) showed all panelists evaluated -myrcene as an unpleasant odor in fresh orange juice using a GC-O detection frequency method. Although th e samples contained high amounts of the remaining terpenes, they had rela tively low intensity along with so me aroma descriptors: fruity (terpinolene), terpeney ( -pinene, -phellandrene, -terpinene and dehydro-p-cymene) and green/grassy (p-cymene) (Table 4-3). p-Cymene and terpinolene had the highest perceived intensity in 8-10, and -terpinene was the lowest in 8-9 x Val4x (Table 4-1). Unlike these aroma active monoterpenes, 39 sesquite rpenes did not produce odor activ ity. In the GC-MS analysis, valencene was the second most abundant compound that constitutes 18.4% and 24.8% of the total content in 8-10 and 8-9 Val4x, respec tively. However, it was not detected in the olfactometric analysis because the content did not exceed its high threshold (4,756 g/L in DOJ) (Plotto et al., 2008). Aldehydes The num ber of aldehydes (12 volatiles) was the highest among the seven identified chemical classes (Table 4-2). Heptanal, ( E ,E )-2,4-nonadienal, ( E ,E )-2,4-decadienal, 2-undecenal and dodecanal were grouped into the green/met allic/fatty aroma category (Table 4-3). ( E ,E )-2,4-

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69 Nonadienal and ( E ,E )-2,4-decadienal were not identified in the five samples by GC-MS because they were below the MS detection limit, yet, th ey could be detected by GC-O. On the other hand, hexanal was the richest aldehyde imparting fresh green/grassy odor unlike fatty (cooked vegetable-like) odors given by these dienals. Jens en et al. (1999) dem onstrated that the odor threshold for (E ,E )-2,4-nonadienal in water (0.0 017 nL/kg) was about 1.47 103 times lower than for hexanal (2.5 nL/kg). ( E ,E )-2,4-decadienal (0.045 nL/kg) had approximately 56 times lower threshold than hexana l. In the present study, ( E ,E )-2,4-nonadienal was the most potent aldehyde in most samples, followed by hexanal, ( E )-2-decenal and octanal. It is evident that GCO analysis can be a powerful method to determ ine the presence of these compounds, using the sensitivity of the human nose, in comparison with GC-MS. A straight-chain saturated aldehyde, octanal, may be an importa nt contributor to citrus and fruity notes. Its aroma intensity was the highest in Fortune M urcott and the lowest in the unknown sample (Table 4-1). Although decanal, as well as octanal and nonanal, has been implicated to contribute to orange flavor (S haw, 1991), its aroma intensity was low in the tangerine samples. The terpenic aldehydes, neral and geranial, which were also believe to be important contributors to ora nge flavor (Ahmed et al., 1978a, 1978b), were not detected by GCO analysis, though they were identified in Fortune Murcott and 8-9 Val4x by using GCMS. The unsaturated aldehydes, (E )-2-pentenal, ( E )-2-heptenal, (E )-2-nonenal, ( E )-2-decenal and 2-undecenal had their own characteristic odors as seen in Table 4-3. All detected aldehydes are degradation products, probably derived from C16 and C18 fatty acids rich in citrus juice sacs (Nordby and Nagy, 1971). Chisholm et al. (2003) reported that 47 aldehydes were found in Clementine peel oil, which contributed over 80% of its aroma using dilution analysis (CHARM analysis). In the present study wh ere juice samples were processed to avoid any peel oil in the

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70 juice, aldehydes contributed 24 to 28% of fresh tangerine aroma, indicating the role of other aroma components. Esters The arom a activity of ethyl butanoate, ethyl 2-methylbutanoate and ethyl hexanoate have also been reported in hand-squ eezed orange juice (T nder et al., 1998; Buettner and Schieberle, 2001b; Arena et al., 2006). The trace amounts of these compounds in 8-9, 8-10, Unknown and Fortune Murcott did not allow their individual identification by MS, unlike the most abundant ethyl acetate. However, the panelists could detect their fruity odors because the thresholds are significantly low: 1.71, 0.08 and 3.3 g/L in DOJ, respectively (Plotto et al., 2008). The content of ethyl butanoate was the hi ghest (2.2%) in 8-9 Val4x, followed by ethyl hexanoate (1.2%), ethyl acetate (0.4%) and ethyl 2-methylbutanoate (0.05%) by GC-MS. Based on the perceived aroma intensity, ethyl butanoate and ethyl 2-methylbutanoa te alone contributed to 22 to 29% of the fruity note in the five samples. Likewise, Buettner and Schieberle (2001b) determined these two compounds were potent odorants of Valencia late orange juice. Ethyl-2methyl butanoate had high fruity/floral aroma inte nsity in all five samples (Table 4-1). On the other hand, ethyl butanoate and et hyl hexanoate had the highest in tensity ratings in 8-9 Val4x, with ethyl hexanoate the highest among the samples. Since ethyl butanoate is an important contributor to desirable orange juice flavor (Shaw, 1991), the higher level of ethyl butanoate and ethyl hexanoate in 8-9 Val4x would contribute to the fruity characteristic of this hybrid (unpublished sensory data). Alcohols Ethanol was relatively rich in the samples but showed low arom a intensity. Shaw (1991) inferred that it may give a lift to other ar omas, without contributing to its own aroma. A terpene alcohol, linalool, accounted for 1.2 to 5.3% of the total content by GC-MS, described as

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71 floral with a strong intensity. This compound makes a pos itive contribution to orange flavor in combination with several other orange volatiles (Shaw 1991). It had the lowest aroma intensity in the unknown sample (Table 4-1). Two isomeric ( Z )and ( E )-carveol, derived from limonene by limonene-6-hydroxylase (Bouwmees ter et al., 1998), had aroma ac tivity with different odors: minty/pencil/piney/fruity /floral and green/rubber/ sulfury, respectively. (Z )-carveol also had the lowest intensity in the unknown sample (Tab le 4-1). These compounds were found in coldpressed oils of Natsudaidai (a natural hybrid of pummelo) (L an-Phi et al., 2006), two mandarin cultivars and Minneola tangelo (Njoroge et al., 2005). With low aroma intensities and multiple descriptors, it is possible that several compounds co-eluted at this time in our samples. Ketones 1-Octen-3-one was the single m ost potent aliphatic ketone, having a mushroom odor. Camphor and d-carvone, cyclic terpene ketones w ith a terpeney odor, also showed aroma activity with relatively high intensity in some samp les. Despite the peculiar green buchu oil odor imparted by camphor, it has not been found in ma ny citrus fruits: e.g., two mandarin cultivars (Frizzo et al., 2004) and Pon tianak orange peel oil (Dharmawan et al., 2009). The weak spicy/woody odor of nootkatone, which level is usually high in grapef ruit and pummelo, was detected in all samples. All of the above ketones were perceived with similar intensities in the five samples (Table 4-1). Alphaand -ionone were detected and are important components to contribute to the floral note, in addition to lin alool mentioned above (M ahattanattawee et al., 2005). Interestingly, one panelist could not detect -ionone at all, confirming that some people may have specific anosmia to that compound (P lotto et al., 2006). Beta-damascenone was not identified by GC-MS, but had high aroma intens ity in some samples with apple sauce/fruity odor. This compound and -ionones showed the highest intensity in 8-9 Val4x (Table 4-1). Since the seed parent 8-9 had low intensity of the ionones, tetraploid Valenci a orange may play

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72 an important role in the production of these floral norisoprenoids. The three C13 norisoprenoids are degradation products from putative carotenoid precursors, and -carotene, and cryptoxanthin and neoxanthin (Mahattawanatawee et al., 2005). Thus, their content may be one of the critical factors to cont rol the desirable odor and flavor at fruit maturity. Although the ionones had similar aroma intensities in Fortu ne Murcott and in 8-9 Val4x, that of damascenone was significantly lower in For tune Murcott. Aroma intensity of -ionone was very low in 8-9, 8-10 and unknown sample. Phenol A high level of thym ol was found by GC-MS in 8-9, 8-10 and 8-9 Val4x sharing common genetic background from Clementine and Minneola (a hybrid between Duncan grapefruit and Dancy tangerine). Thymol was f ound in Dancy tangerine peel oil and Sicilian mandarin peel oil (Shaw, 1979), and Temple tangor essence oil (Moshonas and Shaw, 1983). Wilson and Shaw (1981) reported that thymol and methyl N-methylanthranilate, in addition to terpinene and -pinene, were necessary components for mandarin aroma in cold-pressed oil. Furthermore, the elevated levels of thymol and methyl N-methylanthranila te in mandarin peel oil are a discriminative factor from orange peel oil (Rouseff and Prez-Ca cho, 2007). In this study, thymol was found at the highest concentration by GC-MS and aroma intensity (Table 4-1) in 8-9, where it might contribute to specific tangerine aroma. Ether 1,8-Cineole (eucalyptol), a m onoterpene cycl ic ether, was a strong aroma active compound with a low threshold (9 g/L in DOJ Plotto, unpublished data). This compound may have negative impact on tangerine arom a, since its odor was a characteristic green and sulfury. GCMS chromatograms showed it coeluted with d-lim onene, and its peak area (relative content) was

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73 not quantified. However, because of its character istic green/minty odor, it was clearly identified from the limonene by GC-O. 1,8-Cineole was found in grapefruit oil (Lin and Rouseff, 2001), Clementine peel oil (Chisholm et al., 2003) an d orange essence oil (H gnadttir and Rouseff, 2003) by using GC-O. However, its aroma activity ha s not been clearly reported in citrus juice. Unknown Compounds Fourteen compounds could not be identified by either FI D or GC-MS. Although m ost compounds had low intensity of less than 5, they are not negligible because they may interact with other aroma volatiles in the juice. Comp ared with their RIs and aroma descriptors in published data, four compounds can be tentatively identified: me thional (No. 9), furaneol (No. 20), sotolon (No. 26), trans-4,5-epoxy-( E )-2-nonenal (No. 39) and wine lactone (No. 46) (Hinterholzer and Scieberle, 1998; Buettner and Schieberle, 2001a, 2001b; Chisholm et al., 2003; Hgnadttir and Rouseff et al., 2003; Valim et al., 2003; P rez-Cacho et al, 2007). Each compound has been described with distinctive odors: methional (p otato, cooked potato), furaneol (burnt sugar, caramel), sotolon (geranium, green, metallic, flor al), trans-4,5-epoxy-( E )-2-nonenal (caramel, spicy, seasoning, mushroom) and wine lact one (sweet, spicy, dill, floral, metallic). In addition, a few aroma-active peaks were described as sulfur or rubber. These are likely to be sulfur compounds with very low odor threshold. Sulfur compounds are not detected by the FID type of detector, and might be in too low concen trations to be detected by GC-MS. It is known that processing and storage easily cause the de velopment of skunky (sulfury) off-flavor in mandarin juice (Shaw, 1991). It is possible these compounds resulted from storage of the juice. In citrus juice, they are derived from amino acids such as cystein, cystine and methionine (PrezCacho and Rouseff, 2008).

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74 Tangerine Aroma Profiles As shown in Figure 4-1, a tota l intensity of arom a active p eaks (119 peaks) was used to represent the relative importance of each aroma cat egory. The total intensity of all categories was 181, 174, 149, 197 and 197 in 8-9, 8-10, Unknown, For tune Murcott and 8-9 Val4x, respectively. All samples presented thr ee aroma categories as top notes: fruity, green/metallic/fatty, and terpeney. Among 30 separa te aroma active peaks in the fruity category, ethanol, ( E )-2-pentenal, ethyl bu tanoate, ethyl 2-methylbutanoate ethyl hexanoate, terpinolene, unknown (No. 28 and 48) and -damascenone (Table 4-3) contri bute 73 to 92% of its total intensity. Moreover, the other ar oma categories of tangerine arom a can be primarily explained by the compounds in Table 4-3. 8-9 and 8-10, siblings from the same cross, had different aroma volatile composition and aroma profiles. For instance, ethyl butanoate and ethyl 2-methylbutanoate had higher intensity in 8-9 than 8-10, even though not si gnificant (Table 4-1), contribu ting to a fruitier note to the former. -Phellandrene and -terpinene, described as terpeney, also showed higher intensity in 89 than 8-10. On the other hand, green/grassy compounds, ( E )-2-heptenal and p-cymene, had higher intensity in 8-10 than 8-9 (Figure 41 and Table 4-1). In addition, the unknown compound (No. 21) and terpinolene, described as burned/mu shroom and fruity/green/toasted, respectively, showed higher intensity in 8-9 and 8-10 than th e other samples. Their fruits were grown under the same environmental condition, and harvested at the same date. Thus, genetic effect may play an important role to differentiate their aromas With respect to the terpeney category, it was higher in 8-9 than in 8-10 (Figure 4-1). Mo re fresh terpeney odors of 8-9 might balance undesirable fatty odor, mainly caused by ( E ,E )-2,4-nonadienal and ( E ,E )-2,4-decadienal. When juice samples are consumed, aroma perception is affected by the presence of soluble solids (e.g., sugars and acids) and the corresponding change of gas-liquid partition coefficient of volatile

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75 compounds (Friel et al., 2000). Bald win et al. (2008) reported that the addition of acids with volatiles increased perception of overall toma to aroma using deodorized tomato puree. The higher acidity in 8-9 (1.57% of titratable acidity TA) might also contribute to the aroma and flavor differences from 8-10 ( 1.27% of TA) (data not shown). The unknown sample was the least aromatic amo ng the five tangerine samples. The total volatile content was 3.4 to 6.8 times lower than the other four samples by GC-MS (Chapter 3). Interestingly, three homologous aldehydes, hexanal (C6), heptanal (C7) and octanal (C8) were the most abundant volatiles, following d-limonene. The green/metallic/fatty category, which is mostly the characteristic odor of aldehydes, acco unted for 34% of its aroma. The abundance of aldehydes in addition to low fruity/floral com pounds could contribute to a peculiar pumpkin, fatty aroma and flavor of this sample as desc ribed by a sensory trained panel (data not shown). In addition to those aroma volatiles, the highest sugar content (15 Brix) and lowest acidity (0.71% TA) among the five samples added to its unique flavor (data not shown). Fortune Murcott shar ed a similar aroma profile to 8-9, in terms of total intensities of the major aroma categories, fruity and terpene y, but had more floral peaks, mostly from and ionone. The unknown compound (No. 8) with a citrusy/fruity odor, in addition to -pinene, was detected only in 8-9 and Fortune Murcott (Table 4-1), and might contri bute to their desirable aroma. Fortune Murcott containe d aldehydes in high quality and quantity (Table 3-5 and 3-6), and hence its overall aroma may smell slightly more green, metallic and/or fattier. The aroma profile of 8-9 Val4x (2n = 3x = 27) may be distinguished from that of 8-9 because of its additional genetic background fr om orange. The differences between the two samples mostly stand in the fruity and terpen ey categories and may be associated with the content of esters and monotepenes. Indeed, the total intens ity of esters (ethyl butanoate, ethyl 2-

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76 methylbutanoate, ethyl hexanoate) in 8-9 Val4 x was about 1.6 times higher, and that of monoterpenes ( -pinene, -phellandrene, -terpinene, dehydro-p-cymene) was about 1.8 times lower than that in 8-9 (Table 4-1). Green/gra ssy, citrus, mushroom and other categories are responsible for about 34% of thei r aromas, indicating that they are more likely to contribute to tangerine aroma as background notes.

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77Table 4-1. Consensus of aroma active compounds in five tangerin e hybrids determined by GC-MS a nd GC-O using Osme analysis No.* Compound RTa LRIb Descriptors Aroma Intensityc 8-9 8-10 Unknownd F Me 8-9 Val4x 1 Ethanol 2.08 535 ethanol, alcohol 1.0 2.4 0.7 1.6 1.9 2* Unknown 3.69 652 sulfury, metallic, herb 0 b 0 b 2.3 a 0.9 b 0.3 b 3 ( E)-2-Pentenal 4.91 730 fruity, floral 3.2 2.1 3.8 2.7 3.4 4 Hexanal 5.72 776 green, grassy 5.5 5.1 5.2 5.7 4.9 5* Ethyl butanoate 5.80 781 fruity 3.5 ab 2.5 b 1.3 b 3.1 ab 6.0 a 6 Ethyl 2-methylbutanoate 7.01 844 fruity, floral 5.3 3.9 5.1 5.3 5.5 7 Heptanal 8.31 904 green, fatty, vegetable 4.5 4.8 5.3 4.4 5.1 8* Unknown 8.37 906 citrusy, fruity 2.1 a 0 b 0 b 3.5 a 0 b 9 Unknown 8.52 913 metallic, cooked vegetable 4.3 5.2 5.3 3.9 6.5 10* ( E)-2-Heptenal 9.57 957 green, grassy, rubber 1.8 bc 3.9 ab 4.6 a 1.7 bc 1.0 c 11 1-Octen-3-one 10.51 993 mushroom 4.3 4.7 6.2 5.4 6.0 12 -Myrcene 10.65 998 green, metallic 7.3 7.4 7.0 7.4 8.0 13 -Pinene 10.85 1005 green, piney 1.0 0 0 1.9 0 14* Ethyl hexanoate 11.07 1013 fruity, floral 1.0 b 0.6 b 0.3 b 0 b 4.8 a 15* Octanal 11.19 1017 citrus, fruity 5.2 ab 4.4 b 3.3 b 6.6 a 4.7 ab 16* p-Cymene 11.63 1033 green, rubber 0.3 b 2.6 a 0.5 b 0.5 b 0.2 b 17 -Phellandrene 12.05 1048 minty, piney, terpene 5.6 4.0 4.1 4.8 4.4 18* 1,8-cineole 12.22 1054 green, herb, rubber 2.8 b 8.6 a 8.4 a 7.8 a 8.3 a 19* -Terpinene 12.78 1072 minty, piney, terpeney, fruity 5.3 a 2.6 bc 3.0 bc 4.5 ab 1.5 c 20 Unknown 13.04 1081 burned sugar, caramel 0.7 2.0 2.0 2.0 2.0 21* Unknown 13.30 1090 burned, mushroom 3.6 a 2.5 a 0.7 b 0.3 b 0.2 b 22* Unknown 13.48 1096 burned, musty 3.6 a 0 c 2.3 ab 0.7 bc 3.5 a 23* Terpinolene 13.63 1101 fruity, green, toasted 2.9 ab 4.2 a 0.3 c 1.9 bc 1.1 bc 24 Dehydro-p-cymene 13.76 1105 green, minty, plant, apple 3.0 1.7 2.1 3.0 2.5 25* Linalool 13.87 1109 floral 6.7 a 7.4 a 4.4 b 7.0 a 5.9 ab 26 Unknown 14.21 1120 burned sugar, caramel, musty 2.0 2.3 3.3 4.4 3.9 27 Unknown 14.90 1143 green, metallic, rubber, sulfury 3.7 5.3 3.3 5.5 4.0 28 Unknown 15.11 1149 fruity, musty, floral, green 2.5 3.9 2.8 4.2 3.7 29 ( E)-2-Nonenal 15.32 1156 cucumber, perfumey 0.4 1.6 2.9 1.2 3.4

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78Table 4-1. Continued No.* Compound RTa LRIb Descriptors Aroma Intensityc 8-9 8-10 Unknownd F Me 8-9 Val4x 30 Unknown 15.44 1160 musty, minty, floral 4.2 5.3 4.6 5.4 5.4 31 Camphor 15.61 1166 buchu oil, green tea, hay, musty 3.3 3.6 3.2 6.3 4.7 32* Unknown 15.87 1175 musty, scotch tape 0.9 b 0.3 b 0 b 2.1 a 0 b 33 Decanal 16.34 1191 apple, green 2.1 1.8 0.6 1.1 2.0 34 ( E, E )-2,4-Nonadienal 16.88 1209 fatty, vegetable, noodle 6.9 7.4 5.0 6.3 7.3 35* ( Z )-Carveol 17.19 1220 floral, lemon, minty, pencil 4.7 a 3.0 ab 1.1 b 3.8 a 3.6 a 36 ( E)-Carveol 17.53 1232 green, rubber, sulfury 3.4 3.6 4.2 2.5 1.6 37 d-Carvone 17.68 1238 minty 5.4 3.9 4.6 5.8 4.9 38 ( E)-2-Decenal 17.83 1243 minty, pe ncil, piney, fruity, floral 5. 7 a 4.6 ab 3.6 b 5.1 ab 6.0 a 39* Unknown 18.64 1274 green, rubber, sulfury 2.4 b 3.0 ab 0.2 c 4.2 a 2.6 ab 40* Thymol 18.86 1282 medicinal 4.2 a 2.9 ab 0.5 b 1.1 b 1.9 ab 41 ( E, E )-2,4-Decadienal 19.42 1305 fatty, vegetable 4.5 4.2 2.5 4.2 4.3 42 2-Undecenal 20.92 1370 metallic, musty 4.7 2.4 4.1 5.1 5.2 43* -Damascenone 21.07 1377 apple sauce, fruity, floral 5.6 ab 5.9 ab 4.0 b 1.4 c 7.3 a 44 Dodecanal 21.43 1395 green, rubber 0.8 1.2 0.5 2.8 1.0 45* -Ionone 22.08 1427 floral, perfumey, soap 0.9 b 0.7 b 0.4 b 3.3 a 3.2 a 46* Unknown 22.82 1467 fermented, musty, butter 0.3 c 2.2 ab 0 c 1.8 b 3.4 a 47* -Ionone 23.40 1499 floral, violet 2.1 b 4.1 a 3.9 a 4.2 a 5.0 a 48* Unknown 24.14 1544 sulfury, fruity, floral 1.2 abc 0 c 1.0 bc 2.7 a 2.3 ab 49 Nootkatone 28.06 1843 spicy, woody 1.8 2.1 1.0 1.8 2.8 Peaks for which there was a difference between samples for that aroma intensity by ANOVA a Retention time b Linear Retention Index on the HP-5 column c Averages of three panelists by three rep lications. Numbers followed by the same letter within a row are not significantly diffe rent using the LSD test ( = 0.05). d Three samples with unknown parentage were analyzed by GC-MS. Sample p was used for this analysis. e 'Fortune' 'Murcott'

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79Table 4-2. Tangerine aroma active compounds in 8 chemical classes Monoterpenes Aldehydes Esters Alcohols Ketones Phenol Ether Others -Myrcene ( E)-2-Pentenal Ethyl butanoate Ethanol 1-Octen-3-one Thymol 1,8-Cineole 14 Unknowns -Pinene Hexanal Ethyl 2-methylbutanoate Linalool Camphor p-Cymene Heptanal Ethyl hexanoate ( Z )-Carveol d-Carvone -Phellandrene ( E)-2-Heptenal ( E)-Carveol -Damascenone -Terpinene Octanal -Ionone Terpinolene ( E)-2-Nonenal -Ionone Dehydro-p-cymene Decanal Nootkatone ( E, E )-2,4-Nonadienal ( E)-2-Decenal ( E, E )-2,4-Decadienal 2-Undecenal Dodecanal

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80Table 4-3. List of tangerine aroma ac tive compounds in 8 aroma categories fruity green, metallic, fatty terpeney gree n, grassy citrus mushroom floral other Ethanol Unknown (No. 2) -Pinene Hexanal Unknown (No. 8) 1-Octen-3-one Linalool Unknown (No. 20) ( E)-2-Pentenal Heptanal -Phellandrene ( E)-2-Heptenal Octanal Unknown (No. 21) -Ionone Unknown (No. 26) Ethyl butanoate Unknown (No. 9) -Terpinene p-Cymene -Ionone ( E)-2-Nonenal Ethyl 2-methylbutanoate -Myrcene Dehydro-p-cymene 1,8-cineole Thymol Ethyl hexanoate Unknown (No. 22 ) Camphor Decanal Nootkatone Terpinolene Unknown (No. 27) ( Z )-Carveol Unknown (No. 28) Unknown (No. 30) d-Carvone -Damascenone Unknown (No. 32) ( E)-2-Decenal Unknown (No. 48) ( E, E )-2,4-Nonadienal ( E)-Carveol Unknown (No. 39) ( E, E )-2,4-Decadienal 2-Undecenal Dodecanal Unknown (No. 46)

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81 0 10 20 30 40 50 60 70Total aroma intensity 8-9 8-10 Unknown F M8-9 Val4x fruity green / metallic / fatty terpeney green / grassy citrus mushroom floral other Figure 4-1. Aroma profiles of five tangerine hybrids. Each bar is the sum of intensities in a specific category.

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82 CHAPTER 5 SUMMARY AND CONCLUSIONS The qualitative and quantita tive differences of aroma volatile composition were observed among 20 tangerine hybrids and five commercial cultivars. Among 193 aroma volatiles identified in the hybrids using GC-MS, only a sma ll number of volatiles accounted for more than 90 % of the total content. Volatiles in lower quantity were widely distributed among samples, and were classified mainly as terpene hydrocarbons and oxygenated compounds, such as aldehydes, esters, alcohols and ketones. PCA based on relative pe ak areas clearly differentiated three selections (9-4 Blood4x, Temple and Sanguinelli) with greater volatile levels; Sanguinelli is a sweet or ange and the other two have sweet orange as a direct progenitor. In addition, a second PCA using the content of 11 chem ical classes revealed that most hybrids with more mandarin/tangerine genetic c ontributions were distinguished from the other samples due to their lower volatile quality and/or quantit y. The cluster analysis based on volatile presence/absence grouped samples into five clus ters, each having effect s of genetic background on volatile composition. Different siblings of the same parentage (8-9 Murcott, Robinson Fairchild, Fallglo Fairchild, or 8-9 and 8-10) s howed different volatile composition, implying complex genetic controls for aroma volatile production. Nevertheless, hybrids with sweet orange in their background appeared to produce more sesquiterpenes and esters. Aroma active volatiles in five selected jui ce samples were analyzed by GC-O using the time intensity (Osme) method. The choice of samp les analyzed by GC-O was determined from GC-MS and sensory studies of 20 tangerine hyb rids. Although a total of 150 volatiles were identified by GC-MS, only 49 aroma active peaks were found in a consensus of three panelists. Nineteen volatiles were potent ar oma compounds with perceived inte nsity of more than 5 (on a 0 to 10 point scale) in at least one sample. The ol factometric analysis also revealed compounds that

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83 were not detected by GC-MS. These compounds were camphor, ( E ,E )-2,4-nonadienal, ( Z )carveol, ( E )-carveol, ( E E )-2,4-decadienal, -damascenone and -ionone, with associated descriptors of buchu oil, fatty, fl oral, green, fatty, apple sauce and floral, respectively. Moreover, ( E ,E )-2,4-nonadienal, ( E E )-2,4-decadienal and -damascenone were not identified in any of the other 20 samples by GC-MS. On the other hand, many sesquiterpenes were detected by GC-MS, but none of these had any odor-ac tivity. The top notes of tangerin e hybrids were mainly from terpene hydrocarbons, aldehydes and esters. The ot her chemical classes (alcohol, ketone, phenol and ether), associated with various descriptors, may also contribute to the overall aroma and flavor. Most of these compounds or odor-active peaks were found in all samples, but in different intensity levels, explaining sens ory differences that might be found between samples. GC-O results confirm partially the sens ory characteristics of some samp les (for example, a hybrid of unknown origin with pumpkin/fatty aroma and fl avor; 8-9 Val4x with an orange aroma); however, it is recognized that sugars, acids and other non-volatile compounds also contribute to flavor. This study provides useful information on arom a volatile profiles and sensory quality for future tangerine breeding efforts. Further research is needed to understand what constitutes a desirable combination of tangerine aroma activ e compounds. Moreover, further biochemical and genetic research could lead to a better unders tanding of the metabolic pathways and genes associated with aroma volatile formation. An increasing number of citrus protein and DNA sequence (e.g., expressed sequence tags, EST) en tries are available in public databanks. However, only a few citrus terpene synthases and their cDNAs have been reported, and the formation of other major chemical groups (aldeh yde, ester, alcohol and ketone) is not yet well understood. Since it is likely that many genes control and influen ce the complexities of citrus

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84 aroma and flavor, quantitative tra it locus (QTL) analysis may be a pplied to identify particular regions of the genome linked to volatile production. In addition to specific gene coding regions, regulatory regions ( cisand trans -acting QTLs) might likewise aff ect quantitative differences of odor active volatiles, resulting in th e variety of tangerine overall aromas. Citrus aroma formation occurs as fruits ripen, and hence microarray te chnology could be used to elucidate chronological changes of gene expression involved with vola tile production. With these combined techniques, the development of molecular markers associated with citrus aroma and flavor should accelerate long-term tangerine breeding programs, by enabli ng selection of individua l hybrids at an early stage which possess a greater likelih ood of producing high quality fruit.

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85 APPENDIX A OPTIMIZATION OF SPME FOR GC-MS AND GC-O ANALYSIS Table A-1. Optimization of volatile sampling Panelist Treatment Rating Mean Standard deviation A 1a 3 3 8 4.67 2.89 2b 6 9 7 7.33 1.53 3c 8 7 9 8 1 B 1a 3 3 7 4.33 2.31 2b 9 5 7 7 2 3c 7 8 9 8 1 C 1a 3 4 5 4 1 2b 2 6 6 4.67 2.31 3c 2 5 4 3.67 1.53 a 30 min fiber equilibrium and 3 min fiber exposure b 30 min fiber equilibrium and 30 min fiber exposure c 30 min fiber equilibrium and 60 min fiber exposure

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86 LIST OF REFERENCES Acree, T. E., J. Barnard, and D. G. Cunningham 1984. A procedure for the sensory analysis of gas chromatographic effluents. Food Chem. 14: 273-285. Aharoni, A., M. A. Jongsma, and H. J. Bouw meester. 2005. Volatile science? Metabolic engineering of terpenoids in plan ts. Trends Plant Sci. 10: 594-602. Ahmed, E. M., R. A. Dennison, R. H. Doughe rty, and P. E. Shaw. 1978a. Flavor and odor thresholds in water of selected orange ju ice components. J. Agr. Food Chem. 26: 187-191. Ahmed, E. M., R. A. Dennison, and P. E. Sh aw. 1978b. Effect of selected oil and essence volatile components on flavor quality of pumpout orange juice. J. Agr. Food Chem. 26: 368-372. Albach, R. F., and G. H. Redman. 1969. Compositi on and inheritance of flavanones in citrus fruit. Phytochemistry. 8: 127-143. Allegrone, G., F. Belliardo, and P. Cabella. 2006. Co mparison of volatile concentrations in handsqueezed juices of four different lemon varieties. J. Agr. Food Chem. 54: 1844-1848. Araki, C., and H. Sakakibara. 1991. Changes in the volatile flavor compounds by heating Satsuma mandarin ( Citrus unshiu Marcov) juice. Agric. Biol. Chem. 55: 1421-1423. Arena, E., N. Guarrera, S. Campisi, and C. Nicolosi Asmundo. 2006. Comparison of odour active compounds detected by gas-chromatogr aphy-olfactometry between hand-squeezed juices from different orange va rieties. J. Food Chem. 98: 59-63. Arthur, C. L., and J. B. Pawliszyn. 1990. Solid-p hase microextraction with thermal desorption using fused silica optical fibers. Anal. Chem. 62: 2145-2148. Baldwin, E. A., J. W. Scott, C. K. Shewmake r, and W. Schuch. 2000. Flavor trivia and tomato aroma: Biochemistry and possible mechanisms for control of important aroma components. HortScience 35: 1013-1022. Baldwin, E. A., K. Goodner, A. Plotto. 2008. In teraction of volatiles, sugars and acids on perception of tomato aroma and flavor descriptors. J. Food Sci. 73: S294-S307. Barboni, T., F. Luro, N. Chiaramonti, and J. M. Desjobert, A. Muselli, and J. Costa. 2009. Volatile composition of hybrids Citrus juices by headspace solid-phase micro extraction/gas chromatography/mass spectrometry. Food Chem. 116: 382-390. Barrett, H. C., and A. M. Rhodes. 1976. A numerical taxonomic study of affi nity relationships in cultivated Citrus and its close relatives Syst. Bot. 1: 105-136. Bartle, K. D., and P. Myers. 2002. History of gas chromatography. Trends Anal. Chem. 21: 547557.

PAGE 87

87 Bayer, R. J., D. J. Mabberley, C. Morton, C. H. M iller, I. K. Sharma, B. E. Pfeil, S. Rich, R. Hitchcock, and S. Sykes. 2009. Molecular phylogeny of the orange subfamily (Rutaceae: Aurantioideae) using nine cpDNA sequences. Am. J. Bot. 96: 668-685. Bazemore, R., K. Goodner, and R. Rouseff. 1999. Volatiles from unpasteurized and excessively heated orange juice analyzed with solid phase microextraction and GC-olfactometry. J Food Sci. 64: 800-803. Berry, R. E., C. J. Wagner, Jr., and M. G. Moshonas. 1967. Flavor studies of nootkatone in grapefruit juice. 32: 75-78. Biolatto, A., A. M. Sancho, R. J. C. Cantet, D. R. Gemes, and N. A. Pensel. 2002. Use of nootkatone as a senescence indicator for Rouge La Toma cv. grapefruit (Citrus paradisi Macf.). J. Agric. Food Chem. 50: 4816-4819. Blazquez, C. H. 1967. Ortanique, a new orange-tange rine cross. Proc. Fla. State Hort. Soc. 80: 331-337. Bouwmeester, H. J., J. Gershenzon, M. C. J. M. Konings, and R. Croteau. 1998. Biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway. I. Demonstration of enzyme activities and their changes with development. Plant Physiol. 117: 901-912. Buettner, A., and P. Schieberle. 1999. Characteriza tion of the most odor-active volatiles in fresh, hand-squeezed juice of grapefruit (Citrus paradisi Macfayden). J. Agr. Food Chem. 47: 5189-5193. Buettner, A., and P. Schieberle. 2001a. Evaluati on of key aroma compounds in hand-squeezed grapefruit juice ( Citrus paradisi Macfayden) by quantitation and flavor reconstitution experiments. J. Agr. Food Chem. 49: 1358-1363. Buettner, A., and P. Schieberle. 2001b. Evaluation of aroma differences between hand squeezed juices from Valencia late and Navel orange s by quantitation of key odorants and flavor reconstitution experiments. J. Agr. Food Chem. 49: 2387-2394. Buettner, A., M. Mestres, A. Fischer, J. Guas h, and P. Schieberle. 2003. Evaluation of the most odour-active compounds in the pe el oil of Clementines ( Citrus reticulata Blanco cv. Clementine). Eur. Food Res. Technol. 216: 11-14. Bylaite, E., A. S. Meyer. 2006. Characterisation of volatile aroma compounds of orange juices by three dynamic and static headspace gas chromatography techniques. Eur. Food Res. Technol. 222: 176-184. Cadwallader, K. R., and Y. Xu. 1994. Analysis of volatile components in fresh grapefruit juice by purge and trap/gas chromatography. J. Agr. Food Chem. 42: 782-784. Cameron, J. W., and H. B. Frost. 1968. Gene tics, breeding, and nucellar embryony. p. 325-370. In W. Reuther, L. D. Batchelor, H. J. Webber (eds.). The Citrus Industry. Volume 2. University of California Pr ess, Berkeley, CA, USA.

PAGE 88

88 Chao, C. C. T., P. S. Devanand and B. R. Cao. 2004. Identification of satsuma mandarin ( Citrus unshiu ) cultivars in California using Amplifie d Fragment Length Polymorphism (AFLP) markers. Acta Hort. 634: 159-165. Chisholm, M. G., J. A. Jell, and D. M. Ca ss Jr. 2003. Characterization of the major odorants found in the peel oil of Citrus reticulata Blanco cv. Clementine using gas chromatography olfactometry. Flavour Fragr. J. 18: 275-281. Coleman, R. L., and P. E. Shaw. 1972. Analysis of tangerine essence oil and aroma oil. J. Agr. Food Chem. 20: 1290-1292. Coletta Filho, H. D., M. A. Machado, M. L. P. N. Targon, M. C. P. Q. D. G. Moreira, and J. Pompeu, Jr. 1998. Analysis of the genetic diversity among mandarins ( Citrus spp.) using RAPD markers. Euphytica. 102: 133-139. Corn lio, M. T. M. N., A. R. S. Figueir a, K. G. B. Santos, R. Carvalho, W. S. Soares Filho, and M. Guerra. 2003. Chromosomal relationships among cultivars of Citrus reticulata Blanco, its hybrids and related species. Plant Syst. Evol. 240: 149-161. Defilippi, B. G., A. A. Kader, and A. M. Dandekar. 2005. Apple aroma: alcohol acyltransferase, a rate limiting step for ester biosynthesis, is regulated by ethylene. Plant Sci. 168: 11991210. Delahunty, C. M., G. Eyres, and J. P. Dufour 2006. Gas chromatography-olfactometry. J. Sep. Sci. 29: 2107-2125. del Ro, J. A., A. Ortuo, D. Garca-Puig, I. Porras, A. Garca-Lidn, and F. Sabatert. 1992. Variations of nootkatone and valencene levels during the development of grapefruit. J. Agr. Food Chem. 40: 1488-1490. Demole, E., P. Enggist, and G. Ohloff. 1982. 1-pMenthene-8-thiol: A powe rful flavor impact constituent of grapefruit juice ( Citrus purudisi Macfayden). Helv. Chim. Acta. 65: 17851794. Dharmawan, J., S. Kasapis, P. Curran, and J. R. Johnson. 2007. Characterization of volatile compounds in selected citrus fruits from Asia Part I: freshly-squeezed juice. Flavour Fragr. J. 22: 228-232. Dharmawan, J., S. Kasapis, P. Sriramula, M. J. Lear, and P. Curran. 2009. Evaluation of aromaactive compounds in Pontianak orange peel oil ( Citrus nobilis Lour. Var. microcarpa Hassk.) by gas chromatography-olfactometry, aroma reconstitution, and omission test. J. Agr. Food Chem. 57: 239-244. Dornelas, M. C., and P. Mazzafera. 2007. A geno mic approach to characterization of the Citrus terpene synthase gene family. Genet. Mol. Biol. 30: 832-840. Dravnieks, A., and A. ODonnell. 1971. Princi ples and some techniques of high-resolution headspace analysis. J. Agric. Food Chem. 19: 1049-1056.

PAGE 89

89 Dreyer, D. L., M. V. Pickering and P. Cohan. 1972. Distribution of limonoids in the Rutaceae. Phytochemistry. 11: 705-713. Elmaci, Y., and T. Altug. 2005. Flavor Characte rization of three mandari n cultivars (Satsuma, Bodrum, Clementine) by using GC/MS and fla vor profile analysis techniques. J. Food Qual. 28: 163-170. Esen, A., and R. W. Scora. 1975. Distribu tion of enzymatic browning of young shoot homogenates in the Aurantioideae. Amer. J. Bot. 62: 1078-1083. Falco, L. D., G. de Revel, J. P. Rosier, and M. T. Bordignon-Luiz. 2008. Aroma impact components of Brazilian Cabernet Sauvignon wines using detection frequency analysis (GColfactometry). Food Chem. 107: 497-505. Fang, D. Q, and M. L. Roose. 1997. Identification of closely related citrus cultivars with intersimple sequence repeat markers. Theor. Appl. Genet. 95: 408-417. Food and Agriculture Organization of the United Nations. 2009. FAOSTAT. Accessed 1 September 2009. < http://faostat.fao.org/>. Federici, C. T., D. Q. Fang, R. W. Scora, a nd M. L. Roose. 1998. Phylogenetic relationships within th e genus Citrus (Rutaceae) and related genera as revealed by RFLP and RAPD analysis. Theor. Appl. Genet. 96: 812-822. Friel, E. N., R. S. T. Linfor th, and A. J. Taylor. 2000. An empirical model to predict the headspace concentration of vol atile compounds above solutions containing sucrose. Food Chem. 71: 309-317. Frizzo, C. D., D. Lorenzo, and E. Dellacassa. 2004. Composition and seas onal variation of the essential oils from two mandarin cultivars of southern Brazil. J. Agr. Food Chem. 52: 3036-3041. Frydman, A., O. Weisshaus, M. Bar-Peled, D. V. Huhman, L. W. Sumner, F. R. Marin, E. Lewinsohn, R. Fluhr, J. Gressel, and Y. Eyal. 2004. Citrus fruit bitter flavors: Isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitt er flavonoids of citrus. Plant J. 40: 88-100. Futch, S. H., and L. K. Jackson. 2003a. Murcott (Honey Tangerine). Fact sheet HS174. Horticultural Sciences Department, Florida C ooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Futch, S. H., and L. K. Jackson. 2003b. Orlando Tangelo. Fact sheet HS175. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, Un iversity of Florida. Futch, S. H., and L. K. Jackson. 2003c. Sunburst Tangerine. Fact sheet HS168. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, Un iversity of Florida.

PAGE 90

90 Gancel, A. L., D. Oll, P. Ollitrault, F. Luro, J. M. Brillouet. 2002. Leaf and peel volatile compounds of an interspecific citrus somatic hybrid [ Citrus aurantifolia (Christm.) Swing + Citrus paradisi Macfayden]. Flavour Fragr. J. 17: 416-424. Gancel, A. L., P. Ollitrault, Y. Froelicher, F. To mi, C. Jacquemond, F. Luro, and J. M. Brillouet. 2005. Citrus somatic allotetraploid hybrids exhibit a differential reduction of leaf sesquiterpenoid biosynthesis compared with their parents. Flavour Fragr. J. 20: 626-632. Gancel, A. L., J. Grimplet, F. X. Sauvage P. Ollitrault, and J. M. Brillouet. 2006. Predominant expression of diploid mandarin leaf proteome in two citrus mandarin-derived somatic allotetraploid hybrids. J. Agr. Food Chem. 54: 6212-6218. Garruti, D. S., M. R. B. Franco, M. A. A. P. da Silva, N. S. Janzantti, and G. L. Alves. 2003. Evaluation of volatile flavour compounds from cashew apple ( Anacardium occidentale L) juice by the Osme gas chromatography/olfact ometry technique. J. Sci. Food Agr. 83:14551462. Gmitter, F. G., and X. Hu. 1990. The possible ro le of Yunnan, China, in the origin of contemporary Citrus species (Rutaceae). 44: 267-277. Gmitter, F. G., J. W. Grosser, W. S. Castle and G. A. Moore. 2007. A comprehensive citrus genetics improvement programme. p. 9-18. In: I. H. Khan (ed.). Citrus Genetics, Breeding and Biotechnology. CAB International, Wallingford, Oxfordshire, UK. Goldschmidt, E. E. 1977. Galactolipids and phospholip ids of orange peel and juice chromoplasts. 1977. Phytochemistry. 16: 1046-1047. Goodner, K. L., R. L. Rouseff, H. J. Hofsommer. 2001. Orange, mandarin and hybrid classification using multivariate statistics base d on carotenoid profiles. J. Agr. Food Chem. 49: 1146-1150. Goodner, K. L., R. L. Rouseff, H. J. Hofso mmer. 2008. Practical retention index models of OV101, DB-1, DB-5, and DB-Wax for flavor and fragrance compounds. LWT-Food Sci. Technol. 41: 951-958. Gottwald, T. R., J. H. Graham, and T. S. Sc hubert. 2002. Citrus canker: The pathogen and its impact. Online. Plant Health Progress. DOI: 10.1094/PHP-2002-0812-01-RV. Guen, S. L., C. Prost, and M. Demaimay. 2000. Critical Comparison of three olfactometric methods for the identifica tion of the most potent odor ants in cooked mussels ( Mytilus edulis ). J. Agr. Food Chem. 48: 1307-1314. Haring, H. G., F. Rijkens, H. Boelens, and A. van der Gen. 1972. Olfactory studies on enantiomeric eremophilane sesquiterpe noids. J. Agr. Food Chem. 20: 1018-1021. Hinterholzer, A., and P. Schieb erle. 1998. Identification of the mo st odour active volatiles in fresh, hand-extracted juice of Valencia late oranges by odour dilution techniques. Flavour. Fragr. J. 13: 49-55.

PAGE 91

91 Hodgson, R. W. 1967. Horticultural Varieties of Citrus. p. 431591. In: W. Reuther, H. J. Webber, and L. D. Batchelor (eds.). The Citrus Industry. Volume 1. University of California Press, Berkeley, CA, USA. Hgnadttir, A., and R. L. R ouseff. 2003. Identification of ar oma active compounds in orange essence oil using gas chromatographyol factometry and gas chromatographymass spectrometry. J. Chromatogr A. 998: 201-211. Iezzoni, A., and M. P. Pritts. 1991. Applications of principal components analysis to horticultural research. HortScience. 26:334-338. Jackson, L. K., and S. H. Futch. 2003a. Danc y Tangerine. Fact sheet HS169. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, Un iversity of Florida. Jackson, L. K., and S. H. Futch. 2003b. Fallglo Tangerine. Fact sheet HS173. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, Un iversity of Florida. Jackson, L. K., and S. H. Futch. 2003c. Minne ola Tangelo. Fact sheet HS171. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, Un iversity of Florida. Jackson, L. K., and S. H. Futch. 2003d. Robins on Tangerine. Fact sheet HS178. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, Un iversity of Florida. Jackson, L. K., and S. H. Futch. 2003e. Te mple Tangor. Fact sheet HS181. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, Un iversity of Florida. Jensen, K., M. A. Petersen, L. Poll, and P. B. Brockhoff. 1999. Influence of variety and growing location on the development of off-fla vor in precooked vacuum-packed potatoes. J. Agr. Food Chem. 47: 1145-1149. Jones, M. G., J. Hughes, A. Tregova, J. Milne, A. B. Tomsett, and H. A. Collin. 2004. Biosynthesis of the flavour precursors of onion and garlic. J. Exp. Bot. 55: 1903-1918. Jordn, M. J., C. A. Margara, P. E. Shaw, and K. L. Goodner. 2003. Volatile components and aroma active compounds in aqueous essen ce and fresh pink guava fruit puree ( Psidium guajava L.) by GC-MS and multidimensional GC/G C-O. J. Agr. Food Chem. 51: 14211426. Jouquand, C., C. K. Chandler, K. L. Goodner, a nd A. Plotto. 2008. Optimization of strawberry volatile sampling by direct gas chromatography olfactometry. Proc. Fla. State Hort. Soc. 121: 260-264.

PAGE 92

92 Khan, I. A., and W. J. Kender. Citrus Breeding: Introduction and Objectives. p. 1-8. In: I. H. Khan (ed.). Citrus Genetics, Breeding and Biotechnology. CAB International, Wallingford, Oxfordshire, UK. Kerbiriou, P., A. Plotto, K. Goodner, E. Baldwi n, and F. G. Gmitter, Jr. 2007. Distribution of aroma volatiles in a population of tangerine hybrids. Proc. Fla. State Hort. Soc. 120: 267275. Kirchner, J. G., J. M. Miller, R. G. Rice, G. J. Keller, and N. M. Fox. 1953. Volatile watersoluble constituents of grapefru it juice. J. Agr. Food Chem. 1: 510-512. Lan-Phi, N. T., C. Nishiyama, H. K. Choi, a nd M. Sawamura. 2006. Evaluation of characteristic aroma compounds of Citrus natsudaidai Hayata (Natsudaidai) cold-pressed peel oil. Biosci. Biotechnol. Bi ochem. 70: 1832-1838. Lan-Phi, N. T., T. Shimamura, H. Ukeda, a nd M. Sawamura. 2009. Chemical and aroma profiles of Yuzu ( Citrus junos ) peel oils of different cultivars. Food Chem. 115: 1042-1047. Lea, P., T. Ns, and M. Rdbotten. 1997. Replicates in Sensory Analysis. p. 56-62. In: P. Lea, T. Ns, M. Rdbotten (eds.). Analysis of Varian ce for Sensory Data. John W iley & Sons, New York, NY, USA. Lecanu, L., V. Ducruet, C. Jouquand, J. J. Gr atadoux, and A. Feigenbaum. 2002. Optimization of headspace solid-phase microextraction (SPME) for the odor analysis of surface-ripened cheese. J. Agr. Food Chem. 50: 3810-3817. Lewinsohn, E., Y. Sitrit, E. Bar, Y. Azulay, M. Ibdah, A. Meir, E. Yosef, D. Zamir, and Y. Tadmor. 2005. Not just colors carotenoid de gradation as a link between pigmentation and aroma in tomato and watermelon fru it. Trends Food Sci. Technol. 16: 407-415. Lin, J., and R. L. Rouseff. 2001. Characterizatio n of aroma-impact compounds in cold-pressed grapefruit oil using timeintensity GColfact ometry and GCMS. Flavour Fragr. J. 16: 457-463. Linssen, J. P. H., J. L. G. M. Janssens, J. P. Roozen, and M. A. Posthumus. 1993. Combined gas chromatography and sniffing port analysis of volatile compounds of mineral water packed in polyethylene laminated p ackages. Food Chem. 46: 367-371. Lcker, J., M. K. E. Tamer, W. Schwab, F. W. A.Verstappen, L. H. W. van der Plas, H. J. Bouwmeester, and H. A. Verhoeven. 2002. Monoterpene biosynthesis in lemon ( Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases. Eur. J. Biochem. 269: 3160-3171. Macleod, W. D., Jr., and N. M. Buigues. 1964. Ses quiterpenes. I. Nootkatone, a new grapefruit flavor constituent. J. Food Sci. 29: 565-568. Mahattanatawee, K., R. Rouseff, M. F. Va lim, and M. Naim. 2005. Identification and aroma impact of norisoprenoids in orange juice. J. Agr. Food Chem. 53: 393-397.

PAGE 93

93 Maruyama, T., M. Ito, and G. Honda. 2001. Mo lecular cloning, functio nal expression and characterization of (E)-farnesene synthase from Citrus junos Biol. Pharm. Bull. 24 1171-1175. Matich, A., and D. Rowan. 2005. Pathway analysis of branched-chain ester biosynthesis in apple using deuterium labeling and enantioselectiv e gas chromatography-mass spectrometry. J. Agr. Food Chem. 53: 7198-7203. Mayne, S. T. 1996. Beta-carotene, carotenoids, a nd disease prevention in humans. FASEB J. 10: 690-701. McDaniel, M. R., R. Miranda-Lopez, B. T. Watson, N. J. Micheals, and L. M. Libbey. 1990. Pinot noir aroma: A sensory/gas chromatogra phic approach. p. 23-36. In: G. Charalambous (ed.). Flavors and Off-Flavors. Elsevier Scie nce Publishers, Amsterdam, The Netherlands. McGarvey, D. J., and R. Croteau. 1995. Terpenoid metabolism. Plant Cell. 7: 1015-1026. Merle. H., M. Morn, M. A. Blzquez and H. Bo ira. 2004. Taxonomical contribution of essential oils in mandarins cultivars. Biochem. Syst. Ecol. 32: 491-497. Minh Tu, N. T., L. X. Thanh, A. Une, H. Ukeda, and M. Sawamura. 2002. Volatile constituents of Vietnamese pummelo, orange, tangerine and lime peel oils. Flavour Fragr. J. 17: 169174. Moore, G. A. 2001. Oranges and lemons: Clues to the taxonomy of C itrus from molecular markers. Trends Genet. 17: 536-540. Moshonas, M. G., and P. E. Shaw. 1972. Analys is of volatile constituents from tangerine essence. J. Agr. Food Chem. 20: 70-71. Moshonas, M. G., and P. E. Shaw. 1974. Quantitative analysis of tangerine peel oil. J. Agr. Food Chem. 22: 282-284. Moshonas, M. G., and P. E. Shaw. 1983. Composition of aqueous essence and essence oil from Citrus temple J. Agr. Food Chem. 31: 334-336. Moshonas, M. G., and P. E. Shaw. 1987. Quantita tive analysis of orange flavor volatiles by direct-injection gas chrom atography. J. Agr. Food Chem. 35: 161-165. Moshonas, M. G., and P. E. Shaw. 1990. Flavor evaluation of concentr ated aqueous orange essences. J. Agr. Food Chem. 38: 2181-2184. Moshonas, M. G., and P. E. Shaw. 1994. Quantita tive determination of 46 volatile constituents in fresh, unpasteurized orange juices using dynamic headspace gas chromatography. J. Agr. Food Chem. 42: 1525-1528.

PAGE 94

94 Moshonas, M. G., and P. E. Shaw. 1997. Quantitati on of volatile constituen ts in mandarin juices and its use for comparison with orange juices by multivariate analysis. J. Agr. Food Chem. 45: 3968-3972. Nicolosi, E., Z. N. Deng, A. Gentile, S. La Malfa, G. Continella and E. Tribulato. 2000. Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor. Appl. Genet. 100: 1155-1166. Nisperos-Carriedo, M. O., and P. E. Shaw. 1990. Comparison of volatile flavor components in fresh and processed orange juices. J. Agr. Food Chem. 38: 1048-1052. Njoroge, S. M., H. Koaze, M. Mwaniki, N. T. Minh Tu, and M. Sawamura. 2005. Essential oils of Kenyan citrus fruits: volatile components of two varieties of mandarins ( Citrus reticulata ) and a tangelo ( C paradisi C tangerina ). Flavour Fragr. J. 20: 74-79. Nordby, H. E., and S. Nagy. 1969. Fatty acid pr ofiles of citrus juice and seed lipids. Phytochcmistry. 8: 2027-2038. Nordby, H. E., and S. Nagy. 1971. Fatty acid prof iles of orange and ta ngor juice sac lipid. Phytochemistry. 10: 615-619. Ortuo, A., D. Garca-Puig, M. D. Fuster, M. L. Prez, F. Sabater, I. Porras, A. Garca-Lidn, and J. A. Del Ro. 1995. Flavanone and nootkato ne levels in different varieties of grapefruit and pummelo. J. Agr. Food Chem. 43: 1-5. Pawliszyn, J., B. Pawliszyn, and M. Pawliszyn. 1997. Solid phase microextraction (SPME). The Chemical Educator. 2: 1-7. Prez, A. G., R. Olias, P. Luaces, and C. Sanz. 2002. Biosynthesis of strawberry aroma compounds through amino acid metabolism. J. Agr. Food Chem. 50: 4037-4042. Prez-Cacho, P. R., K. Mahattanatawee, J. M. Smoot, and R. Rouseff. 2007. Identification of sulfur volatiles in canned ora nge juices lacking orange flav or. J. Agric. Food Chem. 55: 5761-5767. Prez-Cacho, P. R., and R. L. Rouseff. 2008. Fr esh squeezed orange juice odor: A review. Cri. Rev. Food Sci. Nutr. 48: 681-695. Prez-Lpez, A. J., and A. A. Carbonell-Barrachina. 2006a. Volatile odour components and sensory quality of fresh and processed mandarin juices. J. Sci. Food Agric. 86: 2404-2411. Prez-Lpez, A. J., D. Saura, J. Lorente, and A. A. Carbonell-Barrachina. 2006b. Limonene, linalool, -terpineol and terpinen-4-ol as quality control parameters in mandarin juice processing. Eur. Food Res. Tech. 222: 281-285. Pino, J. A., R. Marbot, and L. Chang. 2000. Multivariate analysis for classification of commercial grapefruit juice products by volatile constituents. J. Food Qual. 23: 261-269.

PAGE 95

95 Plotto, A., M. McDaniel, and J. P. Mattheis. 2000. Characterizati on of changes in Gala apple aroma during storage using Osme analysis, a gas chromatography-olfactometry technique. J. Am. Soc. Hortic. Sci. 125: 714-722. Plotto, A., C. A. Margara, K. L. Goodner, R. Goodrich, and E. A. Baldwin. 2004. Odour and flavour thresholds for key aroma components in an orange juice matrix: terpenes and aldehydes. Flavour Fragr. J. 19: 491-498. Plotto, A., K.W. Barnes, and K. L. G oodner. 2006. Specific anosmia observed for -ionone, but not for -ionone: Significance for flavor rese arch. J. Food Sci. 71: S401-S406. Plotto, A., C. A. Margara, K. L. Goodne r, and E. A. Baldwin. 2008. Odour and flavour thresholds for key aroma components in an or ange juice matrix: este rs and miscellaneous compounds. Flavour Fragr. J. 23: 398-406. Pollien, P., A. Ott, F. Montigon, M. Baumga rtner, R. Muoz-Box, and A. Chaintreau 1997. Hyphenated headspace-gas chromatography-sn iffing techniques: Sc reening of impact odorants and quantitative aromagram comparisons. J. Agr. Food Chem. 45: 2630-2637. Radford, T., K. Kawashima, P. K. Friedel, L. E. Pope, and M. A. Gianturco. 1974. Distribution of volatile compounds between the pulp and serum of some fruit ju ices. J. Agr. Food Chem. 22: 1066-1070. Rega, B., N. Fournier, and E. Guichard. 2003. Solid phase microextracti on (SPME) of orange juice flavor: Odor representa tiveness by direct gas chromatography olfactometry (D-GCO). J. Agr. Food Chem. 51: 7092-7099. Reineccius, G. 2005. Flavor Analysis. p. 33-72. In: G. Reineccius (ed.). Flavor Chemistry and Technology. CRC Press, Boca Raton, FL, USA. Ringnr, M. 2008. What is principal co mponent analysis? Nature Biotechnol. 26: 303-304. Rouseff, R., and P. R. Prez-Cacho. 2007. Citr us Flavour. p. 117-134. In: R. G. Berger (ed.). Flavours and Fragrances Chemistry, Biopro cessing and Sustainability. Springer-Verlag Berlin Heidelberg, Germany. Rowan, D. D., A. J. M., S. Fielder, and M. B. Hunt. 1999. Biosynthesis of straight-chain ester volatiles in red delicious and granny smith a pples using deuterium-labeled precusors. J. Agr. Food Chem. 47: 2553-2562. Sawamura, M., and T. Kuriyama. 1988. Quantitative determination of volatile constituents in the pummelo ( Citrus grandis Osbeck forma Tosa-buntan). J. Agr. Food Chem. 36: 567-569. Sawamura, M., K. Shichiri, Y. Ootani, and X. H. Zheng. 1991. Volatile constituents of several varieties of pummelos and characteristics among Citrus species. Agric. Biol. Chem. 55: 2571-2578.

PAGE 96

96 Sawamura, M., N. T. M. Tu, Y. Onishi, E. Og awa, and H. S. Choi. 2004. Characteristic odor components of Citrus reticulata Blanco (Ponkan) cold-presse d oil. Biosci. Biotechnol. Biochem. 68: 1690-1697. Schaffer, R. J., E. N. Friel, E. J. F. Souleyre K. Bolitho, K. Thodey, S. Ledger, J. H. Bowen, J. H. Ma, B. Nain, D. Cohen, A. P. Gleave, R. N. Crowhurst, B. J. Janssen, J. L. Yao, and R. D. Newcomb. 2007. A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the fi nal step in each bios ynthetic pathway. Plant Physiol. 144: 1899-1912. Schwab, W., R. Davidovich-Rikanati, and E. Lewinsohn. 2008. Biosynthesis of plant-derived flavour compounds. Plant J. 54: 712-732. Scora, R. W., and M. N. Malik. 1970. Chemical ch aracterization of citrus as a tool in phylogeny. Taxon 19: 215-228. Scora, R. W. 1975. On the history and origin of Citrus Bull. Torrey Bot. Club. 102: 369-375. Sharon-Asa, L., M. Shalit, A. Frydman, E. Bar, D. Holland, E. Or, U. Lavi, E. Lewinsohn, and Y. Eyal. 2003. Citrus fruit flavor and ar oma biosynthesis: isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound va lencene. Plant J. 36: 664-674. Shaw, P. E. 1979. Review of quantit ative analyses of citrus essent ial oils. J. Agr. Food Chem. 27: 246-257. Shaw, P. E., and C. W. Willson, III. 1981. Importance of nootkatone to the aroma of grapefruit oil and the flavor of grapefruit juice. J. Agr. Food Chem. 29: 677-679. Shaw, P. E., and C. W. Wilson, III. 1982. Volatile sulfides in headspace gases of fresh and processed citrus juices. J. Agr. Food Chem. 30: 685-688. Shaw, P. E. 1991. Fruits II. p. 305-327. In: H. Ma arse, (ed.). Volatile Compounds in Foods and Beverages. Marcel Dekk er, New York, NY, USA. Shaw, P. E., M. G. Moshonas, C. J. Hearn, a nd K. L. Goodner. 2000. Volatile constituents in fresh and processed juices from grapefruit a nd new grapefruit hybrids. J. Agr. Food Chem. 48: 2425-2429. Shaw, P. E., K. L. Goodner, M. G. Moshona s, and J. Hearn. 2001. Comparison of grapefruit hybrid fruit with parent fruit based on compos ition of volatile components. Sci. Hortic. 91: 71-80. Shimada, T., T. Endo, H. Fujii, M. Hara, T. Ueda, M. Kita, and M. Omura. 2004. Molecular cloning and functional characte rization of four monoterpe ne synthase genes from Citrus unshiu Marc. Plant Sci. 166: 49-58.

PAGE 97

97 Shiota, H. 1993. New esteric components in the volatiles of banana fruit ( Musa sapientum L.). J. Agr. Food Chem. 41: 2056-2062. Soost, R. K., and M. L. Roose. 1996. Citrus. p. 25 7-323. In: J. Janicks, J. N. Moore (eds.). Fruit Breeding. Volume 1. Tree and Tropical Fruits. John W iley & Sons, New York, NY, USA. Steffen, A., and J. Pawliszyn. 1996. Analysis of flavor volatiles using headspace solidphase microextraction. J. Agr. Food Chem. 44: 2187-2193. Stevens, S. S. 1957. On the psychophys ical law. Psychol. Rev. 64: 153-181. Swingle, W. T., and P. C. Reece. 1967. The botany of Citru s and its wild relatives of the orange subfamily (family Rutaceae, subfamily Aurantio ideae). p. 190-430. In: H. J. Webber, L. D. Batchelor (eds.). The Citrus Industry. Volume 1. University of California Press, Berkeley, CA, USA. Talon, M., and F. G. Gmitter, Jr. 2008. Citrus genomics. Int. J. Plant Genomics. 2008: 1-17. Tanaka, T. 1977. Fundamental discussion of Citrus classification. Stud. Citrol. 14: 1-6. Tholl, D. 2006. Terpene synthases and the regulati on, diversity and biologi cal roles of terpene metabolism. Curr. Opin. Plant Biol. 9: 297-304. Tominaga, T., and D. Dubourdieu. 2000. Identifi cation of cysteinylate d aroma precursors of certain volatile thiols in passion fruit juice. J. Agr. Food Chem. 48: 2874-2876. Tnder, D., M. A. Peterson, L. Poll, and C. E. Olsen. 1998. Discrimination between freshly made and stored reconstituted orange juice usi ng GC odour profiling and aroma values. Food Chem. 61: 223-229. Tressel, R., and F. Drawert. 1973. Biogenesis of banana volatiles. J. Agr. Food Chem. 21: 560565. Ullrich, F., and W. Grosch. 1987. Identification of the most intense volatile flavour compounds formed during autoxidation of linoleic aci d. Z. Lebensm. Unters. Forsch. 184: 277-282. Umano, K., Y. Hagi, K. Nakahara, A. Shoji, and T. Shibamoto. 1992. Volatile constituents of green and ripened pineapple (Ananas comosus [L.] Merr). J. Agr. Food Chem. 40: 599603. U.S. Department of Agriculture. 1997. United Stat es Standards for Grades of Florida Tangerines. Accessed 1 Semptember 2009. . U.S. Department of Agriculture. 2009. C itrus Summary 2007-08. Accessed 1 September 2009. .

PAGE 98

98 U.S. Department of Agriculture-Nutrition Coordinating Center (U SDA-NCC). 1998. USDANCC Carotenoid Database for U.S. Foods. Accessed 1 September 2009. < http://www.nal.usda.gov/fnic/foodcomp/Data/car98/car98.html>. Valim M. F., R. L. Rouseff, and J. Lin. 2003. Gas chromatographic-olfactometric characterization of aroma compounds in two t ypes of cashew apple nectar. J. Agr. Food Chem. 51: 1010-1015. van Ruth, S. M. 2001. Methods for gas chromatography-olfactometry: A review. Biomol. Eng. 17: 121-128. van Ruth, S. M. 2004. Evaluation of two ga s chromatographyolfactometry methods: The detection frequency and perc eived intensity method. J. Chromatogr. A. 1054: 33-37. Webbber, H. J. 1967. History and Development of the Citrus Industry. p. 1-39. In: W. Reuther, H. J. Webber, L. D. Batchelor (eds.). Th e Citrus Industry. Volume 1. University of California Press, Berkeley, CA, USA. Weenen, H., W. E. Koolhaas, and A. Apriya ntono. 1996. Sulfur-containi ng volatiles of durian fruits ( Durio zibethinus Murr.). J. Agr. Food Chem. 44: 3291-3293. Wilson, C. W., III., and P. E. Shaw. 1978. Synthesis of nootkatone from valencene. J. Agr. Food Chem. 26: 1430-1432. Wilson, C. W., III., and P. E. Shaw. 1981. Importan ce of thymol, methyl N-methylanthranilate, and monoterpene hydrocarbons to the aroma and flavor of mandarin co ld-pressed oils. J. Agr. Food Chem. 29: 494-496. Wutscher, H. K., N. P. Maxwell, and A. V. Shull. 1973. Bower, a new mandarin variety for Texas. J. Rio Grande Valley Hort. Soc. 27: 40-43. Yajima, I., T. Yanai, M. Nakamura, H. Sakakibara, and K. Hayashi. 1979. Compositions of the volatiles of peel oil and juice from Citrus unshiu. Agric. Biol. Chem. 43: 259-264. Zellner, B. D., P. Dugo, G. Dugo, and L. M ondello. 2008. Gas chromatographyolfactometry in food flavour analysis. J. Chromatogr A. 1186: 123-143.

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99 BIOGRAPHICAL SKETCH Takayuki Miyazak i was born in Arida city, Wa kayama prefecture, Japan. His hometown is well known as a center of citrus production, es pecially Satsuma mandarin. He attended the Laboratory of Horticultural Crop Physiology in the Department of Life Sciences/Faculty of Bioresources at Mie University and received a Bacheolor of Science in March 2006. After graduation, he attended the Gradua te School of Bioresources at Mie University till July 2006. Takayuki was awarded an ambassadorial scholarsh ip from Rotary Found ation and enrolled in the graduate program of the Horticultural Scie nces Department at University of Florida in August 2006. After taking courseworks in Gainesvill e, he started the rese arch project under the supervision of Dr. Frederick G. Gmitter, Jr., a pr ofessor of citrus breeding and genetics, at the Citrus Research and Education Center in Lake Alfred, Florida. Following the completion of his M.S. program, he will go back to Japan and pursue his career in horticulture.