T.C. Niğde Ömer Halisdemir University Graduate School of Natural and Applied Sciences Department of Agricultural Genetic Engineering

S. B. GÜVEN, 2020 S.B. GÜVEN, T.C. NİĞDE ÖMER HALİSDEMİR UNIVERSITY GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES DEPARTMENT OF AGRICULTURAL GENETIC ENGINEERING

MASTERTHESIS DETERMINATION OF BIOACTIVE PROPERTIES OF SOME SHORT DAY ONION INBRED LINES PRODUCED IN ALATA CLIMATE

SAİME BUSE GÜVEN

HALİSDEMİRUNIVERSITY

NİĞDE ÖMER ÖMER NİĞDE

August 2020

GRADUATE SCHOOL OF NATURAL AND SCHOOL AND APPLIEDNATURAL OF GRADUATE SCIENCES

T.C. NİĞDE ÖMER HALİSDEMİR UNIVERSITY GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES DEPARTMENT OF AGRICULTURAL GENETIC ENGNEERING

DETERMINATION OF BIOACTIVE PROPERTIES OF SOME SHORT DAY ONION INBRED LINES PRODUCED IN ALATA CLIMATE

SAİME BUSE GÜVEN

Master Thesis

Supervisor

Assist. Prof. Dr. Ali Fuat GÖKÇE

August 2020

Saime Buse GÜVEN tarafından Dr. Öğr. Üyesi Ali Fuat GÖKÇE danışmanlığında hazırlanan “Determination of Bioactive Properties of Some Short Day Onion Inbred Lines Produced in Alata Climate” adlı bu çalışma jürimiz tarafından Niğde Ömer Halisdemir Üniversitesi Fen Bilimleri Enstitüsü Tarımsal Genetik Mühendisliği Anabilim Dalı’nda Yüksek Lisans (İngilizce) tezi olarak kabul edilmiştir.

Başkan : Dr. Öğr. Üyesi Ali Fuat GÖKÇE, Niğde Ömer Halisdemir Üniversitesi, Ayhan Şahenk Tarım Bilimleri ve Teknolojileri Fakültesi, Tarımsal Genetik Mühendisliği Bölümü

Üye : Doç. Dr. Zahide Neslihan ÖZTÜRK GÖKÇE, Niğde Ömer Halisdemir Üniversitesi, Ayhan Şahenk Tarım Bilimleri ve Teknolojileri Fakültesi, Tarımsal Genetik Mühendisliği Bölümü

Üye : Dr. Öğr. Üyesi Fatih HANCI, Erciyes Üniversitesi, Ziraat Fakültesi, Bahçe Bitkileri Bölümü

ONAY:

Bu tez, Fen Bilimleri Enstitüsü Yönetim Kurulunca belirlenmiş olan yukarıdaki jüri üyeleri tarafından …./…./2020 tarihinde uygun görülmüş ve Enstitü Yönetim Kurulu’nun …./…./2020 tarih ve …...... sayılı kararıyla kabul edilmiştir.

...... /...../2020

Prof. Dr. Murat BARUT MÜDÜR

THESIS CERTIFICATION

I certify that the thesis has been written by me and that, to the best of my knowledge and belief. All information presented as part of this thesis is scientific and in accordance with the academic rules. Any help I have received in preparing the thesis, and all sources used, have been acknowledged in the thesis.

Saime Buse GÜVEN

SUMMARY

DETERMINATION OF BIOACTIVE PROPERTIES OF SOME SHORT DAY ONION INBRED LINES PRODUCED IN ALATA CLIMATE

GÜVEN, Saime Buse Niğde Ömer Halisdemir University Graduate School of Natural and Applied Sciences Department of Agricultural Genetic Engineering

Supervisor :Assist. Prof. Dr. Ali Fuat GÖKÇE

August 2020, 80 pages

Onion is a widely consumed vegetable all around the world since ancient times. It is rich in nutritional polyphenolic compounds. Flavonoids, particularly quercetin, has been found in substantial amount in this family. The bioactive properties of onions play a crucial role in human health and are now became an important objective in onion breeding programs. In this study, pomological and phytochemical analyses were performed using 19 different short day onion genotypes produced under Alata conditions. Results showed that genotype means of the total soluble solid contents were 5.2 to 8.5 °Brix, while pyruvic acid had wide distribution in the range of 3.277 to 8.828 µM / mL. The total antioxidant capacity varied from 0.07 to 0.10 mM trolox equivalent / 100 mL and total anthocyanin results were between 0.0557 and 0.5556 mg / 100 mL. Total phenolic values were observed within the range of 12.01 and 32.50 mg gallic acid equivalent / 100 mL. Fructose values were found between 1.50 and 2.41 g / 100 g FW. Genotype effects for glucose and sucrose were not significantly different. Quercetin values were observed between 0.06 and 1.94 μg / 100 g FW. The data obtained in this study can further be used for the improvement of onion genotypes and in onion breeding and quality improvement programs.

Keywords: Short day onion, phenolic compounds, antioxidant, anthocyanin, pyruvic acid, sugar, flavonoid, quercetin, pH, total soluble solid content, titratable acidity

ÖZET

ALATA İKLİMİNDE ÜRETİLEN KISA GÜN SOĞAN HATLARINDA BİYOAKTİF ÖZELLİKLERİN BELİRLENMESİ

GÜVEN, Saime Buse Niğde Ömer Halisdemir Üniversitesi Fen Bilimleri Enstitüsü Tarımsal Genetik Mühendisliği Anabilim Dalı

Danışman :Dr. Öğr. Üyesi Ali Fuat GÖKÇE

Ağustos 2020, 80 sayfa

Soğan, eski zamanlardan beri tüm dünyada yaygın olarak tüketilen bir sebzedir. Besleyici polifenolik bileşikler bakımından zengindir. Flavonoidler özellikle kuersetin bu ailede önemli miktarda bulunmuştur. Soğanların biyoaktif özellikleri insan sağlığı üzerinde çok önemli bir rol oynamaktadır ve soğan ıslah programlarında artık önemli bir ıslah amacı haline gelmiştir. Bu çalışmada, Alata koşullarında yetiştirilen 19 farklı kısa gün soğanın pomolojik ve fitokimyasal analizi yapılmıştır. Sonuçlar, suda çözünebilir kuru madde miktarının 5,2 ile 8,5 °Brix, piruvik asit miktarının ise 3,277 ile 8,828 µM / mL aralığında geniş bir dağılımı olduğunu göstermiştir. Toplam antioksidan kapasitesi 0,0719 ile 0,1013 mM troloks eşdeğeri / 100 mL aralığında olurken, toplam antosiyanin değerleri 0,0557 ile 0,5556 mg / 100 mL olarak bulunmuştur. Toplam fenolik değerleri 12,01 ile 32,50 mg gallik asit eşdeğeri / 100 mL aralığında gözlenmiştir. Fruktoz değerleri 1,5000 ile 2,4067 g / 100 g yaş ağırlık arasında gözlenmiştir. Genotiplerin glikoz ve sükroz üzerine etkisi istatistiksel açısından önemsiz bulunmuştur. Kuersetin değerleri ise 0,0633 ile 1,9433 μg / 100 g taze ağırlık arasında gözlenmiştir. Bu çalışmada elde edilen veriler soğan genotiplerinin iyileştirilmesinde ve ayrıca soğan ıslahı ve kalite iyileştirme programlarında kullanılabilir.

Anahtar Sözcükler: Kısa gün soğan, fenolik bileşikler, antioksidan, antosiyanin, pürivik asit, şeker, flavonoid, kuersetin, suda çözünmüş kuru madde miktarı, pH, titre edilebilir asit

ACKNOWLEDGEMENTS

Onion is a vegetable widely found and eaten all over the world, due to, it has a unique flavor and taste, moreover, it is the richest and most commonly consumed source of dietary flavonoids. The main flavonoids in the onion are flavonols. Quercetin is a major flavonols in the onion. Flavonoids are bioactive compounds that are known for their potential healthful effects. They provide protective effects against cancer, coronary heart disease, and age-related degenerative diseases. Besides, the quercetin is antioxidant and antioxidant activity is known as one of the essential medical property. The bioactive properties of onions play a crucial role in human health and are now became an important objective in onion breeding programs. Therefore, in present thesis, 19 different short day onion genotypes performed by pomologic and phytochemical analyses. A breeder may use these genotypes based on their bioactive properties depending on the breeding objectives.

I would like to thank my supervisor Dr. Ali Fuat GÖKÇE who provided me the opportunity to pursue a master degree under his kind supervision. I am deeply indebted to Dr. Ali Fuat GÖKCE for giving me an opportunity to work in his team, for all the knowledge in the area of field, greenhouses, and laboratory work that I have learned from him, and for his very helpful suggestions during my research. I also wish to express my sincere gratitude to Dr. Mustafa ÜNLÜ and Dr. Veysel ARAS for their support in the laboratory work.

I thank Advisory Committee Members Assoc. Prof. Dr. Z. Neslihan ÖZTÜRK GÖKÇE and Dr. Fatih HANCI. Their knowledge, energy, and enthusiasm were critical to this effort.

Special thanks go to TÜBİTAK and Ayhan Şahenk Foundation for providing me partial fellowship during my master degree program. This thesis has been prepared as a part of TÜBİTAK project no 117G023.

vi My deepest appreciation is conveyed to my friends Zehra ÜZER, Arslan ASİM, Muhammad Naeem BUKHARI and Rıdvan ARSLAN for their constant support and encouragement during all my endeavors. Finally, I would like to dedicate this achievement to my mother Necla GÜVEN, my father H. Gürol GÜVEN, and my brother Ç. Çağlar GÜVEN for their encouragement, wishes and contribution during all my endeavors.

vii

TABLE OF CONTENTS

SUMMARY ...... iv ÖZET ...... v ACKNOWLEDGEMENTS ...... vi LIST OF TABLES ...... x LIST OF FIGURES ...... xi SYMBOLS AND ABBREVIATIONS ...... xii CHAPTER I INTRODUCTION ...... 1 CHAPTER II LITERATURE REVIEW ...... 4 2.1 Origin of Allium ...... 4 2.2 Taxonomy of Allium ...... 4 2.3 Ecological Requiments of Onion ...... 5 2.4 General Properties of Onion ...... 6 2.5 Economic Importance of Onion ...... 9 2.6 Importance of Onion for Human Health ...... 10 2.7 Phenolic Compounds in Onion ...... 11 2.7.1 Flavonoid and quercetin compound in onion ...... 13 2.7.2 Anthocyanins in onion ...... 17 2.8 Pungency and Flavor of Onion Bulbs ...... 18 2.9 Antioxidant Quantity in Onion...... 20 2.10 Sugar Content and Total Soluble Solid in Onion Bulbs ...... 20 CHAPTER III MATERIALS AND METHODS ...... 23 3.1 Materials ...... 23 3.2 Methods ...... 24 3.2.1 Pomological analyses ...... 25 3.2.2 Phytochemical analyses ...... 28 3.2.3 Statistical Analyses ...... 37 CHAPTER IV RESULTS AND DISCUSSION ...... 38 4.1 Results of Pomologic Analyses ...... 38 4.1.1 Color measurement ...... 39 4.1.2 Total soluble solid content (TSS) ...... 40

viii 4.1.3 Titratable acidity (TA) ...... 42 4.1.4 pH ...... 43 4.2 Phytochemical Results ...... 44 4.2.1 Pyruvic acid ...... 46 4.2.2 Antioxidant ...... 48 4.2.3 Anthocyanins ...... 50 4.2.4 Total phenolic content (TPC) ...... 51 4.2.5 Sugars (Fructose, Glucose, Sucrose) ...... 53 4.2.6 Quercetin (Flavonoid compound) ...... 55 CHAPTER V CONCLUSION ...... 57 REFERENCES ...... 61 CURRICULUM VITAE ...... 80

LIST OF TABLES

Table 2.1. Leading onion producing countries in 2019 ...... 9

Table 2.2. Onion production area (ha) and quantity (ton) of leading provinces of Turkey in 2019 ...... 10 Table 2.3. °Brix scale of some vegetables and fruits ...... 22 Table 3.1. Short day onion elite lines used in the thesis study ...... 23 Table 3.2. Mobile phases and usage amount by time for HPLC ...... 33 Table 4.1. Analysis of variance table for color measurements (L*, a*, b* values), total soluble solid (TSS), titratable acidity (TA), and pH ...... 38 Table 4.2. Mean separations of scale color, L*, a*, and b* values of the genotypes ..... 39 Table 4.3. Mean separations of total soluble solid content (TSS), titratable acidity (TA, g / mL), and pH values of the genotypes ...... 41 Table 4.4. Analysis of variance table for pyruvic acid, antioxidant, anthocyanin, total phenolic content (TPC), fructose, and quercetin ...... 45 Table 4.5. Mean separations of pyruvic acid, total antioxidant, anthocyanin, phenol and flavonoid values of the genotypes ...... 46 Table 4.6. Mean separations of sugars (fructose, glucose, and sucrose) and quercetin values of the genotypes ...... 54

LIST OF FIGURES

Figure 2.1. Root structure of Allium cepa L...... 7 Figure 2.2. Onion harvesting stage (bending of onion neck) ...... 8 Figure 2.3. Basic skeletal structure of flavonoid compounds ...... 14 Figure 2.4. Essential subgroup of flavonoids chemical structure ...... 14 Figure 2.5. Quercetins structure found in onion ...... 15 Figure 3.1. Onion bulbs used for pomological and phytochemical analyses ...... 24 Figure 3.2. Scale color measurement by chromometre ...... 25 Figure 3.3. Measurement of total soluble solid contents of onion samples by refractometer ...... 26 Figure 3.4. Measurement of titratable acidity of onion samples ...... 27 Figure 3.5. Measurement of pH value of onion samples ...... 28 Figure 3.6. Incubation of sample mixtured for pyruvic acid analysis on water bath ...... 28 Figure 3.7. Onion juice samples were prepared and measured by spectrophotometer ... 29 Figure 3.8. Onion juice samples of short day onion genotypes ...... 30 Figure 3.9. Measurement of total anthocyanins ...... 31 Figure 3.10. Preparation of samples for measurement of the total phenolics ...... 32 Figure 3.11. Total phenolics analysis with spectrophotometer ...... 32 Figure 3.12. Flavonoid chromatogram ...... 34 Figure 3.13. Sugar chromatogram ...... 35 Figure 3.14. Calibration graph of fructose ...... 35 Figure 3.15. Calibration graph of glucose ...... 36 Figure 3.16. Calibration graph of sucrose ...... 36

SYMBOLS AND ABBREVIATIONS

Symbols Descriptions

% Percentage °Brix Brix degree °C Celcius degree µg Microgram µL Microliter cm Centimeter g Gram ℎ° Hue angle

H2SO4 Sulfuric acid ha Hectare kg Kilogram L Liter M Molar mg Miligram min Minute mL Milliliter mM Millimolar nm Nanometer ppm Parts per milion rpm Revolutions per minute sec Second T Ton μM Micromolar 퐶* Chroma value

Abbreviations Descriptions

ACN Acetonitrile ACSOs Alkyl Cystine Sulphoxide

xii ANOVA Analysis of Variance CIE Commission Internationale de I’Eclairage DAD Diode Array Detector DF Dilution factor DNA Deoxyribonucleic Acid DNPH 2,4-Dinitrophenylhydrazine DPPH 2,2-diphenyl-1-picrylhydrazyl DW Dry Weight FAOSTAT Food and Agriculture Organization Statistical FOSs Fructooligosaccharides FRAP Ferric Reducing Ability of Plasma FW Fresh Weight GAE Gallic Acid Equivalent GLM Generalized Linear Model HCL Hydrochloric Acid HPLC High Performance Liquid Chromatography KCl Potassium Chloride LSD Least Significant Difference MeOH Methanol MW Molecular weight NaOH Sodium Hydroxide ODS Octadecylsilyl PDA Photodiode Array Detection pH Potential for Hydrogen R&D Research and Development RID Refractive Index Detectors ROS Reactive Oxygen Species SAS Statistical Analysis Software TA Titratable Acidity TE Trolox Equivalent TEAC Trolox Equivalent Antioxidant Capacity TFA Trifluoroacetic Acid TPC Total Phenolic Content TSS Total Soluble Solid

xiii CHAPTER I

INTRODUCTION

Member crops of the Allium are being grown worldwide. The Allium genus as summarized by Brewster (2008) include diverse, numerous cultivated plant species such as onion (Allium cepa L.), garlic (A. sativum L.), leek (A. ampeloprasum L. porrum group), shallot (A. cepa L. aggregatum group), Japanese bunching onion (A. fistulosum L.), chives (A. schoenoprasum L.), and ramson (A. ursinum L.). Onion is the most common and widely consumed species of Allium genus, distributed to over all areas of the world. Onion was proposed to be the first domesticated in the lands of Afghanistan, Pakistan, Tajikistan and Northern Iran as the primary center (McCollum, 1976; Vvedensky, 1944). The second center was indicated North of Iran, known as Near East Asia, West of India, East of Caspian Sea and Azerbaijan (Baytop and Mathew, 1984).

Onion (A. cepa L.) has a great significance in world's economy. It is generally grown, traded and consumed in almost every countries. It is an appetizing vegetable with many different uses, as well as being consumed as salad in Turkey and other countries since ancient times. Over the years, it became an essential vegetable for great number of dishes because of its unique aroma and flavor. This increased the demand of onion in the recent decates. According to Food and Agriculture Organization Statistical (FAOSTAT) values, China ranks the first among the leading onion producer countries, followed by India, USA, Iran, Egypt, Russia and Turkey. Currently, onion is the second most important crop globally. Worldwide, onion production is 96,773,819 tons, and the area under onion cultivation is approximately 5,039,908 hectares (Anonymous, 2019a).

Onion is biannual vegetable that needs two growing seasons to produce seed. It has a tremendous genotypic variation due to varied photoperiod lengths, widespread growing areas and climatic conditions. There are three onion types depending on the day light requirements; short day onion which requires a day length of 12 hours, intermediate day onion which requires a day length between 12 and 14 hours, and long day onion which requires a day length of over 14 hours (Gökçe and Tekeli, 2015).

1 Among the other traits, bulb color of onion is the most important property because the physical appearance is the first trait that attracts consumers. Some processing conditions in food sector can cause damage to onion flesh color due to varying pH values, amount of titratable acidity, storage conditions (Ahmed and Shivhare, 2001).

People have great interest in natural products in order to treat different diseases. In developed countries, people are concerned with the beneficial aspect of food. Edible onion (A. cepa L.) is consumed abundantly throughout our country. It includes rich polyphenolics that have an important role in the health benefits of humans. It also contains many phytochemicals that possess varying bioactive properties, like antioxidative activity. The phytochemicals in this vegetable fall into two main groups as volatile and non-volatile compounds. Volatile compounds include sulfur groups whereas non-volatile compounds include flavonoids and phenols. Onion is also vegetable rich in alk(en)yl cysteine sulphoxides (ACSOs) in volatile sulfur group (Randle et al., 1995; Thomas and Parkin, 1994). ACSOs are the essential chemicals to give distinct flavor and odor to onion. When the ACSOs enzymatically breakdown, they produce stoichiometric amounts of ammonia and pyruvic acid; therefore, the pyruvic acid is used to measure to directly assess the overall flavor of onion (Randle et al., 1998; Yoo et al., 2006). Pyruvate is used for preventing certain diseases due to blood thinning effect and breaking down fat such as high cholesterol, cataracts, cancer, weight loss and obesity.

Phenolic compounds are also called biological active substances because they have a significant role in health. They are capable of scavenging free superoxide radicals, reducing the risk of cancer and protecting biological systems against the harmful effects of oxidative processes on macromolecules, such as carbohydrates, proteins, lipids and DNA. Therefore, they are reducing the risk of numerous diseases such as cancer, cardiovascular diseases, nervous system disorders, and Alzheimer's and Parkinson's diseases (Yünlü and Kır, 2016).

Phenolic compounds are a widespread secondary metabolites including numerous groups. They are classified according to their carbon skeleton into the subsequent foremost classes; simple phenols, phenolic acids, flavonoids, tannins, lignans, lignins, curcuminoids, coumarins, and stilbenes. Flavonoids are one of the biggest groups of phenolic compounds that are naturally produced. They are important molecules for health

2 benefits due to their antioxidative, antibacterial, and anticarcinogenic properties. The quercetin is the most familiar one of the flavonols that belongs to flavonoid (Wang et al., 2018). It is abundantly found in onion. Quercetin has antioxidant and anti-inflammatory effects which might help reduce inflammation, kill cancer cells, control blood sugar, and help prevent heart disease (Smith et al., 2003).

Anthocyanins are also a subgroup of flavonoids that are water-soluble color pigments giving natural color to fruits and vegetables. With this, they have a in role in plant pollination by the color of the flower. Besides, they have an antioxidant effect, therefore, they have benefits for human health in the treatment of several diseases such as cardiovascular diseases, cardiac spasm, vascular occlusions, artery stiffness, and clot formation (Nizamlıoğlu and Nas, 2010).

Onion is also rich in antioxidants which vary among onion mainly depending upon the flesh color and genotypes. In onion breeding programs, antioxidant effect has an important trait because of its positive affect on human health. The bioactive properties have a great influence on breeding programs to produce better quality onion cultivars.

There are variations in phytochemicals with different bioactive properties of onion depending on the cultivar since there are numerous genetic variations in onion (Sloan, 2000). Plant breeders aim to develop onion cultivars with higher nutritional values. The aim of this thesis is, thereby, to determine bioactive properties of some short day onion elite breeding lines to select onion genotypes with higher nutritional value.

3 CHAPTER II

LITERATURE REVIEW

2.1 Origin of Allium

Onion (Allium cepa L.) is one of the earliest cultivated vegetables in history (Teshika, 2019). There is no clear evidence about the origin of onion. It was first domesticated in the lands of Afghanistan, Pakistan, Tajikistan and Northern Iran (McCollum, 1976; Vvedensky, 1944), then later mountainous parts of Turkmenistan, Anatolia and Near East Asia including Northern Iran, Western India, Eastern Caspian Sea and Azerbaijan (Baytop and Mathew, 1984).

Onion is known to be used for many different purposes in ancient civilizations. It was reported that the onion, which has great importance in every field from past to present, has been transported from Egypt to Europe with the conquering armies. There were texts about medical practice on Allium genus of vegetables especially garlic and onion in some countries such as Egypt, Greece, Rome, China, and India. Onion and similar figures were found in the pyramids left by the Egyptian civilization. It was also used to feed slaves to protect from diseases who were building huge pyramids and it was believed as a holy vegetable, only the people that deserved from the God in Egypt could have it. In Rome, onion was used to feed soldiers before the battlefield to boost their energy and power (Guercio, et al., 2014). Nowadays, it is grown almost anywhere in the world and and important vegetable of every cuisine (Brewster, 2008).

2.2 Taxonomy of Allium

Allium is known as the oldest cultivated crop, besides it is one of the earliest classified crops. In the last century, taxonomic of the Allium has been reclassified several times, and there is still a big discussion about it due to enormous variant close relative plant species in the group. The first classification was depended on the morphological features such as shape, size, color, and pungency of bulb onion. Gökçe (2001) summarized the classification history of onion in his dissertation that the first name of onion and identification varied a lot from Hypocrite's time of 430 B.C. and on McCollum (1976).

4 The first Allium types were listed comprehensively by Carolus Clusis' ''Rariorum Plantarum Historia'' (1601). He also illustrated the various Allium species under the taxonomic heading Victoralis, Scorodoprasum, Moly, Allium sive moly montanum, and Moly Narcissi foliis. Stearn (1944) indicated that the Allium was used instead of onion and its relatives. Additionally, one of the publications ''De allii Genera Naturali Libellus'' (1745) by Swiss botanist Albercht von Haller indicated that 24 Allium species were nomenclatured on the basis of morphological properties. In addition, different types of classifications were based on ovary development. Plants which had more than two ovules in each chamber of the ovary were Melanocrommyum species and other species were Crommyum that included just 2 ovules in ''Phytographia Canariensis'' in 1848 by Philip Barker Webb (Strean 1944). Vvedensky (1944) classified the Allium genus as consisted of four parts, Cepa (bulb onion), Porrum (garlic and leek), Phyllodon (Japanese bunching onion), and Rhiziridium (chive and Chinese chive) with detailed identification of Allium.

Botanical classification of Allium genus that is somehow still valid today depends on the most of the researchers’ studies (Hutchinson, 1934; Hutchinson, 1973). Onion belongs to class Monocotyledones, Superorder is Liliiflorae, Order is Asparagales, Family is Alliaceae, Tribe is Alliae, and genus is Allium (Brewster 2008). Before this classification, Allium was in Liliaceae family (Cronquist, 1968; Cronquist et al., 1977) because of its free ovary and attachment of the other flower parts (Jones and Mann 1963), and the Amaryllidaceae (Hutchinson, 1934; Hutchinson 1973) due to its umbellate inflorescence surrounded by a spathe (Jones and Mann 1963). Nonetheless, some scientist in the year 2020 decided to use as Allioideae (formerly the family Alliaceae), since they divided the family into three subfamilies: Amaryllidoideae, Agapanthoideae, and Allioideae (Gökçe, 2020 personel communication).

2.3 Ecological Requiments of Onion

Onion is a cool climate vegetable relatively resistant to frost, but sensitive to prolonged freezing temperatures. It is grown between all areas in the Southern Hemisphere of Argentina and New Zealand until the 50th parallel, and in the Northern Hemisphere of Canada and Poland up to the 65th parallel. Although onion varieties can adapt to different climates around the world, they grow better in their own climate requirements. Onions adapt to different climatic requirements are referred to as short day for those which needs

5 a day length of less than 12 hours to bulb, intermediate for those requires a day length of 12-14 hours, and long days for those requires a day length over 14 hours (Gökçe and Tekeli, 2015). Short day and long day onions differ in bulb properties, usage, storage and growing seasons at lower or higher allitudes. In case of delay in the seed sowing time, as the plants will not fulfill their day light requirements, they will be harvested without completing vegetative development and the bulbs harvested remain small with low commercial quality.

In Turkey, short day onions are planted during fall season at Mediterranean coastline which is not exceeding cold in winter, intermediate day onions are planted during early spring months, where the winters are cold, and long day onions are planted in March and April. The temperature around 10 to 15 °C are required for good germination of onion seeds. During the development and growth, it is thought that cool weather between 10 and 25 °C rather than high temperature is required and 25 to 30 °C temperature is good for the bulbs to mature and the leaves to dry. Additionally, the high temperature cause sunburn to onion (Gökçe and Tekeli, 2015).

Onion is a biennial vegetable, which requires two years or two seasons to produce seed from seed. The first year is from seed to the bulb, and the second year is from bulb to the seed. In some regions, onion set is produced from the seed, the bulb is taken from the sets, thereafter seed is produced. Hence it requires three growing seasons from set to produce seed depending on the climatic condition (Gökçe and Tekeli, 2015). Although planting from seedling is not common for the commercial onion production, seedling production is done for the breeding lines with limited and valuable seeds (Gökçe et al., 2010; Gökçe et al., 2011).

2.4 General Properties of Onion

The Allium genus has different root system than other plant species as it is thin and white structured. Onion roots are usually singly developed from the bottom of the bulbs (Figure 2.1) and majority of them are found within 30 cm of depth and width (Brewster, 2008; Gökçe and Tekeli, 2015).

Figure 2.1. Root structure of Allium cepa L.

Onion leaves develop inner side of the earlier leaf reciprocally and by tearing the base of the old leaf. The white part of the leaf forms the pseudostem and has a pale structure. The fleshy portions on the leaf base elongate by storing the nutrients and form the bulb onion with interlockings. Height of onion leaves is approximately between 30 to 80 cm. Depending on climate and growing condition, an onion plant develops 8 or 14 leaves before bulb elongation arranged from outside to inside from old to young ones, respectively. Additionally, some onion types have a wax layer on their leaves based on their genotype.

Long day onions, which are also called the winter or storage onions, has a strong smell and pungent taste, and has longer storage periods compared to short day onions. Furthermore, they have higher numbers scales than short and intermediate day onion varieties. They also have higher dry matter content than that of the short day varieties. Contrarily, short day onions, called summer onions, have a mild sweetish flavor, thin scale and high water content with a short storage period (Gökçe et al., 2010; Gökçe et al, 2011).

The flower of onion plant consists of thousands of flowers on the receptacle which are covered by a thin membrane. Onion is a hermaphrodite plant since it has a perfect flower. The height of scapes can be 150 to 200 cm and it depends on the bulb size of onion as one to three scapes can develop from each growth point. The scape number of a bulb

7 onion depends on the center numbers of the bulb. A single onion flower has 6 petals, 6 sepals, 6 male organs and 1 female organ. Since male organs mature before the female organ (protandry), they encourage cross-pollination (Gökçe and Tekeli, 2015).

Onion requires cool season, proper light and well organic soil for good root development. The soil pH is recommended to be slightly acidic (6 to 7). The fertilization is required according to the nutrient status of the soil. During development of the green parts, onion needs moisture and plenty of water, however, when the harvest time approaches, water supply is reduced for bulb maturation (Gökçe and Tekeli, 2015). The harvest time is based on the size of bulb and position of the onion leaves. When the leaves of onion become dry and pseudostems fall sideways (Figure 2.2), they are ready to be harvested. The onion bulbs are generally spread in an open field for sun drying for a certain period of time (depending on the weather condition, until the onion roots becomes dry and brown). This process is called curing, in order both to get rid of various diseases and pests and to ensure full regeneration (Nabi et al., 2013). At the end of this process, the roots and dried leaves are removed from the onion and it became suitable for sale and storage. Storage condition is based on shelf-life of onion beside the variety characteristics. Onions that are considered for longer storage period with high dry matter content and a higher number of scales. They can be stored about 6 to 9 months at 1 to 2 °C with a relative humidity of 70 to 75 % (Gökçe et al., 2011; Mutlu, 2006).

Figure 2.2. Onion harvesting stage (bending of onion neck)

8 2.5 Economic Importance of Onion

The production and consumption of crops from Allium genus has spread to large areas around the world today. The economically important cultivated forms of Allium genus include onion, garlic, leek, shallot, Japanese bunching onion and chives (Brewster, 2008). Onion and garlic are the most important species of Allium genus. They are grown, traded, and consumed in many countries. Onion has a great value globally because of its multifarious uses in every society. It is an appetizing vegetable with many different uses; it is being consumed as food in Turkey and other countries since ancient times. It is consumed as fresh in meals and salads, frozen, onion fries, and its powder is used as spices in some country cuisines (Havey et al., 2004a; McCollum, 1966). Onion became the essential vegetable for great numbers of dishes around the globe. In this way, the demand of onion is increasing day by day. Onion also has an important role in the world economy. It was the third fundamental horticultural crop after potato and tomato in the year of 2019 (Anonymous, 2019a). Developing countries importing onion in the past started local production of onion. It was noticed that onion was the second in production after tomato with the area of 647,114 ha contrary to tomato 607,018 ha (Anonymous, 2019a). The Table 2.1 shows the worldwide onion production in the year of 2019 (Anonymous, 2019a).

Table 2.1 Leading onion producing countries in 2019 (Anonymous, 2019a)

Countries Production (T) Area (ha) Yield (T/ha) China 24,775,344 1,120,209 22,116 India 22,071,000 1,315,000 16,784 United States of America 3,284,420 52,690 62,334 Egypt 2,958,324 81,517 36,291 Iran 2,406,718 61,152 39,356 Pakistan 2,119,675 150,199 14,112 Turkey 1,930,695 59,198 32,614 Bangladesh 1,737,714 178,506 9,734 Russian Federation 1,642,106 60,499 27,142 Mexico 1,572,608 50,168 31,347 Other Countries 32,275,215 1,910,241 21,331 WORLD 96,773,819 5,039,908 19,201.5

9 The Table 2.2 shows the total onion production in the province of Turkey in 2019. Onion production is the highest in Ankara with a quantity of 510,414 tons, and followed by Amasya, Çorum, and Adana with the productions of 248,599, 166,460, and 153,307 tons, respectively (Anonymous, 2019a).

Table 2.2. Onion production area (ha) and quantity (ton) of leading provinces of Turkey in 2019 (Anonymous, 2019a)

Province Area (ha) Production (T) Ankara 97,085 510,414 Amasya 57,330 248,599 Çorum 46,499 166,460 Adana 34,863 153,307 Tokat 42,500 112,232 Bursa 28,631 110,148 Hatay 34,046 108,899 Eskişehir 26,540 106,040 Konya 22,907 74,006 Gaziantep 10,174 27,855 Other Province 126,558 312,735 Total 527,133 1,930,695

2.6 Importance of Onion for Human Health

Nowadays, lots of diseases have arisen and people are trying to find cure by using natural products. In developed countries, people are getting more concerned with the beneficial aspects of food (Sloan, 2000). Allium genus was used for medicine for about 4,000 years and onion and garlic have especially been important to medical area by reducing various health problems such as cardiovascular diseases and were recommended for cure of other diseases (Desjarding, 2008; Havey et al., 2001; Havey et al., 2004a; Havey et al., 2004b; Nicastro et al., 2015). The recommended amount for daily consumption of onion is approximately a half bulb onion or three green onions consumed as raw (about 4 to 7 µg / ml) for a healthy diet (Desjarding, 2008).

10 Onion is grown for its edible parts and is valued for its flavor and therapeutic properties; therefore, it is greatly used in every cuisine and almost every region (Benkeblia, 2005). Its usage is not only limited with the dishes as dehydrated onion powder and onion juice concentrate used as an essential oil, but also, like other Allium genus, onion has a medical importance as being rich source of phytochemical, organosulfur compounds, fructooligosaccharides (FOSs), and flavonoids (Böttcher et al., 2018). Genus Allium is one of the most researched among vegetables and has attracted great interest for the food industry (Yünlü and Kır, 2006).

Nile and Park (2013) reported that onion possesses 89.1 % water, 9.3 % carbohydrates, 1.1 % protein, 0.1 % fat, and also some vitamins and minerals. Carbohydrates such as sucrose, fructose and glucose create dry matter and they are quite important for health (Havey et al., 2004a; Havey et al., 2004b). Fructans in onion content is a substantial soluble dietary fiber source (Kleessen et al. 1997), and it has anticarcinogen effect especially with colorectal cancers (Roberfroid and Delzenne 1998).

Several studies showed that there were high phytocompounds in onion including phenolic acids, flavonoids (quercetin, kaempferol), anthocyanins, and organosulfur compounds. Different onion populations have different amount of phytochemical accumulation. In onion breeding programs, increasing concentrations of health-enhancing compounds for consumers is a desirable property. According to the medical feature of phenolic compounds pointed out in comparison to phytochemicals classes, flavonoid compounds protect plants against external factors like diseases and pests (Havey et al., 2001; Havey et al., 2004a; Teshika, 2019). Since quercetin accumulates in it (Patil et al 1995b; Lombard et al. 2002), onion has strong antioxidant property (Hertog and Hollman 1996). Anthocyanins in the subgroup of flavonoids are responsible of color pigments that give fruits and vegetables their natural color. Antioxidants have been reported to be used in the treatment of many diseases, especially cancer (Nizamlıoğlu and Nas, 2010).

2.7 Phenolic Compounds in Onion

Recently, one of the most frequently studied issues in both medical and scientific research is ways of maintaining a healthy life and preventing diseases. Phenolic compounds, being rich in many plant species, called biological active substances because of their effects on

11 human health, preventing occurrence of many disorders related to oxidative stress that is of great risk to human health like cancer (Baba and Malik 2015), cardiovascular diseases, nervous system disorders (Sas et al. 2007), and Alzheimer's and Parkinson's diseases (Singh and Jialal 2006). One of the most important property of these compounds is their antioxidant activity to retain free radicals from metabolic reactions. The antioxidants taken from vegetables and fruits form a protective shield against harmful substances that enter body (Yünlü and Kır, 2016).

The chemicals are divided into primary and secondary metabolites that are synthesized by plants. The primary metabolites are involved in basic compounds such as fatty acids, sugars, amino and nucleic acids. They are involved in various reactions such as glycolysis or biosynthesis in all cells and are necessary for plant growth and development (Wu and Chappell, 2008). On the other hand, secondary metabolites are structurally more abundant than the primary ones. They include terpenoids, phenolics and alkaloids (Winkel-Shirley, 2002). They are not directly essential for metabolism such as photosynthesis or protein synthesis, but they are necessary for plant's vital activity in the environment. In the biotic and abiotic stress conditions plant produce higher number of secondary products to fight against the negative effects of stresses (Bystrická, et al., 2013; Ornston, et. al., 1979). Secondary metabolites also work as signalling compounds to defend plants against herbivores and viruses. The other important role of secondary metabolites for plants is the protection from UV radiation and oxidants. They are different between populations, individuals and even varied at developmental stages (Wink, 2003).

In plants, phenolics are the most significant and enormous group of bioactive compounds in secondary metabolites (Kim et al., 2003). They have many functions in physiology and cellular metabolism like pollination, and sensory properties (taste, color, aroma). They also protect plants from pest and disease infestation, UV light and oxidative stress (Cheynier, 2012; Winkel-Shirley, 2002). They are quite diverse in their structure involving simple molecules such as gallic acid, and vanilin, as well as polyphenols such as flavonoids, stilbenes, and polymers derived from these groups (Quideau et al., 2011). Phenolics are divided into several groups according to ring number and binding with other elements (Wink, 2003). There are kinds of phenolic compounds that are typical of certain plant families or can only be found in certain plant organs or even only at a certain plant development stage. For example, anthocyanins are pigments that are related with purple,

12 red and blue plant organs, whereas they are also found in flowers and play role in attraction processes for pollination and in seed dissemination (Cheynier, 2012).

Several studies indicated that as compared to other vegetables, onion contains much higher phenolic contents (Velioglu et al., 1998; Vinson et al., 1998) in addition to total flavonoids, antioxidant activity, vitamin C contents and anthocyanins (Majid and Nanda, 2018). Bajaj et al. (1980) indicated that the red variety of onion has low phenolic compound (1.75 mg) than white variety (2.95 mg) in dry matter. Albishi et al. (2013) indicated that the red onion has a change in the phenolic compound due to sprouting, and the inner layer of red onion bulb is 1.4 times lower than that of outer layer of rings, Prakash et al. (2007) also reported similar results. A higher value of total phenol content of onion was observed by Chu et al (2002). They studied total phenolic concent in 10 different vegetables, and as a result, the highest 3 values for phenolic contents were 80.76 mg gallic acid equilavelent (GAE) / 100 mL in broccoli and 79.55 mg GAE / 100 mL of of spinach, followed by 68.90 mg GAE / 100 mL in yellow onion. The lowest one was with 14.95 mg GAE / 100 mL in celery.

2.7.1 Flavonoid and quercetin compound in onion

Flavonoids are one of the biggest groups of phenolic compunds that are naturally produced. There are more than 9,000 flavonoids identified. They are usually found in plants seeds, leaves, fruits and flowers as glycoside forms, whereas aglycon (without sugar part) form is less common (Crozier et al., 1997). Figure 2.3 shows basic skeletal structure of flavonoid compounds (Wang et al., 2018). They are found inside the cells or in great number of plant tissues and on the surface of various plant organs. They protect plants against biotic stresses (disease-pest). Flavonoids have been known as plant pigments for centuries. The polyphenols have been primarily dealt due to their roles in physiology and their effects on color and flavor characteristics of plants in recent years. Furthermore, they have a prominent effect on health due to their antioxidative, antibacterial, and anticarcinogenic properties (Güven et al., 2010; Ross and Kasum, 2002). Flavonoids capture hydroxyl radicals, superoxide anions and lipid peroxy radicals, therefore, they are good antioxidants (Wang et al., 2018). They have important place in human diet as consumption of polyphenol-rich foods reduce risk of diseases caused by reactive oxygen species (ROS). Antioxidants decrease the risk of cancer and

13 cardiovascular diseases by scavenging free radicals (Prakash et al., 2007). According to Robards et al. (1997), it is estimated that approximately 2 % of all carbon photosynthesized by plants are turned into ﬂavonoids or closely associated compounds.

Figure 2.3. Basic skeletal structure of flavonoid compounds (Wang et al., 2018)

Flavonoids consist of six groups that are flavones, flavanols (cathechins and proanthocyanidins), flavanones, flavonols, isoflavonoids (isoflavones), and anthocyanidins in the Figure 2.4 (Balasundram et al., 2006).

Figure 2.4. Essential subgroup of flavonoids chemical structure (Balasundram et al., 2006)

14 In the flavonoid group, flavonols are one of the most widespread and essential ones. Flavonoids consist of kaempferol, myricetin, quercetin and methylated derivative isorhamnetin (Wang et al., 2018) and they are essential pigments in onion scales. Flavonols are present in plant tissues as a glycosylated conjugate form like quercetin-3- glucoside, quercetin-3,4′-diglucoside (Spencer and Crozier, 2012). Figure 2.5 shows the structure of flavonoids found in onion (Lee, et al., 2008).

Figure 2.5. Quercetins structure found in onion (Lee, et al., 2008)

Quercetin is an important flavonol because of its role in decreasing risk of cardiovascular diseases. It is known as an anti-cancer and antioxidant agent, additionally, it reduces the rate of DNA degradation (Smith et al., 2003). Quercetin is found with varying concentration of 15 to 30 mg / kg fresh weight (FW) in lots of fruits and vegetables (Macheix J et al., 1990). The richest sources of quercetin are onion, curly kale, leeks, broccoli, and blueberries, but onion has the highest value up to 1.2 g / kg FW (Manach et al., 2004). Hollman and Katan (1999) reported that onion holds significant position in Dutch diet because it provides approximately 29 % of the flavonoids (Lombard et al., 2005). Hertog et al. (1993) reported that taking enough quercetin corresponds to approximately 16 mg per day. The most important sources of flavonoids are tea (48 % of total intake), onions (29 %), and apples (7 %). According to Hertog et al. (1992), onion has the highest content of quercetin among the 28 vegetables and 9 fruits tested. The

15 comprehensive study of Justesen et al. (1998) indicated that most vegetables have quercetin content almost less than 10 mg / kg FW; however, several vegetables surprisingly have great quercetin amount where red onion (45 mg / g FW) is at top among them, followed by yellow onion (34 mg), spring onion (18 mg), kale (12 mg), cowberry (21 mg) and cranberry (16 mg) per g FW. Among more than 25 various flavonols group in onion, quercetin and derivatives of quercetins are the most dominant pigments. Several researchers concluded that quercetin quantity of bulb onions is higher in the outer scales and rings (Patil and Pike, 1995a; Xihuan, 2011) and varies with color, type and cultivars of onion (Lombard et al., 2005; Patil and Pike, 1995a). According to Roldan-Marin et al. (2009), quercetin aglycone and 2 glycoide (quercetin-4'-O-glucoside and quercetin-3,4'- O-diglucoside) are essential onion flavonols among all forms of the diglucoside and monoglucoside conjugates.

In onion cultivars, quercetin amount varies depending on the genetic and environmental factors. Patil and Pike (1995a) conducted a study to observe quercetin amount of 75 different cultivars of onions, which included red, yellow, pink, and white color onions and reported that yellow onion has the highest quercetin amount and white onion has the lowest. Additionally, they observed that the dry scale of onion has a higher quercetin amount than the edible part of onion.

Hertog et al. (1992) studied quercetin levels of 9 fruits and 28 vegetables. It was reported that quercetin contents in the edible parts of many vegetables per kg were usually lower except for onions (284 to 486 mg), kale (110 mg), broccoli (30 mg), French beans (32 to 45 mg), and slicing beans (28 to 30 mg). Later, Hollman and Arts (2000) reported that among other species of vegetables and fruits, onion per kg has 5 to 10 times higher contents of quercetin (300 mg) than broccoli (100 mg), apples (50 mg), and blueberries (40 mg).

Lachman et al. (2003) reported that red color onion cultivars had the highest value of total flavonoid than white onion cultivars; however, red onion had lowest level of quercetin. Total quercetin content of onion on average was 5 to 10 times higher than that of other vegetables. Patil et al. (1995b) reported that the growing area of onion has an effect on the nutritional benefits as well as quercetin content. Slimestad et al. (2007) also indicated that the flavonoid content observed in red onion variety was higher (415 to 1917 mg /kg

16 FW) as compared to yellow onion variety (270 to 1187 mg / kg FW). In addition, Elhassaneen and Sanad (2009) reported that white cultivars have lower flavonols than red cultivars. Herrmann (1988) reported that yellow onion had more flavonols than red one, while 21 flavonols including kaempherol and quercetin derivatives observed among 18 leeks were less in concentration than edible onion species.

Juaniz et al. (2016) indicated a relationship between antioxidative and antiradical activities and phenolic compounds, and red onion indicated the highest phenolics and flavonoids with highest antioxidant activities than other cultivars. Furthermore, the antioxidant activity showed differences between the layers, where the onion antioxidant contents increased considerably from inner layer to outer layer (Kaur et al., 2009). Beesk et al. (2010) showed that the middle part of onion has highest flavonoid amount than both inner and outer part of onion.

2.7.2 Anthocyanins in onion

Anthocyanins are included in the flavonoid class of phenolic compounds. They are known as water-soluble pigments that give natural color to fruits and vegetables, especially red, violet and blue color. It is important to note that red colors were determined by carotenoid pigments instead of anthocyanin in some fruits. Anthocyanins are present in all parts of the plant, including root, stem, leaf, flower, and fruit. The anthocyanidin aglycones are the glycosides that are released by the hydrolysis (Castaneda-Ovando et al., 2009). Among the six anthocyanidins, cyanidin is the most common as compared to the others including delphinidin, peonidin, pelargonidin, petunidin, and malvidin. The anthocyanins have not been used for the food sector because of the instability against the several factor such as pH, and light, although they are used for the color processing of the jams and juices (Cools et al., 2010). In acidic condition, anthocyanins appear as red but turn blue when the pH increases. The synthesis of anthocyanins in fruit is affected by environmental factors such as light, temperature, nutritional status, disease, and drought. They also function in increased resistance to environmental stress factors (Tetiktabanlar, 2011). In addition, anthocyanins have been reported to be four times more effective than ascorbic acid and α-tocopherol in scavenging ROS. They reduce oxidative damage by chelating iron and copper, reducing the light intensity falling on the chloroplast and scavenging of the ROS species (Gould and Lister, 2006). They have a strong antioxidant

17 effect; therefore, they are used for the treatment of some diseases, especially cancer (Nizamlıoğlu and Nas, 2010), as well as against the infectious diseases. They have also been reported to be effective in cardiovascular disorders (Vazquez-Prieto et al., 2010), heart spasm, vascular occlusions, artery stiffness (Ali et al., 2000), and clot formation (Gonzalez et al., 2009). Hosseini et al. (2017) reported that oxidative stress positively influences fat accumulation and increased body weight. Therefore, less antioxidant levels lead to development of obesity comorbidities.

There are not many studies focusing on the anthocyanin content of onion. Cools et al. (2010) conducted the research to observe effects of different temperatures on an onion variety and they reported that the anthocyanin amount decreased by curing at 28°C than at 20°C. Zhang et al. (2016) compared the white, yellow, and red onions for their anthocyanin amount and reported that red onion cultivars had higher total anthocyanin, flavonoid, and polyphenol content and antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric reducing ability of plasma (FRAP) methods.

2.8 Pungency and Flavor of Onion Bulbs

Onions are consumed quite a lot, and not only for human diet. They are also used to increase flavor of other foods (Dhumal, et al., 2007). Yoo and Pike (2001) observed that the concentration of pyruvic acid is the determinant for the pungency of the flavor of onion bulb which is of great interest for growers and consumers regarding onion and garlic.

Organosulfur compounds are among the important bioactive substances of Allium species. Onions are rich in alkyl cystine sulphoxide (ACSOs) which is related with the pungency and smell of onion (Griffiths et al., 2002). Mechanically damaged onion, during macerating or cutting, produce pungent flavor of onion, which is caused by breakdown of the enzyme alliinase driving with flavor precursors, such as S-alk(en)yl-L-cysteine sulphoxides. Moreover, the hydrolysis reaction that causes enzymatic breakdown which is catalyzed by the S-alk(en)yl-L-cysteine sulphoxides produces stoichiometric amounts of ammonia and pyruvic acid within 6 min (Schwimmer and Weston, 1961).

18 The pyruvic acid production is a scale to alliinase action for the flavor processors and it is indicated to be correlated with the perceived onion pungency. Consumption of highly pungent onion cultivar cause set down of total blood cholesterol, low density lipoprotein, and triglycerides than the milder pungent cultivar (Anthon and Barrett, 2003). There are some specific compounds such as sugars and organic acids that support classification of flavor and aroma of onion. The soluble carbohydrates including sugars support onion flavor such as sweetness. These properties are important for onion breeding (Dhumal et al., 2007).

Pungency level and total soluble solids are observed to be significant properties for onion. Hamilton et al. (1996) indicated that the short day onion pungency was affected by location, year, and cultivar and it was also observed that the less pungency as a 3.9 µM in year 1993 increased one fold as a 7.3 µM in the year 1994. Location influence on the pungency level was also noticed but with a smaller difference of 0.5 µM.

Short day onions have mild pungency flavor (Yoo et al., 2019). Yoo et al. (2006) conducted a study on the pungency of onion and agreed on the effect of genetic and environmental factors on pyruvic acid content. Furthermore, it was observed that the onion bulb size was inversely proportional to the pungency level. Other observations showed no relation with the weight and soluble solid content among the 4 clones grown at Weslaco (Yoo et al., 2006). In contrast, Rodriguez et al. (2009) indicated that there was correlation between Brix, sugar content and weight of onion bulb.

Sulfur nutrition is directly related with pungent flavor of long day onion. Sweet onion gets pungent when grown in soil with higher levels of sulfur (Randle, 1992). In crop rotation after legume harvesting, onion showed high pungency level due to the nitrogen and sulfur fertilization. However, the soluble solid content did not change (Resemann et al., 2004).

Manjunathagowda et al. (2019) conducted a study to observe the performance of the 30 different short day onion cultivars for nutritional quality. The result showed that the highest pyruvic acid amount was 2.31 μM / g.

19 The short day onion pungency increases comparatively during bulb maturation beside dry matter and sugar content. It is considered that there is an association between sweetness and pungency. Therefore, long harvesting time could be problem for sweet onion production (Hamilton et al., 1997).

2.9 Antioxidant Quantity in Onion

Onion has high levels of antioxidant contents. Antioxidant activity is known as one of the essential medical property (Gökçe et al., 2010). Studies have shown that antioxidant content of onion was affected by genotype and phenotype on the qualitative and quantitative aspects. Antioxidant content varies due to the differences in chemical compounds showing enormous variations in A. cepa L. (Mogren et al., 2007; Patil et al., 1995b; Yang et al., 2004). Yang et al. (2004) reported that the phenolics and flavonoids had important role in antioxidant capacity among 10 different onion varieties with a comparative analysis.

A study was conducted to estimate the total phenolic and total antioxidant contents of a wide range of onion cultivars by Gökçe et al. (2010). “Ferric reducing ability of plasma” (FRAP) and “Trolox equivalent antioxidant capacity” (TEAC) methods were used to analyze total antioxidant capacities. As a result of this study, in both methods, the red onion had higher antioxidant activity than yellow and white onions, whereas yellow onion had the highest phenolic compound amount. Furthermore, they indicated that the FRAP, and TEAC had a positive relation with soluble solids.

Sidhu et al. (2019) observed that environmental conditions were effective on the phenolic content during growth and, as a result, the antioxidant capacity and total phenolic compounds were decreased from outer part of onion to inner part of onion. Benkeblia (2005) reported that there were higher antioxidant and phenolic contents in red and purple cultivars as compared to green, yellow, red, and purple onion.

2.10 Sugar Content and Total Soluble Solid in Onion Bulbs

The physical appearances of vegetables and fruits are the most important criteria such as color, size and shape (Opara and Pathare, 2014). With the increase of living standards,

20 the emphasis is excessively given to the quality of vegetables and fruits such as flavor that is associated with total soluble solid content (TSS) (mainly sugars, followed by pectin, organic acids and amino acids), titratable acidity (TA), total soluble solids / titratable acidity (TSS / TA) ratio and texture (Chen and Opara, 2013a; Chen and Opara, 2013b). According to the consumer, desire for sweetness is the unchanging property of vegetables and fruits. Therefore, sugar and sweetness are important for plant research area (Hong et al., 2014).

Onion bulbs can accumulate significant amounts of nonstructural carbohydrates (Jaime et al., 2001), as well as, small amounts of dissolved vitamins, fructans, proteins, pigments, phenolics, and minerals, which are referred to as TSS (Chope et al., 2006; Kader, 2008a; Kader, 2008b). TSS are the quality criteria for indicating sweetness of fresh and processed horticultural food products. High TSS is required for dehydration industry in the production of flakes and powders for spices and are related with bulb characteristics.

The research on TSS shows that dry matter content of solutions fundamentally contains sucrose. TSS and its component carbohydrates are measured by Brix scale hydrometer, a refractometer, high-performance liquid chromatography (HPLC), and enzymatic assays (Ma et al., 2014; Revanna et al., 2013). The “degrees Brix” (°Brix) is unit of TSS and it is equivalent to percentage (%) (Dongare et al., 2014).

Sugar content is related with the quality of product; however, high sugar content does not mean higher sweetness. There are other substances that affect flavor and taste. °Brix gives objective results in determination of flavor and sweetness (Table 2.3).

It can help for variety selection, harvest scheduling, and other aspects of crop production including irrigation, fertilization, and post-harvest management (Terry et al., 2005). However, this correlation is not clear since other substances also affect TSS, except sugar (Chope et al., 2006; Crowther et al., 2005). Kader et al. (2003) reported that TSS was an indicator of sugar in blueberries and strawberries; however, there was no clear correlation observed between them. Their hypothesis was high content of anthocyanins and phenolic compounds are responsible to increase TSS by contributing up to 32 % measured by refract light of refractometer.

21 Table 2.3. °Brix scale of some vegetables and fruits (Anonymous 2020)

Poor Average Good Excellent Onion 4 6 8 10 Carrots 4 6 12 18 Broccoli 6 8 10 12 Celery 4 6 10 12 Sweet Potatoes 6 8 10 14 Tomatoes 4 6 8 12 Squash 4 8 12 14 Strawberries 6 10 14 16 Raisins 60 70 75 80 Raspberries 6 8 12 14

Sugar content is related with the sweetness of onion bulb and the sugar is affected by the pungency of onion flavor (Lee et al., 2009b). However, Terry et al. (2005) observed that high dry matter content of onion had more accumulation of fructans; however, low dry matter content of onion had the glucose and fructose. Moreover, during bulb formation, they could not find any certain relationship between dry matter concent and sugars. Crowther et al. (2005) reported that TSS is not an indicator of sugars, since TSS value includes not only sugars, but also salts, organic acids, and proteins. Contrarily, Chope et al. (2007) reported that onion, had higher sugar content, that showed 2-fold higher TSS value. That is there is not enough data in onions showing direct relationship between °Brix and TSS values. Manjunathagowda et al. (2019) studied performance of 30 different short day onion cultivars for their nutritional quality. The results showed that reducing sugars, non-reducing sugars, and total sugars amounts were 3.83 %, 3.52 %, and 6.45 %, respectively.

22 CHAPTER III

MATERIALS AND METHODS

3.1 Materials

Nineteen short day onion elite lines (Table 3.1) were used from GAF-MTN gene pool that was developed by Dr. Ali Fuat GÖKÇE during his long-term onion breeding program started at the University of Wisconsin-Madison campus between the years 1998-2002, continued at Uludağ University Agricultural Faculty between the years 2002-2006, and thereafter at MTN Seed Co. Ltd. in Çepni village of Bandırma district of Balıkesir province.

Table 3.1. Short day onion elite lines used in the thesis study

Genotype Number Source Color K18 GAF-MTN Yellow K19 GAF-MTN Yellow K20 GAF-MTN Yellow K25 GAF-MTN Yellow K26 GAF-MTN Yellow K28 GAF-MTN Yellow K35 MTN Yellow K37 MTN Yellow K38 MTN Yellow K39 MTN White K40 MTN Yellow K41 MTN Yellow K42 MTN Yellow K51 Commercial Domestic Yellow K52 CommercialDomestic Yellow K53 Commercial Domestic Yellow K58 Commercial Import Yellow K59 Commercial Import Yellow K60 Commercial Import Yellow

23 3.2 Methods

The experiment was carried out at the experimental fields of Alata Horticultural Research Institute of Alata, Erdemli district of Mersin province. Short day onion breeding lines were grown in Alata climate conditions between October to June, 2019. The onions were harvested when pseudostems fall down, left for a week to dry, pseudostems were cut of and the bulbs were cured under shelter for 10 days and moved to the laboratory for analyses.

Pomological and phytochemical analyses were performed using 10 representative onion bulbs as three replicates for each variable without any bulb storage (right after harvest) (Figure 3.1). Bulb scales were removed, and onions were chopped roundly. Onion pieces were blended by a standard food blender for 2 to 3 min. The onion puree was filtered through Whatman filter paper (No. 2), and the juice was stored at -18 °C until phytochemical analyses. For pomological analyses fresh onion juice obtained after filtering was used.

Figure 3.1. Onion bulbs used for pomological and phytochemical analyses

24 3.2.1 Pomological analyses

3.2.1.1 Color measurement

The scale color of onion bulb was determined as L*, a* and b* values using a Minolta portable chromameter (Konica – Minolta, CR- 400, Japan) from 3 to 5 spots of bulb scales as defined by CIE (Commission Internationale de I’Eclairage) (Figure 3.2). The hue angle (ℎ°) and chroma (퐶*) values were calculated by following formulas (3.1, 3.2) using L*, a*, b* data after calibration with standard white plate.

퐶*= (3.1)

ℎ° = ( ) (3.2)

Figure 3.2. Scale color measurement by chromometre

L* (lightness) shows that how much light is reflected from the measurement surface; in other words, it indicates the change in the lightness and darkness of the color from black to white (0 = White; 100 = Black). A* indicates color changes from red (+60) to green (-

25 60), whereas b* indicates color changes from yellow (+60) to blue (-60). The hue angle indicates the quality of the color (00 = red pink, 900 = yellow, 1800 = green, 2700 = blue). Chroma value indicates the vitality of the color where a value of 0 indicates a gray- achromatic (colorless) color, but as the value increases the viability of the color increases (Lucas et al., 2018).

3.2.1.2 Total soluble solids content (%)

Total soluble solid contents of short day onion bulbs were determined by the method described by Randle and Bussard (1993). A few drops of fresh juice were placed on an Atago Pal-1 Digital Refractometer 0-53 Brix-Pal-1 and data was expressed as °Brix (Figure 3.3).

Figure 3.3. Measurement of total soluble solid contents of onion samples by refractometer

3.2.1.3 Amount of titratable acidity (%)

Titratable acidity was calculated as citric acid according to the method described by Cemeroglu (2010). For titratable acidity, 1 mL of fresh onion juice and 9 mL of distilled water were mixed in a glass beaker, and 3-5 drops of phenolphthalein was added. To ensure to make the mixture homogenized, the glass jars were gently shaken. After that,

26 titration was carried out by adding 0.1 N NaOH dropwise until the solution color turned into pink-purple color (Figure 3.4). The amount of 0.1 N NaOH added was noted when the color changed. Amount of titratable acidity was determined by the following formula (3.3) (Cemeroğlu, 2010).

(3.3)

Figure 3.4. Measurement of titratable acidity of onion samples

3.2.1.4 pH

The pH value was measured by a Thermo Scientific Orion Star A111 pH meter directly from fresh onion juice (Figure 3.5).

Figure 3.5. Measurement of pH value of onion samples

3.2.2 Phytochemical analyses

3.2.2.1 Determination of pyruvic acid

The pyruvic acid analysis was based on the method of Schwimmer and Weston (1961). 0.1 mL homogenized onion samples were taken and 3 mL of 0.0125 % of 2,4- dinitrophenylhydra-zinc (DNPH) in 2 N HCI were mixed in the tubes. The mixture was incubated on water bath at 40 °C for 10 min (Figure 3.6).

Figure 3.6. Incubation of sample mixtured for pyruvic acid analysis on water bath

28 After incubation, 8 mL of 0.6 N NaOH was added and 300 µL of final mixture was transferred into a microcuvette for spectrophotometric measurement by the Agilent Technologies Cary 60-UV-Vis Spectrophotometer at 420 nm wavelength. The absorbance of solution was blanked with 0.0125 % of 2,4-dinitrophenylhydra-zinc reagent (Figure 3.7). Pyruvic acid concentration was calculated using sodium pyruvate standard curve prepared from dilutions of 20 mmol / L pyruvic acid solution (0, 0.5, 1, 2, 4, 6, 8, 10 µmol / mL) treated with the same procedure given above.

Figure 3.7. Onion juice samples were prepared and measured by spectrophotometer

3.2.2.2 Determination antioxidant activity

The antioxidant activity of onion juice was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl-hydrate) free radical scavenging method as previously described by Klimczak et al. (2007). Five mL of onion juice sample was taken and centrifuged (Beckman Coulter Mikrojuge-16 microfuge machine) at 4 °C at 5000 rpm for 5 min. 0.1 mL extract was

29 taken and mixed with 3.9 mL DPPH solvent (2.36 mg / 100 mL methanol) (Figure 3.8). 0.1 mL of distilled water treated the same way was used as the reagant blank. 300 µL prepared sample was transferred to a microcuvette and analyzed by using Agilent Technologies Cary 60-UV-Vis Spectrophotometer at a wavelength of 515 nm for 30 min. The antioxidant activity was expressed as the percentage of decline of the absorbance after 1 min based on control (reagent blank) corresponding to the percentage of DPPH. The obtained absorbance values were calculated with Trolox (10 to 100 µM / L) standard slope table as presented by Trolox equivalent / g fresh fruit (Ozgen et al., 2006).

Figure 3.8. Onion juice samples of short day onion genotypes

3.2.2.3 Total anthocyanin determination

The total anthocyanin analysis was conducted by pH differential method provided by Giusti and Wrolstad (2001) with some modifications. The method is based on the anthocyanin structural transformation which occurs with a change in pH between 1.0 and 4.5. One mL of onion juice was taken and diluted with 24 mL pH 1.0 buffer (0.025 M potassium chloride; 1.86 g KCL in 1 L distilled water, pH adjusted with 1 N HCl). At the same time, 1 mL onion juice was diluted with 0.4 M sodium acetate 24 mL buffer pH 4.5

(32.81 g sodium acetate in 1 L distilled water, pH adjusted with 1 M H2SO4) (Figure 3.9). Samples were then vortexed for 20-30 sec. 300 µL sample was taken from each tube and transferred to a microcuvette to measure the absorbance against a blank (distilled water) at both 520 nm and 700 nm by Agilent Technologies Cary 60-UV-Vis Spectrophotometer. Total amount of anthocyanin results was expressed as µg of cyanidin-

30 3-glucoside equivalent in g fresh weight (mg / 100 mL) basis according to the following formula (3.4).

A x MW x Df x 100 Total monomeric anthocyanin amount = (3.4) ε x L

A (absorbance value): (A520nm-A700nm) pH 1.0- (A520nm-A700nm) pH 4.5 MW: Molecular weight (449.2 g / mol) Df: Dilution factor 휀: Molar absorption coefficient (26,900) L: Microcuvette thickness (1 cm)

Figure 3.9. Measurement of total anthocyanins

3.2.2.4 Determination of total phenolics

The analysis of total phenolics amount was based on the method of Singleton and Rossi (1965), Singleton et al. (1999) and Biçgel (2008). Two mL onion juice sample was taken and mixed with 8 mL solvent (80 % methanol). After this, the samples were centrifuged at 5000 rpm for 5 min, 50 µL supernatant was taken and mixed with 100 µL Folin-

31 Ciocalteu solvent. 1500 µL distilled water was added to the mixture and incubated for 10 min at room temperature (Figure 3.10). 50 µL of 20 % (w / v) sodium carbonate was added and incubated in dark for 2 hours. When phenolic substance was turned into blue color, 300 µL was loaded to a microcuvette by pipette. Total phenolic amount was measured at 765 nm by Agilent Technologies Cary 60-UV-Vis Spectrophotometer (Figure 3.11). The result was expressed as mg gallic acid equivalent (GAE) on 100 mL basis.

Figure 3.10. Preparation of samples for measurement of the total phenolics

Figure 3.11. Total phenolics analysis with spectrophotometer

32 3.2.2.5 Flavonoid analysis (Quercetin)

Flavonoid analysis was based on the method of Koh et al. (2009). Frozen onion samples were weighted to get 3 g and 9 mL of solvent (80 % MeOH with trifluoroacetic acid to pH 2.5) which was added. The samples were vortexed for 10 to 15 sec. After this, samples were sonicated for 30 min, and they were centrifuged at 12000 rpm for 90 sec by Beckman Coulter Mikrojuge-16 microfuge machine. Then supernatant was filtrated by 0.45 µM membrane filter and 10 µL was injected into vials by injector. The analysis was carried out at 30 °C by HPLC-PDA (Shimadzu-20AD, Japan) using ODS-3 column at a wavelength of 272 nm and flavonoid amount was determined according to standard flavonoids which were quercetin, kaempferol, luteolin, myricetin, isorhamnetin (Figure 3.12). The condition of usage HPLC and used mobile phases were as follows; The mobile phase comprised of 0.05 % trifluoroacetic acid (TFA) in water (A), 0.05 % TFA in methanol (B), and 0.05 % TFA in acetonitrile (ACN). The linear gradient was used as flow rate of 1.0 mL / min which were as given in the Table 3.2. Total analysis time was 71 min.

Table 3.2. Mobile phases and usage amount by time for HPLC

Time (min) Mobile Phase A (%) Mobil Phase B (%) Mobil Phase C (%) 0.01 90.0 6.0 4.0 5.00 85.0 9.0 6.0 30.00 71.0 17.4 11.6 60.00 0.0 85.0 15.0 71.00 90.0 6.0 4.0

33 mAU 272nm,4nm (1.00) 60

40 3/Luteolin/46.339/959684

2/Quercetin/45.508/660335 4/Kaempferol/48.431/738447

35 5/Isorhamnetin/48.940/685048 1/Myrecetin/41.581/539718 30

0 42.0 43.0 44.0 45.0 46.0 47.0 48.0 49.0 min

Figure 3.12. Flavonoid chromatogram, retention time of myriecetin, quercetin, luteolin, kaempferol, and isohamnetin were 41.581, 45.508, 46.339, 48.431, and 48.940, respectively

3.2.2.6 Sugar analysis (Glucose, Fructose, Sucrose)

The most important soluble carbohydrates in onion includes sucrose, fructose and glucose, and they were analyzed by HPLC based on the method explained by Bartolome et al. (1995).

One mL of the onion juice was taken and diluted with 1 mL ultra-distilled water in the 2 mL Eppendorf tubes. After that, samples were vortexed for 10-15 sec, and centrifuged at 14000 rpm for 90 sec by Beckman Coulter Mikrojuge-16 microfuge machine. Then, the supernatant was filtrated by 0.45 µM membrane filter and 20 µL was injected into vials by injector. The analysis was carried out at 40 °C by HPLC-RID (Shimadzu-20AD, Japan with refractive index detector) using NH2 (4.6X250 mm) column and total sugar amount was determined according to standard glucose, fructose and sucrose (Figure 3.13). Soluble sugars in the samples was calculated comparing peak area which was determined with respect to retention time of standard sugars graphs (Figures 3.14, 3.15, 3.16). Results of sugar contents were expressed as g / 100 g FW. The condition of usage of HPLC and used mobile phases are as follows. The mobile phase comprised of 80 % acetonitrile and 20 % ultra-distilled water. The linear gradient was used as flow rate of 1.2 mL / min (isocratic). Total analysis time was 18 min.

34 mV Detector A 500

450

400

350 fruktoz/8.398/7298055

300 sakkaroz/16.407/7680994 250

200 glikoz/10.470/4161016

150

100

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

Figure 3.13. Sugar chromatogram, retention time of fructose, glucose and sucrose were 8.398, 10.470 and 16.407, respectively

Conc. 5

0 0 1000000 2000000 3000000 4000000 5000000 6000000 Area

Figure 3.14. Calibration graph of fructose

35 Conc.

0 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 Area

Figure 3.15. Calibration graph of glucose

Conc.

0 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 Area

Figure 3.16. Calibration graph of sucrose

36 3.2.3 Statistical Analyses

In this study, data were analyzed by Tukey's Studentized Range via SAS software (SAS, 2005). GLM procedure was used for analysis of variances (ANOVA), and the means of samples were calculated by TABULATE procedure. Tukey’s Studentized Test is implemented for the pairwise comparisons of the genotype means for those variables if their genotypes p value is ≤ 0.05 after ANOVA. For a given variable, genotype means were grouped using Tukey Least Significance Difference (LSD) values calculated at p ≤ 0.05 to found if there were any significant differences among the genotype means of dependent variables (phenolic acid, flavonoid, anthocyanin, antioxidant, pyruvic acid, TA, pH, sugar, soluble solid content).

37 CHAPTER IV

RESULTS AND DISCUSSION

4.1 Results of Pomologic Analyses

The results of analysis of variance of 4 variables (color measuremt (L*, a*, b* values), total soluble solid acid (TSS), titratable acidity (TA), and pH) are given in the Table 4.1. The Table 4.1 shows that the genotype effects for the b* value of color measurement was not significant (p > 0.05), whereas for the other measurements were found to be significant (p ≤ 0.05).

Table 4.1. Analysis of variance table for color measurements (L*, a*, b* values), total soluble solid (TSS), titratable acidity (TA), and pH

Variables Source DF Sum of Squares Mean Square F Value Pr > F L* Genotypes 18 277.3378 15.4076 2.43 0.0107 Error 38 241.4012 6.3527 Corrected Total 56 518.7391 a* Genotypes 18 6.1388 0.3410 1.96 0.0406 Error 38 6.6247 0.1743 Corrected Total 56 12.7635 b* Genotypes 18 134.5450 7.4747 0.94 0.5391 Error 38 301.6368 7.9378 Corrected Total 56 436.1818 TSS Genotypes 18 43.3533 2.4085 201.89 <0.0001 Error 38 0.4533 0.0119 Corrected Total 56 43.8066 TA Genotypes 18 1.4647 0.0814 39.80 <0.0001 Error 38 0.0777 0.0020 Corrected Total 56 1.5424 pH Genotypes 18 0.3098 0.0172 118.21 <0.0001 Error 38 0.0055 0.0001 Corrected Total 56 0.3154

38 4.1.1 Color measurement

The Table 4.2 shows that there were significant differences for the L* variable at p ≤ 0.05 level among the means of short day onion genotypes used in the study. The a* value results showed significant differences, while the b* value results did not reveal any significant differences for all the genotypes tested at p ≤ 0.05 level. The L* values were between 49.6 to 58.5, and K60 was the only breeding line in the lowest group of 19 different short day onion genotypes tested. The a* values were between 0.143 to 1.653 and the genotype K40 was in the lowest group among 19 different short day onion genotypes (Table 4.2).

Table 4.2. Mean separations of scale color, L*, a*, and b* values of the genotypes

Genotypes L* a* b* K18 56.8 ab** -1.140 ab** 13.08 K19 54.6 ab -1.040 ab 15.30 K20 53.7 ab -0.587 ab 12.58 K25 56.3 ab -0.143 a 13.16 K26 55.6 ab -0.997 a b 14.44 K28 56.3 ab -1.073 ab 13.24 K35 58.5 a -0.677 ab 12.92 K37 56.9 ab -0.617 ab 15.95 K38 55.7 ab -0.873 ab 13.04 K39 51.6 ab -0.443 ab 10.36 K40 56.6 ab -1.653 b 13.40 K41 56.1 ab -1.197 ab 13.44 K42 57.4 a -1.200 ab 16.60 K51 55.0 ab -1.213 ab 13.06 K52 51.4 ab -0.997 ab 11.83 K53 56.9 ab -0.790 ab 11.85 K58 55.6 ab -0.967 ab 12.25 K59 54.5 ab -0.823 ab 12.61 K60 49.6 b -0.690 ab 10.71 LSD at 0.05 7.76 1.285 8.67 *Chromameter describes color in three coordinates: L, lightness, from 0 (black) to 100 (white); a, from -60 (green) to 60 (red); and b, from -60 (blue) to 60 (yellow) **Within columns, means followed by the same letter are not significantly different (LSD at 0.05)

39 In the Table 4.2 showed that the commercial short day onion genotypes (K51, K52, K53, K58, K59 and K60) had similar values with the rest of the short day onion genotypes for L* and a* value.

Color is an important quality trait in onion cultivars. In this study, color composition was measured according to the values of L* (lightness and darkness of color), a* (red to green color change), and b* (yellow to blue color change) of short day onion breeding lines. Among the 19 short day onion genotypes used in the study, genotype number K39 is the white scale color and rest of them are yellow (Table 3.1).

Gökçe et al. (2010) studied the color value of the different colored onions and they observed that the highest value of 69.0 from the yellow color onion cultivar for L*, then measured 67.2 from the white color onion cultivar and 35.3 from the red color onion cultivar. For the a* value, the red onion cultivar had the highest value of 33.5, the yellow onion cultivar had -1.4 and the white onion cultivar had the lowest value of -2.6. For the b* value, the red onion cultivar had the lowest value (-2.7) and the white onion cultivar was 4.4, where the highest value was observed as 7.5 from the yellow onion cultivar. The data observed in thesis study were the similar result with Gökçe et al. (2010). The yellow color onion genotypes were higher than the white one with respect to the L* values. All the onion breeding lines used in this study was among the short day groups, thereby, the reason for not having any significant difference might be related with the fact that short day onion scales have few layers than long day genotypes.

4.1.2 Total soluble solid content (TSS)

In this study, TSS showed significant (p ≤ 0.05) differences among the short day onion genotype means (Table 4.3), and the mean values were between 5.2 to 8.50 °Brix. The genotype K38 was in the highest group and the genotypes K37, K58, and K59 were in the lowest group among the 19 different short day onion genotypes. In the Table 4.3 showed that the commercial short day onion genotypes (K51, K52, K53, K58, K59 and K60) were less value than the rest of the short day onion genotypes for TSS value.

There are some important quality charactertics of onion bulbs and TSS is one of them (Resemann et al., 2004). The soluble solid content is known as indicator of sweetness in

40 onion (Enciso et al., 2009). The observed results are in agreement with other studies in the literature. For example, Rodriguez Galdon et al. (2009) reported Brix degree between 4 and 8 °Brix. Similar results were found by Zambrano et al. (1994), where they reported TSS value of onion bulbs between 5.39 and 5.84 °Brix. Roldan-Marin et al. (2009) observed TSS as 6 °Brix in his study. Caruso et al. (2014) observed 7.7 °Brix in short day onions that were planted on 1st of February which was later increased to 8.1 °Brix on 18th of March in the same planted onion.

Table 4.3. Mean separations of total soluble solid content (TSS), titratable acidity (TA, g / mL), and pH values of the genotypes

Genotypes TSS TA pH K18 6.60 e* 1.256 cd* 5.88 ef* K19 7.16 d 1.418 ab 5.90 de K20 8.10 b 1.134 def 5.87 efg K25 5.70 ijk 0.891 h 5.92 d K26 5.73 hij 1.134 def 5.83 hijk K28 7.66 c 1.053 efg 5.80 jkl K35 6.20 fg 1.067 efg 5.80 klm K37 5.50 jkl 1.013 fgh 5.96 b K38 8.50 a 1.539 a 5.92 bcd K39 6.00 ghi 1.310 bc 5.78 lm K40 6.26 efg 0.972 gh 5.85 fghi K41 6.13 fg 1.282 bc 5.76 m K42 6.40 ef 0.972 gh 5.84 ghij K51 6.30 efg 1.053 efg 5.87 efgh K52 5.96 ghi 1.053 efg 5.82 ijk K53 6.06 fgh 1.121 def 5.95 bc K58 5.36 kl 1.040 efg 6.00 a K59 5.20 l 1.175 cde 5.84 fghij K60 6.10 fg 1.013 fgh 5.69 n LSD at 0.05 0.34 0.139 0.04 *Within columns, means followed by the same letter are not significantly different (LSD at 0.05)

41 In contrast to the previously summarized studies, there are some reports with higher TSS values in onion. For example, Manjunathagowda et al. (2019) conducted a study on the performance of the short day onions where they observed higher TSS values for short day onion cultivars with a °Brix value between 13.5 and 15. Comparing to the other studies, Pöhnl et al. (2017) observed that TSS value was the highest with 18.8 °Brix and the lowest with 9.7 °Brix among the 20 different onion cultivars they studied. Vagen and Slimestad (2008) observed TSS value between 6.8 to 12.3 °Brix among the 15 different varieties of onion. Sekara et al. (2017) reported TSS contents between 6.2 and 13.3 °Brix and a similar range was also observed by Lai et al. (1994). They reported that TSS value of the short day onion bulb was between 5.6 and 10.2 °Brix.

Yoo et al. (2006) observed TSS values between 5.4 and 7.5 °Brix in onion genotypes they studied. Additionally, they reported that TSS had no specific relationship between the pyruvic acid and bulb weight, so although TSS is the sum of organic content, the pungency of onion is not related with TSS. Another study supporting our results reported by Lee et al. (2009b). They observed that TSS value in short day onion cultivars was between 6.2 and 9 °Brix, in addition, there was no exact relationship with pungency level and TSS. Resemann et al. (2004) observed TSS value between 6.5 °Brix and 17 °Brix among the 16 onion cultivars. Besides this, they also reported that there was a relationship between pungency and TSS content. Similar result was reported by Lee and Suh (2009a). They observed positive association between pungency level and TSS. Another supportive study was conducted by Randle and Lancaster (2002). They reported a positive association between pungency and total soluble solids due to the effects on matter related to soluble solids.

In this study, genotype number K38 was in the highest group in both analysis, pyruvic acid and TSS (Tables 4.3 and 4.5). Genotype number K58 and K59 were in the lowest group in both analysis, pyruvic acid and TSS (Tables 4.3 and 4.5).

4.1.3 Titratable acidity (TA)

Table 4.3 showed that TA was significantly different (p ≤ 0.05) among the short day onion genotypes. The mean separation values and pairwise comparison of 19 short day onion genotypes for TA is given in the Table 4.3. The mean TA values were in a short range

42 between 0.972 and 1.539 g / mL. The genotypes K19 and K38 were ranked in the highest group and the genotypes K37, K40, K42, and K60 were in the lowest group among the 19 short day onion genotypes (Table 4.3). In the Table 4.3 showed that the commercial short day onion genotypes (K51, K52, K53, K58, K59 and K60) had similar results than the rest of the short day onion genotypes for TA value. Some of the short day onion genptype had higher value than the commercial short day onion genotype such as K18, K19, K38, K39, and K41, however, some of them had lower value than commercial short day onion genotypes such as K25, K40, and K42.

Roldan-Marin et al. (2009) observed around 2.00 g / mL TA values of the onion juice in his study. Although this study was in agreement with the observed data, there was not a certain comparison because of the lack of information of onion type used by Roldan- Martin et al. (2009). Forney et al. (2010) observed the sulfur effect on TA of yellow onion bulb. They explained that with the application of 30 and 50 kg / ha sulfur, respectively, it increased from 0.121 to 0.124 and 0.128 g / mL. The initial value of 0.121 g / mL reported by Forney et al. (2010) was similar result to the results obtained in this thesis study; however, similarly, the onion type was also missing for a reliable comparison. Caruso et al. (2014) in their study found that four transplanting times (1st February, 16st February, 3rd March, 18th March) affected TA of short day onions. It was reported that TA increased from 1.97 to 2.23 g equivalent of citric acid per 100 g FW in the period of 1st of February to 18th of March, respectively. Their reported initial result (1.97 g / mL) was similar to values observed in this study of 19 short day onion genotypes with a transplanting time of October.

4.1.4 pH

Results of pairwise pH mean comparison among the 19 different short day onions are shown in the Table 4.3, and a significant (p ≤ 0.05) difference was observed among the short day onion genotypes. The mean pH values were in the short range with 5.69 and 6.0. The genotype K58 was in the highest group, whereas the genotype K60 was in the lowest group among the 19 short day onion genotypes. Except the genotype K39 (white color), rest of the genotypes were similar (yellow color), therefore the similar pH value was an expected result.

43 Observed data showed that there was no similarity between TA and pH value. TA is known as total acidity, whereas pH is effective acidity related to the concentration of dissociated hydrogen ions. Genotype K38 was in the highest group for TA, whereas, the genotype K58 was in the highest group for pH. Genotypes K25, K37, K40, K42 and K60 were in the lowest group for TA, whereas, genotype K60 was in the lowest group for pH. Therefore, there was no relationship between TA and pH values evaluated in 19 short day onion genotypes (Table 4.3).

The highest pH value of short day onion genotypes was the in commercial group (K60), however, in the Table 4.3 showed that the commercial short day onion genotypes (K51, K52, K53, K58, K59 and K60) had similar values with rest of the short day onion genotypes for pH values. pH value is an important criterion for food sector such as storage, shelf-life, and any treatment (like heat treatment) for making souce, marmelate, like onion powder. Besides, ratio between pH and sugar is other important matter to consumption. Although, there was no specific information of the onion type (long or short day), similar result was observed by Roldan-Marin et al. (2009) as a pH value of 5.0 right after harvest. Berno et al. (2014) reported that pH value was affected by the storage temperature. The pH value was decreased from 5.5 to 4.0 at the end of the shelf-life at 15 °C. Although it is not possible to make comparison according to storage temperature because in this thesis variables were evaluated directly after harvest, the initial pH value (5.5) was similar among the 19 short day onion genotypes. Rodriguez Galdon et al. (2009) reported the pH values were between 5.14 to 5.84, but did not provided onion type. On the other hand, Roberts and Kidd (2005) observed pH values between 3.25 and 3.35.

4.2 Phytochemical Results

The results of analysis of variance of 6 variables (pyruvic acid, antioxidant, anthocyanin, total phenolic content (TPC), fructose, and quercetin) are given in the Table 4.4. Table 4.4 shows that the genotype effects for the glucose and sucrose analyses were not significant with a p-value > 0.05, whereas the genotype effects for the variables were found to be significant at p ≤ 0.05 level.

44 Table 4.4. Analysis of variance table for pyruvic acid, antioxidant, anthocyanin, total phenolic content (TPC), fructose, and quercetin

Variables Source DF Sum of Mean F Value Pr > F Squares Square Pyruvic Genotypes 18 102.7172 5.7065 303.14 <0.0001 Acid Error 38 0.7153 0.0188 Corrected 56 103.4326 Total Antioxidant Genotypes 18 0.0036 0.0000 50.65 <0.0001 Error 38 0.0001 0.0000 Corrected 56 0.0037 Total Anthocyanin Genotypes 18 0.9732 0.0541 2.73 <0.0045 Error 38 0.7517 0.0198 Corrected 56 1.7249 Total TPC Genotypes 18 1279.3146 71.073035 23.63 <0.0001 Error 38 114.3022 3.007952 Corrected 56 1393.6168 Total Fructose Genotypes 18 2.4278 0.1349 2.48 0.0091 Error 38 2.0642 0.0543 Corrected 56 4.4920 Total Glucose Genotypes 18 1.92502 0.10695 1.07 <0.4150 Error 38 3.79820 0.09995 Corrected 56 5.72322 Total Sucrose Genotypes 18 7.7574 0.43097 1.49 <0.1486 Error 38 11.0027 0.28954 Corrected 56 18.7601 Total Quercetin Genotypes 18 19.3347 1.07415 3.14 <0.0015 Error 38 12.9801 0.34158 Corrected 56 32.3148 Total

45 4.2.1 Pyruvic acid

In this study, pungency levels of 19 short day onion elite breeding lines were observed. The aim of the analyses was to find the pungency level of onion genotypes used. Results of pairwise mean comparison among the 19 different short day onions are shown in the Table 4.5, and there was a significant (p ≤ 0.05) genotype effects with respect to pyruvic acid amount of short day onion breeding lines analysed.

Table 4.5. Mean separations of pyruvic acid, total antioxidant, anthocyanin, phenol and flavonoid values of the genotypes

Genotypes Pyruvic Acid1 Antioxidant2 Anthocyanin3 Phenol4 K18 5.242 de5 0.0893 bcd5 0.1252 ab5 16.32 cde5 K19 6.112 c 0.0932 b 0.1392 ab 22.79 b K20 6.646 b 0.0886 cde 0.1948 ab 12.89 e K25 3.277 i 0.0843 def 0.1670 ab 12.69 e K26 3.972 h 0.0843 def 0.2227 ab 14.85 de K28 6.470 bc 0.0868 def 0.5566 a 16.91 cde K35 3.347 i 0.0908 bc 0.1670 ab 12.40 e K37 4.302 h 0.1007 a 0.1670 ab 12.89 e K38 8.828 a 0.0839 defg 0.3618 ab 19.26 bcd K39 4.877 ef 0.0812 fghi 0.2227 ab 18.67 bcd K40 4.975 ef 0.0719 ghij 0.4175 ab 15.24 de K41 4.182 h 0.0767 hij 0.2922 ab 15.34 de K42 4.182 h 0.0776 hij 0.0557 b 16.61 cde K51 3.347 i 0.0762 ij 0.1113 b 12.01 e K52 4.737 fg 0.0747 j 0.3896 ab 18.28 bcd K53 4.344 gh 0.1013 a 0.0835 b 12.89 e K58 4.056 h 0.0834 efgh 0.1809 ab 15.24 de K59 4.056 h 0.0806 fghi 0.3479 ab 20.83 bc K60 5.417 d 0.0893 bcd 0.0835 b 32.50 a LSD at 0.05 0.422 0.0061 0.4329 5.34 1Pyruvic Acid (μmol / mL), 2Total Antioxidant (mM TE / 100 mL) 3Total Anthocyanin (mg / 100 mL) 4Total Phenol (mg GAE / 100 mL) 5Within columns, means followed by the same letter are not significantly different (LSD at 0.05)

46 The mean pyruvic acid contens of the genotypes were in broad range between 3.277 and 8.828 μM / mL (Table 4.5). The genotype K38 was in the highest group and the genotypes K25, K35 and K51 were in the lowest group among the 19 different short day onion genotypes. The mean pyruvic acid values of the genotypes were generally between 3.277 and 6.646 μM / mL except the genotype K38 that showed the highest value as 8.828 μM / mL (Table 4.5).

Pungent flavor is one of the main preferences for consumer. The taste can change from person to person, therefore there is not any certain information about the range of pungent level preferred for a person. However, in Texas, USA, onion industry indicated that the scale of pyruvic acid is 4.5 µM / mL or less means mild pungency (Anonymous, 2017). Almost in similar way, the Vidalia Onion Industry in Georgia, USA, reported that for sweet onion the scale must be 5 μM / mL or less and other scales showed that the level of pyruvic acid between 0 and 3.5 μM / mL are fairly sweet, from 3.6 to 5 µM / mL are sweet and mild, from 5.1 to 7.5 µM / mL are pretty pungent, and those over 7.6 µM / mL are extra pungent flavor (Anonymous, 2019b). According this information, the genotypes K25 (3.277 μmol / mL), K35 (3.347 μmol / mL), and K51 (3.347 μmol / mL) were in the group of fairly sweet, genotypes K26 (3.972 μmol / mL), K58 (4.056 μmol / mL), K59 (4.056 μmol / mL), K41 (4.182 μmol / mL), K42 (4.182 μmol / mL), K37 (4.302 μmol / mL), K53 (4.344 μmol / mL), K52 (4.737 μmol / mL), K39 (4.877 μmol / mL), and K40 (4.975 μmol / mL) were in the sweet and mild group, whereas K18 (5.242 μmol / mL), K60 (5.417 μmol / mL), K19 (6.112 μmol / mL), K28 (6.470 μmol / mL), and K20 (6.646 μmol / mL) were pretty pungent, and K38 (8.828 μmol / mL) was in the extra pungency level group. In the Table 4.3 showed that the commercial short day onion genotypes (K51, K52, K53, K58, K59 and K60) were in the mild level of pungency. It is important to note that, although generally accepted, Yoo et al. (2012) indicated that the scale was not specifically valid to determine the pungency of onion and onion quality standards. Lee et al. (2009b) reported that pungent flavor depends on the personal perception, and as a general 3.5 µM / mL or less were considered mild, while more than 5 µM / mL were considered strongly pungent.

Manjunathagowda et al. (2019) observed the pyruvic acid levels of short day onion genotypes was between 1.72 and 2.31 µM / mL, Elhassaneen and Sanad (2009) observed

47 the different pungency levels in the two Egyptian onion varieties, where red color bulb was higher than white color bulb of onion, 11.37 and 8.24 µM / mL, respectively.

Lee et al. (2009b) conducted a study on characterization of the short day onions according to 3 pungency levels (low, medium, and high). Among 9 different onion varieties, 8.3 and 1.9 µM / mL were highest and the lowest amounts, respectively. The low range was between 1. 9 to 2.8 μM / mL; medium level was between 4.8 to 5.4 µM / mL and the highest level was between 7.2 to 8.3 µM/ mL. After these grouping, an onion which had 1.9 µM / mL of pyruvic acid was considered to have low flavor, and an onion having 8.3 µM / mL of pyruvic acid was considered to have high flavor.

Crowther et al. (2005) conducted a study to estimate the association between pungency and taste of onion. It was determined by panelists and the results indicated that between 7.15 and 10.62 µM / mL were for strong taste of onion, between 4.19 and 6.74 µM / mL were for mild taste and between 2.31 and 3.34 µM / mL were sweet taste of onion; however, there was no relationship between the long day and short day onion cultivars. The expected result was the lowest pungency level in short day onion genotypes because of the higher water inclusion during growing period due to environmental conditions including rains. Enciso et al. (2009) worked on the relationship between irrigation and pungency level of yellow short day onion, the values were observed between 3.9 to 4.4 µM / mL onion juice.

4.2.2 Antioxidant

The aim of the analysis was to determine the total antioxidant capacity of 19 short day onion breeding lines used in the thesis. Results of pairwise mean comparison among the 19 different short day onions are shown in the Table 4.5. A significant (p ≤ 0.05) genotype effect for antioxidant content was observed among the onion genotypes as given in the Table 4.4. In this study, the mean total antioxidant capacity values were between 0.0719 and 0.1013 mM TE / 100 mL (Table 4.5). The genotypes K37 and K53 were in the highest group and the genotypes K40, K41, K42, K5, and K52 were in the lowest grup among 19 different short day onion genotypes. In the Table 4.5 showed that the commercial short day onion genotypes (K51, K52, K53, K58, K59 and K60) were similar result with the

48 rest of the short day onion. The commercial short day onion of K53 was in the highest antioxidant level, however, K51, and K52 were in the lowest antioxidant level.

Recent studies reported the importance of antioxidant contents in food for health benefits. Antioxidants can enhance the self-defense mechanism of cells by inhibiting oxidative reaction, therefore, they can reduce significant diseases such as neurodegeneration, type II diabetes, and cardiovascular diseases (Lee et al., 2004; Griffiths et al., 2002). Therefore, the antioxidant capacity determination is important property for health benefits.

Lu et al. (2011) conducted a study by following three different methods (TEAC, DPPH and FRAP) for the determination of the total antioxidant capacity of 4 different onion cultivars: however, there was no information about the day length requirements of onion cultivars used. FRAP method showed the highest antioxidant capacity. In each method red onion cultivars had the highest capacity of antioxidant, followed by yellow, white, and sweet onion cultivars. They reported that DPPH method resulted in TA values between 0.0719 and 0.1013 mM TE / 100 mL. Lu et al. (2011) observed that red onion had 0.0052 mM TE / 100 mL, yellow onion 0.0045 mM TE / 100 mL, white onion had 0.0030 mM TE / 100 mL and sweet onion had 0.0014 mM TE / 100 mL. However, the same onion cultivars were also analyzed by TEAC, where red onion resulted in 0.0281 mM TE / 100 mL, yellow 0.0152 mM TE / 100 mL, white 0.0118 mM TE / 100 mL, and sweet onion 0.0105 mM TE / 100 mL. These results reported by Lu et al. (2011) were lower than the current study results, the reason could be that storage condition before analyses, while they were measured after freeze-drying, in this study measurements were performed without freeze-drying but by freezing the onion juice.

Gökçe et al. (2010) studied total antioxidant capacity of different colored onion cultivars by both FRAP and TEAC methods. Red onion cultivar had the highest antioxidant capacity in both methods as 0.0154 and 0.0093 mM TE / 100 mL in TEAC and FRAP, respectively. Yellow onion cultivar was higher than white color onion cultivar, where FRAP method resulted in 0.0098 and 0.0056 mM TE / 100 mL, respectively, and TEAC method 0.0147 mM TE / 100 mL versus 0.0087 mM TE / 100 mL. Additionally, they reported that some yellow onion genotypes could have higher antioxidant contents than red onion cultivar due to their high total phenolic contents. Although Gökçe at al. (2010) compared the scale color of onion without emphasizing the day length effect on the

49 antioxidant capacity, yellow color of onion genotype value was lower (0098 mM TE / 100 mL) than the results obtained in this thesis study with yellow onion genotypes (0.0747 mM TE / 100 mL). The difference could be due to the sample preparation for the analysis.

Yang et al. (2004) observed the total antioxidant capacity of 10 different onion cultivars and a shallot. The highest value was 0.0455 mM TE / 100 mL for the shallot, and the lowest value was 0.005 mM TE / 100 mL of a yellow onion cultivar. However, there was no significant differences in antioxidant capacity based on the color such as yellow, red and pink. The same result was observed in this study where there was one white color of onion genotype (K39) and the rest of them were yellow onion genotypes and the results showed that there was no relationship between antioxidant capacity and scale color of onion.

4.2.3 Anthocyanins

The aim of the anthocyanin measurements was to determine the total anthocyanin capacity of 19 short day onion genotypes used as the plant material in the study. Results of pairwise mean comparison among the 19 different short day onions are shown in the Table 4.5. As shown in Table 4.4, there was a significant genotype effects (p ≤ 0.05) for the anthocyanin capacity of the onion genotypes studied. The mean total anthocyanin capacity values were between 0.5556 and 0.0557 mg / 100 mL (Table 4.5). The genotypes K42, K51, K53, and K60 were in the lowest group. Besides, the commercial short day onion genotypes (K51, K52, K53, K58, K59 and K60) had similar result with the rest of the short day onion. Most of the short day onion genotypes studied were in the highest group and. It is important to note that the high value of total anthocyanin was desired because of the health benefit.

Anthocyanins are water-soluble pigments of polyphenolic compounds and they are responsible for cyanic colors ranging from salmon pink to red, violet to dark blue in most flowers, fruits, leaves, and stems. Beside this, anthocyanin has a role in health medicine and in plant by attracting insects for pollination, seed dispersal, and protection of plant against pest attacks (Strack and Wray, 1989). Several studies showed that red fruits and vegetables had high level of antioxidants due to the high concentration of anthocyanins

50 (Ozgen et al., 2009). That is, anthocyanin is one of the most important property in plant life and health benefit.

Zhang et al. (2016) conducted a study to observe total anthocyanin contents of yellow, red, and white onion varieties. Result of this study indicated that total anthocyanin contents of the red cultivars were higher in amount as compared to yellow and white cultivars that were 29.99, 9.64, and 0.75 mg / 100 g, respectively. However, Geetha et al. (2011) observed total anthocyanin content in three different onion varieties and they found that methanol extracts of red, small and big onion peel were 80 mg / g, 30 and 40 mg / g, respectively. In acidified methanol extracts the total anthocyanin contents were 109 mg / g, 58 and 35 mg / g, respectively.

Anthocyanin has the antioxidant property, therefore, in present study, short day onion genotypes that have the highest value of anthocyanin should also have the highest in antioxidant capacity as expected.

4.2.4 Total phenolic content (TPC)

The aim of the analyses was to determine the TPC of 19 short day onion genotypes used in the study. Results of pairwise mean comparison among the 19 different short day onion breeding lines are shown in the Table 4.5. A significant (p ≤ 0.05) genotype effect was observed for the TPC as given in the Table 4.4. The mean values were between 12.0 and 32.50 mg GAE / 100 mL (Table 4.5). The genotype K60 was in the highest group, four genotypes were in the mid group (K38, K39, K52, and K59), while 13 genotypes out of the 19 short day onion genotypes were in the lowest group (Table 4.5). In the 6 commercial short day onion genotypes, K60 was in the highest group, K52, and K59 in the mid group, however, K51, K53 and K58 were in the lowest group. As a general, the commercial short day onion genotypes were similar with the rest of them.

Phenolic compounds are necessary for the plants as they constitute redox properties accountable for antioxidant activity. Hydroxyl groups in plant extracts are accountable for facilitating free radical scavenging. As a basis, phenolic contents were measured using the Folin–Ciocalteu reagent in each extract. The results were expressed in gallic acid equivalents (GAE) per gram fresh extract weight or volume (Aryal et al., 2019).

The observed results were lower than some of the already available reports in the literature. Murkute and Gorrepati (2018) analyzed 34 different short day onions for different biochemical parameters for the determination of storage periods. As a result of the determination of TPC by Folin–Ciocalteau method, the highest TPC value was 79.36 mg GAE / 100 mL followed by 79.26 mg GAE / 100 mL and 42.86 mg GAE / 100 mL. The lowest value was 41.06 mg GAE / 100 mL followed by 41.23 mg GAE / 100 mL and 42.86 mg GAE / 100 mL. Chu et al. (2002) found TPC as 68.90 mg GAE / 100 mL of a yellow onion. They reported that the TPC and antioxidant capacity were not in strong correlation with each other. In the present study the genotypes K37 and K53 were in the highest total antioxidant capacity value; however, the genotype K60 was in the highest TPC value so there was also no correlation between these properties for 19 short day onion genotypes studied supporting the report of Chu et al. (2002).

Vinson et al. (1998) studied TPC of 23 different vegetables by dry weight and fresh weight. Among them, red onion had higher TPC (41.0 µM / g DW and 4.0 µM / g FW) than yellow onion (22.0 µM / g DW and 2.4 µM / g FW). The reason for the difference in values was explained as that red onion had more phenolic anthocyanins conferring the reddish color of the flesh. Elhassaneen and Sanad (2009) observed the differences in TPC of the two Egyptian onion varieties where the red onion had higher TPC than the white one. In the present study, genotype K39 was white in color and rest of the genotypes were yellow color and top four genotypes with respect to TPC were yellow colored genotypes (K60,K19, K59 and K38), while the fifth was the white color onion (K39). That is, there was no correlation between the color and TPC value. It is important to mention that TPC not only related with the color, but besides is also affected by genotype and environment on the accumulation (Kaur et al., 2009).

Zhang et al. (2016) analyzed TPC of yellow, red, and white onions. Result of this study showed TPC content of yellow cultivar had higher amount followed by red and white, with 741.84, 622.27, and 389.69 mg GAE / 100 mL, respectively. Another supportive study conducted by Gökçe et al. (2010). They studied different colored onion cultivars to determine total phenolics, the highest value was 3.7 mg GAE / 100 mL in yellow onion followed by 2.2 mg GAE / 100 mL in red and the lowest one was 1.1 mg GAE / 100 mL of white color onion cultivar. While, Lu et al. (2011) conducted a study to determine TPC

52 in 4 different onion genotypes and they observed the highest value in red onion as 4.28 mg GAE / 100 mL, followed by white (2.69 mg GAE / 100 mL), yellow (1.64 mg GAE / 100 mL), and sweet (1.42 1.64 mg GAE / 100 mL) onion cultivars.

4.2.5 Sugars (Fructose, glucose, sucrose)

The aim of the analyses was to find the sugar contents of 19 short day onion genotypes. The Table 4.4 shows that the genotype effects for the fructose were significant with a p value of 0.009 while the genotype effects for the glucose (p < 0.415) and sucrose (p < 0.149) were not significant in the ANOVA table. Nonetheless, pairwise genotype mean comparisons showed that there are some genotypes have significantly higher mean values than that of the other genotypes for the glucose and sucrose (Table 4.6). Since the ANOVA test results were not significant, not to speculate, the pairwise mean separation results were not explained herein.

Results of pairwise mean comparison among the 19 different short day onion breeding lines for the fructose are shown in the Table 4.6. The fructose mean values were between 1.5000 and 2.4067 g / 100 g FW (Table 4.6). The genotypes K25, K35, K37, K53, K58, K59, and K60 were in the highest group and the genotypes K38, K39, K40, and K41 were in the lowest group among the 19 short day onion genotypes (Table 4.6).

In the Table 4.6 showed that the 6 commercial short day onion genotypes (K51, K52, K53, K58, K59, and K60). In the 6 of commercial genotypes, 5 of them were in the highest group (K52, K53, K58, K59, and K60) besides, the fructose values were almost similar between the commercial short day onion genotypes, and the rest of 13 short day elite onion genotypes.

There are various factors responsible for the quality of onion, the carbohydrate composition is one of them. Storage life is an effective property for the consumer, and it is related with the carbohydrate level of the onion bulb. There are fundamental nonstructural carbohydrates that are sucrose, fructose and glucose in onion bulb (Shiomi, 1989; Terry, et al., 2005). Sweet flavor is based on the sugar and pyruvate accumulation into the bulb at up to 25 °C temperature (Lee and Suh, 2009a). Sugar is used as a sensory for sweetness (Terry et al.2005).

Table 4.6. Mean separations of sugars (fructose, glucose, and sucrose) and quercetin values of the genotypes

Genotypes Fructose1 Glucose2 Sucrose3 Quercetin4 K18 1.9500 bcde5 2.8167 abc5 0.5867 cd5 1.3200 abcd5 K19 1.9033 cde 2.6833 abc 0.7567 bcd 0.4667 def K20 1.9833 bcde 2.5533 c 1.5600 ab 0.7667 cdef K25 2.0400 abcd 2.5667 bc 1.3800 abcd 1.0000 abcde K26 1.9833 bcde 0.0843 def 1.5600 ab 1.1167 abcd K28 1.9800 bcde 2.6167 bc 1.2167 abcd 0.5033 def K35 2.2400 abc 2.9367 abc 0.9500 abcd 1.9433 a K37 2.0667 abcd 2.8433 abc 1.4867 ab 1.0400 abcd K38 1.8233 def 2.6300 bc 1.6400 ab 0.6000 def K39 1.8200 def 2.6867 abc 1.2767 abcd 1.8280 bcd K40 1.5000 f 2.6233 bc 1.7400 a 1.0900 abcd K41 1.6533 ef 2.8467 abc 1.4233 abc 1.5240 de K42 1.9600 bcde 2.7600 abc 0.8367 bcd 1.6000 abc K51 1.9833 bcde 2.9367 abc 0.8367 bcd 1.7567 ab K52 2.1700 abcd 3.0567 abc 1.0267 abcd 1.7200 abc K53 2.3000 ab 2.8167 abc 1.4867 ab 1.3033 abcd K58 2.4067 a 2.9167 abc 1.2467 abcd 0.9767 bcde K59 2.1000 abcd 3.1800 a 0.5067 d 0.4033 def K60 2.0567 abcd 3.0800 ab 0.7633 bcd 0.0633 ef LSD at 0.05 0.3852 0.5226 0.8894 0.9660 1Fructose (g / 100 g FW) 2Glucose (g / 100 g FW) 3Sucrose (g / 100 g FW) 4Quercetin (μg / g FW) 5Within columns, means followed by the same letter are not significantly different (LSD at 0.05)

Kim et al. (2017) reported the correlation between total sugar, sucrose, and glucose with pyruvic acid in Vidalia (sweet and hot short day) onions by treatment of nitrogen and sulfur. Among the sugar content, fructose and glucose concentrations were more than sucrose concentration. In low fertilization, the sweet onion had 2.69 g / 100 g FW and in high fertilization, the ratio was increased to 5.98 g / 100 g FW. While in low fertilization, hot onion had 2.50 g / 100 g FW and in high fertilization, the ratio was increased to 2.66 g / 100 g FW.

Lee et al. (2009b) conducted a study on 9 different short day onion cultivars. As a result, sucrose, fructose, and glucose contents were varied from 4.7 to 8.1, 9.3 to 18.5 and 32.3 to 48.6 mg /g FW, respectively. Beside this, the total sugar content was between 49.9 to 74.5 mg / g FW and, in conclusion, there was no association between pungency level and total sugar content either for sucrose, fructose, or glucose. Kim et al. (2017) reported that sugar was affected by the environment, therefore, the different content of fructose between Lee at al. (2009b) and our study can be explained by several factors associated with the environment such as the climate, and the soil type. Caruso et al. (2014) conducted a study to observe the effects of transplanting time and density of onion on several factors. As a result, they reported that there was no relation between transplanting time and the onion bulb glucose (27.7 g / 100 g of the bulb DW) and fructose contents (21.4 g / 100 g of the bulb DW). However, sucrose content was observed to be increased in March planted onion. Caruso et al. (2014) also indicated that warmer temperatures may function in increasing sucrose levels. The present study was conducted at Alata / MERSİN climate, which might be the reason for most of the onion breeding lines studied having different sucrose levels compared to the literature.

4.2.6 Quercetin (Flavonoid compound)

The aim of the analyses was to determine the quercetin belonging to flavonoids in 19 short day onion breeding lines. Results of pairwise mean comparison among the 19 different short day onion genotypes are shown in the Table 4.6. The quercetin values were affected significantly by the genotypes with a p value of 0.0015 as given in the Table 4.4. The quercetin mean values varied in a range from 0.0633 to 1.9433 μg / g FW (Table 4.6). Ten genotypes among the 19 short day onion genotypes were in the highest group and five of them were in lowest group. In the 6 commercial short day onion genotypes, K51, K52, and K53 were in the highest group, however, K59 and K60 were in the lowest group. The elit short day onion genotypes had almost similar result for quercetin analyses (Table 4.6).

The flavonoids belong to the polyphenolic compounds, which have antimicrobial and free radical scavenging properties, and moreover, they have a high antioxidant capacity (Chu et, al., 2000; Prakash, et al., 2007). Therefore, they have a great potential for health-

55 promoting activities such as reducing the risk of cardiovascular diseases and cancer. The most abundant compound among the flavonoids is quercetin, which protects the DNA strand (Chu et, al., 2000; Prakash, et al., 2007). Trammell and Peterson (1976) reported that short day onion cultivars had lower amounts of flavonoid than long day cultivars.

Herrmann (1976) indicated that colored onion cultivars had higher flavanoid contents than white onion cultivars. Studies indicated that red colored onion had higher level of flavonoids because of the presence of anthocyanins (Griffiths et al., 2002; Rodrigues et al., 2003; Elhassaneen and Sanad, 2009). Yoo et al. (2010) compared the quercetin amounts of short and long day yellow onion cultivars. The results of both short and long day onions were close to each other. The 4 different long day yellow onion cultivars had 5.8, 3.1, 2.4 and 2.1 μg / g FW, while 3 different short day yellow onion cultivars had 2.2, 1.9, and 1.8 μg / g FW. In our study, yellow onion genotype K35 (1.9433 μg / g FW) was in the highest group followed by the white onion genotype K39 (1.8280 μg / g FW) and genotype K39 showed that a higher value than rest of the 17 yellow onion genotypes. Yoo et al. (2010) reported results of short day yellow onions and there was higher quercetin value than this present study, which might be related with the genotype effect or the cultivation techniques.

56 CHAPTER V

CONCLUSION

Onion (Allium cepa L.) is a vegetable widely found and consumed all over the world, and it is the richest source of dietary polyphenolic compounds. Phenolic compounds are quite crucial for health, especially the flavonoid group. Onion contains plenty of quercetin, which prevents numerous diseases. Moreover, sulfur-containing phytochemicals and pyruvic acid were abundantly detected in onions, and they were observed to be effective in preventing certain diseases due to their blood-thinning effect. Bioactive properties such as antioxidant activity and amounts of compounds with health benefit differ according to onion color and genotype. For this purpose, 19 different short day onion varieties were observed on the basis of their bioactive properties in order to define their composition to be used in further breeding studies.

Short day onions are known to have higher water holding capacity than long day onion genotypes and TSS is known as indicator of sugar (Crowther et al., 2005). Therefore, the expected results were to obtain high TSS and high sucrose together in the same genotype. However, our results did not support this relationship probably due to cultivation climate and soil effects. The genotypes K37 (5.50), K58 (5.36), and K59 (5.20) were in the lowest group for TSS (Table 4.3) whereas, the genotypes K25 (2.0400 g / 100 g FW), K35 (2.2400 g / 100 g FW), K37 (2.0667 g / 100 g FW), K53 (2.3000 g / 100 g FW), K58 (2.4067 g / 100 g FW), K59 (2.1000 g / 100 g FW), and K60 (2.0567 g / 100 g FW) were in the highest group for fructose (Table 4.6). Additionally, among the sugar compound only fructose was significantly different (p < 0.009), whereas sucrose and glucose were not significantly different (p < 0.149) and (p < 0.415), respectively (Table 4.4). Although a relationship was expected between TSS and sugar content, it is valuable to mention that TSS includes not only sugar, but also pectin, organic acids and amino acids.

Acidity in foods is one of the most important properties in terms of taste and processing. For example, many processes to be applied to foods should be arranged according to the acidity of the food. It is impossible to determine the heat treatment conditions that must be applied to a food without knowing its pH. At the same time, acidity and pH determination is frequently required in the production of vegetable preserves such as

57 various sauces, ketchups, artichokes and okra, as well as in the monitoring of acid fermentations, and in the production of jam and marmalade. On the other hand, since acidity has an important role in the formation of flavor balance of many foods, it is necessary to constantly determine acidity during cultivation. For example, since the sugar / acid ratio in fruits is the most important factor in taste, ripening of the fruits is often monitored in this way. Regarding acidity, two concepts stand out as real (effective) acidity (pH) and titratable acidity (total acidity). Actual (effective) acidity is the acidity determined by pH. Effective acidity is related to the concentration of dissociated hydrogen ions. It is used to describe the degree of acidity, in other words, the strength of the acidity is an expression of hydrogen ion activity in the environment. This acidity, which is measured by a pHmeter, is not related with titratable acidity Berno et al. (2014). In the present study result of TA and pH value were no supportive of this fact as genotypes K19 (1.418 g / mL) and K38 (1.539 g / mL) were in the highest group for TA and genotype K58 (6.00) was in the highest group and genotype K60 (5.69) was in the lowest group for pH value (Table 4.3). Therefore, there was not a certain relationship between TA and pH value.

The onion flavor and aroma are related with some factors like sugar, pH, TSS and pungency level. Pyruvic acid is a known indicator of pungency level of onion (Griffiths et al., 2002). It is known for its benefit for human health, besides, onion is an edible vegetable therefore the aroma and flavor is important for consumption. In the present study, the genotype K38 (8.828 μM / mL) was in the highest group for pyruvic acid (Table 4.5). Although, the taste can change person to person, in general, low pungency level is more desirable for cooking. There was not an universally accepted scale for pungency level of onion but based on the scale provided by Vidalia Onion Industry in Georgia, USA, the genotypes K25 (3.277 μM / mL), K35 (3.347 μM / mL), and K51 (3.347 μM / mL) was in the group of fairly sweet, the genotypes K26 (3.972 μM / mL), K58 (4.056 μM / mL), K59 (4.056 μM / mL), K41 (4.182 μM / mL), K42 (4.182 μM / mL), K37 (4.302 μM / mL), K53 (4.344 μM / mL), K52 (4.737 μM / mL), K39 (4.877 μM / mL), and K40 (4.975 μM / mL) were in the sweet and mild group, and K18 (5.242 μM / mL), K60 (5.417 μM / mL), K19 (6.112 μM / mL), K28 (6.470 μM / mL), and K20 (6.646 μM / mL) were in the pretty pungency, whereas K38 (8.828 μM / mL) was in the extra pungency level group.

58 Anthocyanin is a water-soluble pigment responsible from the red, purple and blue color in vegetables and fruits (Tetiktabanlar, 2011). It also has the antioxidant property therefore, expected result was observing a parallel value between anthocyanin and antioxidant levels. The genotypes K37 (0.1007 mM TE / 100 mL) and K53 (0.1013 mM TE / 100 mL) were in the highest group for antioxidant, however, except the genotypes K42 (0.0557 mg / 100 mL), K51 (0.1113 mg / 100 mL), K53 (0.0835 mg / 100 mL), and K60 (0.0835 mg / 100 mL), rest of the 19 short day onion breeding lines used in the study were in the highest group for anthocyanin (Table 4.5). As a result, in this study, there was no certain relationship between antioxidant capacity and anthocyanin amount of short day onions studied. Besides, the quercetin has a strong antioxidant property, therefore the expected result was to observe high antioxidant capacity and quercetin amount in the same genotypes analyzed in this study. However, in the present study, the genotypes K18 (1.3200 μg / g FW), K25 (1.0000 μg / g FW), K26 (1.1167 μg / g FW), K35 (1.9433 μg / g FW), K37 (1.0400 μg / g FW), K40 (1.0900 μg / g FW), K42 (1.6000 μg / g FW), K51 (1.7567 μg / g FW), K52 (1.7200 μg / g FW), K53 (1.3033 μg / g FW) were in the highest group for quercetin (Table 4.6), whereas the genotypes K37 (0.1007 mM TE / 100 mL) and K53 (0.1013 mM TE / 100 mL) were in the highest group for antioxidant (Table 4.5). Although most of the lines were not in agreement in terms of relationship between quercetin and antioxidant capacity, the genotype K37 and K53 was observed to be in the highest group for both properties. It is wrothy to mention that antioxidant capacity has numerous compounds such as vitamin E, vitamin C, vitamin A, and flavonoids besides quercetin, which might explain this observation (Balasundram et al., 2006). Another expected result was to have higher anthocyanin amount and antioxidant capacity in yellow onion genotypes compared to the white onion line used in the study, but there was no relationship between the scale color with anthocyanin and antioxidant capacity.

Chu et al. (2000) reported that onion genotypes with yellow scale color had higher content of TPC than the red onions. In present study, only the genotype K39 had white scale color and rest of genotypes had yellow scale color. According to the Table 4.5, the genotype K60 was in the highest group and thirteen of the rest were in the lowest group among the 19 different short day onion genotypes. The top four genotypes (K60, K19, K59, K38) were yellow in color and the fifth was the white color genotype (K39).

59 According to each variable results showed similarity between the 13 short day onion genotypes with commercial short day onion genotypes (K51, K52, K53, K58, K59, and K60).

As a final conclusion, the genotypes K38 for TSS; K19 and K38 for TA; K58 for pH; K38 for pyruvic acid; K37 and K53 for antioxidant; all the genotypes except K42, K51, K53, and K60 for anthocyanin; K60 for TPC; K25, K35, K37, K53, K58, K59, and K60 for fructose; K18, K25, K26, K35, K37, K40, K42, K51, K52, and K53 for quercetin variables had the highest mean values. A breeder may use these genotypes based on their bioactive properties depending on the breeding objectives.

Besides, other objectives of breeder could be for cooking in this case, the flavor and taste of onion are related to the pungency level, sugar content also, TSS and pH. Therefore, the highest value could be considered K25, K35, K37 for fructose, K38 for TSS, and K37 for pH. The pungency level can be change person to person but as a general can be chosen a low level of pungent flavor.

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CURRICULUM VITAE

Saime Buse GÜVEN was born on April 21, 1995 in Istanbul. She completed her primary education from Yalçın Eskiyapan School, Ankara and secondary education from Fethiye Gazi School, Fethiye. She completed high school education from Fethiye Anatolian High School. She graduated from faculty of Ayhan Şahenk Agriculture Science and Technologies, Department of Agricultural Genetic Engineering, Nigde, TURKEY in 2018. She had been in Poland for education under ERASMUS program. She had also been to Germany for internship in IPK (Leibniz Institute of Plant Genetics and Crop Plant Research) in Cell Structure Department via ERASMUS program in 2016. She was trainee in Plant Biotecnology Lab of Ankara Biotechnology Institute in 2017. She started her master degree program at Ayhan Şahenk Agriculture Science and Technologies, Department of Agricultural Genetic Engineering, Nigde, TURKEY under the supervision of Dr. Ali Fuat GÖKÇE. During her master degree program, she joined TUBİTAK project as a researcher and she worked on Determination of Bioactive Properties of Some Short Day Onion Inbred Lines Produced in Alata Climate. Her thesis was conducted at Alata Horticultural Research Institute by herself. She knows English.