Selection high sucrose soybean lines from a mutation polulation derive fron ems treatment

Sucrose content in soybean seeds is desired to be high because it is a sweetness-imparting component, it helps extends acceptance for derived food products.. We developed EMS –mediated

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

DUONG THI BICH HUONG

SELECTION HIGH SUCROSE SOYBEAN LINES FROM A MUTATION POPULATION DERIVE FROM EMS TREATMENT

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

DUONG THI BICH HUONG

SELECTION HIGH SUCROSE SOYBEAN LINES FROM A MUTATION POPULATION DERIVE FROM EMS TREATMENT

Ph.D Duong Van Cuong _

Ph.D Pham Bang Phuong

Thai Nguyen, 2016/08/20

Thai Nguyen University of Agriculture and Forestry

mutation population derive from EMS treatment

Dr Duong Van Cuong

Dr Pham Bang Phuong

Abstract:

Soybean is one of the most important crops Soybean seeds contain about 35% carbohydrate Sucrose content of soybean carbohydrate range from 2.5 – 8.2%, raffinose ranges from 0.1-0.9%, stachyose ranges 1.4-4.1% Sucrose content in soybean seeds is desired

to be high because it is a sweetness-imparting component, it helps extends acceptance for derived food products To improve of quality carbohydrate, the breeding programs primary focused on increasinge sucrose content Mutagenesis is used to study gene function and obtain new genetic resources for plant breeding to find a new genetic resources We developed EMS –mediated mutant population to select high sucrose concentration lines by megazyme kit to analyze sucrose content Selection of 710 out of 3777 mutant lines derived from a mutation population which was treated EMS 0.3% Finishing the preliminary analysis, as a results we obtained 390 lines with sucrose content of larger than that of Pungsanamul The remaining

soy-320 lines were lower Selected 31 soybean lines have larger than that sucrose content Pungsanamul were selected and reanalyzed Sucrose concentration of all 31 those lines were larger than that Pungsanamul sucrose content PE 1472 was confirmed as a unique germplasm with the highest sucrose content (21.216 g/100g) PE 831(16.818 g/100g) were confirmed with extremely low concentrations Pungsanamul was identified around 16.122g/100g The identified germplasm with high sucrose profiles will be valuable in breeding specialty soybeans for improved sugar content The selection of 31 mutant lines with high sucrose profiles will facilitate in a large-scale soybean breeding program

Keywords

High sucrose, EMS, soybean mutaion

ACKNLOWLEDGMENTS

The firstly, I would like to express the gratitude and profound thanks to special person who always admire and respect That is my wonderful supervision Professor, Jeong-Dong Lee You was so kind, love the students, caring, sharing, listening, and always willing to do what's best for students Thank you for having the faith in myself and giving me a gold opportunity to internship during the duration Exchange student’s program in your lab Here I have had many great experiences and learned a lot of experience from the learning environment and the social environment In deep my heart, you are a greatest teacher

The secondly, I would also like to give acknowledgements to Park Cheolwoo The day to day operations during my research and life would have been nearly impossible without the guidance Park Cheolwoo, he was by my side every step of the way and provided countless suggestions on how to better my studies I am very thankful for your time, help and support throughout my graduate studies

The thirdly, I would to thank my advisor Dr Nguyen Minh Phuc, Master Huyn

Jo although different major and university but whenever they always helped, supported, and guided carefully for their guidance and feedback during my academic research They were crucial elements to the success of the entire research team They were always there to bounce ideas off of and give a helping hand wherever they could

I have benefited from the work of the groups around Korea over the years and I have enjoyed the friendship and collegiality of the students, staff, and others who collaborated in the group’s projects during this time A big thanks goes out to all my member in my lab Sovetgul Asekova, Bota, Min Su Kim, Jae-eun Jeong, Dong Ho Lee and member in other lab Everyone there was more than willing to help my with my research in any way they could

Another great thanks goes out to my parents, Binh and Loan who raised me into the young lady I am today I would not have been able to accomplish so much in my undergraduate studies without their love, support, and guidance, encouragement and motivation

I would like to start off by thanking God for good health and all the great

possibilities that he has put into my life I have been very fortunate in my life and been

allowed the opportunity for higher education

Lastly, I would like to be special big thank my great supervisor Dr Pham Bang

Phuong and Dr Duong Van Cuong at Thai Nguyen University of Agriculture and

Forestry who recommended me to be today I have had a big chance to learn and

conducting this topic Especially, Dr Pham Bang Phuong always oriented and create

the most favorable conditions for my learning path Anytime I have difficulty in

writing thesis who gave the best advice to support the shared interest enthusiastically

help to anyone Another great thanks goes to all teacher in my faculty: Biotechnology

and Food taught knowledge and communication have valuable experience

Many thank you and best regards

Student

Duong Thi Bich Huong

CONTENTS

ACKNLOWLEDGMENTS ii

CONTENTS iv

LIST OF FIGURES v

LIST OF TABLES vi

LIST OF ABBREVIATION vii

I INTRODUCTION 1

1.1 Soybean 1

1.2 The sucrose 4

1.2.1.Structure 5

1.2.2.The function of sucrose 6

1.2.2.1 The role of sucrose in the body 6

1.2.2.2 The role of sucrose in food product 9

1.2.3.Trade and economic 12

1.2.4.Situation research 15

1.2.5.Chemical Mutagenesis 16

1.3 Research Objective 17

II MARERIAL AND METHODS 18

2.1Material and equipment 18

2.1.1 Plant Material 18

2.1.2 Equipment and chemical 18

2.2 Method 20

2.2.1 Carbohydrates extraction 20

2.2.2 Sucrose quantification by the GOPOD/invertase method (enzymatic method) 21

2.2.3 Calculation 27

III RESULTS AND DISCUSSION 28

3.1 The soybean seeds treated by EMS 28

3.2 Determination carbohydrates extraction 29

3.3 Preliminary evaluation of the sugar content 29

3.4 Selection of high sucrose content lines 34

IV CONCLUSION 41

REFERENCES 42

APPENDICES 46

LIST OF FIGURES

Figure 1: Soybean plant 1

Figure 2: Soybean seeds 2

Figure 3: Chemical structure of sucrose 5

Figure 4: The structure of glycogen enables its rapid mobilization into free glucose to power cells 8

Figure 5: World raw sugar price for Calendar years 1969-2014 13

Figure 6: The chart showed global sugar consumption to again outpace production, 2009/2010-2016/2017 14

Figure 7: Schema displayed procedure of plant material 19

Figure 8: Soybean powder were ground and filtered by standard testing sieve 20

Figure 9: Extracted the sugars from sample using ethanol 50% in oven (A) and after centrifugal finish (B) 21

Figure 10: The Megazyme Sucrose/D-Glucose Test Kit 22

Figure 11: A The carbohydrates extraction poured invertase and GOPOD in oven at 50°C 25 Figure 12: The chart displaying standard curve of D-Glucose standard solution 27

Figure 13: The Pungsanamul 28

Figure 14: The Pungsanamul were treated 0.3%EMS 28

Figure 15: The soybean solution was extracted by 50% ethanol 29

Figure 16: The chart shows the percentage of sucrose content of the mutant soybean lines 30

Figure 17: The chart was shown the percentage of sucrose content both 48 mutant lines 33

Figure 18: The chart showed sucrose content of 31 mutant lines 38

:

LIST OF TABLES

Table 1: Sucrose concentration both the 48 mutant lines and Pungsanamul were

determined by the GOPOD/invertase method 31 Table 2: The first replications, the sucrose concentrations % (g/100 g) The value of

glucose content was measured at 510 nm, µg /ml value, µg/mg value were determined by the GOPOD/invetase method in seed of 31 different soybean lines from a mutation population derived from EMS treatment and a control line (Pungsanamul) 35 Table 3: The second replications, the sucrose concentrations % (g/100 g) The value of

glucose content was measured at 510 nm,µg /ml value, µg/mg value were determined by the GOPOD/invertase method in seed of 31 different soybean lines from a mutation population derived from EMS treatment and a control line (Pungsanamul) 36 Table 4: Mean two replications of sucrose concentrations in % (g/100 g) were

determined by the GOPOD/invetase method In seed of 31 different soybean lines from a mutation population derived from EMS treatment and the control sample Pungsanamul 37

LIST OF ABBREVIATION

ATP Adenosin triphosphate

EMS Ethyl Methane Sulphonate

ELISA Enzyme-Linked ImmunoSorbent Assay

FAO Food and Agriculture Organization

FDA Food and Drug Administration

G6DH Glucose-6- phosphate dehydrogenase (or G6PDH)

GOPOD Glucose oxidase peroxidase

HPAE-PAD High-Performance Anion-Exchange Chromatography-Pulsed

Amperometric Detection HPLC High pressure liquid chromatography

NADPH Nicotinamide adenine dinucleotide phosphate

NIRS Near-infrared reflectance spectroscopy

NSP Nonstarch polysaccharides

PCR Polymerase Chain Reaction

RMP Revolutions Per Minute

Pungsan Pungsanamul

SBP Sucrose blinding protein

TLC Thin layer chromatography

USA United States Dollar

USD The United States of America

USDA United States Department of Agriculture

I INTRODUCTION 1.1 Soybean

Cultivated soybean [G max (L.) Merr] is a diploidized tetraploid (2n=40), in the family Leguminosae, the subfamily Papilionoideae, the tribe Phaseoleae, the genus Glycine Wild and the subgenus Soja (Moench) It is an erect, bushy herbaceous annual that can reach a height of 1.5 meters Three types of growth habit can be found amongst soybean cultivars: determinate, semi-determinate and indeterminate (Bernard

and Weiss, 1973) (Figure 1)

Figure 1: Soybean plant Cultivation is successful in climates with hot summers, with optimum growing

conditions in mean temperatures of 20 to 30 °C (68 to 86 °F); temperatures of below

20 °C and over 40 °C (68 °F, 104 °F) stunt growth significantly They can grow in a wide range of soils, with optimum growth in moist alluvial soils with a good organic

content For best results, though, an inoculum of the correct strain of bacteria should

be mixed with the soybean (or any legume) seed before planting and take 80–120 days from sowing to harvesting (https://en.wikipedia.org/wiki/Soybean, 2016)

In addition to its rich protein and oil content 40%, 20 % respectively, soybean

seed also contains approximately 33% carbohydrate, up to 16% of which are soluble sugars (Hymowitz and Collins, 1974) Mature soybean contain trace amount of monosaccharides such as glucose and arabinose, and measurable number of disaccharides and oligosaccharides, with sucrose in the range of 2.5 – 8.2%, raffinose

in the range 0.1-0.9%, stachyose in the range 1.4-4.1% (Hymowitz et al 1972)

(Figure 2)

Figure 2: Soybean seeds

Soybean is a quantitative short day plant and hence flowers more quickly under short days (Garner and Allard, 1920)

Demand for soybeans continued and still is continuing to grow on a worldwide basis following below:

World soybean production has increased by 76% from 6.5 billion bushels in

2000 to 11.3 billion bushels in 2014 Brazil and Argentina increased soybean production by 138% and 98%, respectively, during the same time period The United States increased soybean production by 44% between 2000 and 2014 while soybean production increased by 137% in the ROW region Most of that increase took place in other South American countries (Richard and Won, 2015)

The world soybean production in the year of 2016 was approximately 313, 26 million metric tons The USA is the largest soybean with producer with 106, 93 million metric tons The other major production countries such as Brazil, Argentina and Paraguay contributed about 97, 56.5 and 8 8 million metric tons, respectively (USDA, 2016)

Soybeans increased 24.75 USD/BU or 2.18% from 1159.25 on Friday June 17

to 1134.50 in the previous trading session Soybeans gained 182.8 USD/BU or 18.71 percent during the last 12 months from 976.50 USD/BU in June of 2015 (http://www.tradingeconomics.com/commodity/soybeans, 2016)

Soybean are used for food, pharmaceutical, cosmetic, bio-fuel industries It is rich in protein carbohydrates, saponins, phytic acid and oil but has lower levels of calcium It has wide range of therapeutic as well as pharmaceutical applications It has sedative, anti-spasmodic, diaphoretic, anti-pyretic properties, with hormonal balancing effects, can be used to prevent breast cancer, prostate cancer, endometrial cancer and baldness and has great benefit to the liver and circulation It acts as a component of easy release jelly and also as an agent that provides good hardness and disintegrating property

In tofu, soymilk, natto, and many other soy food products, desirable sugars including glucose, fructose, and sucrose contribute to the favorable sweet taste and are ready-todigest, while raffinose and stachyose are indigestible and cause undesirable flatulence and diarrhea (Rackis, 1975)

In China, Japan, and Korea, soybeans are also used in industrial products, including oils, soap, cosmetics, resins, plastics, inks, crayons, solvents, and clothing

Soybean oil is the primary source of biodiesel in the United States, accounting for 80%

of domestic biodiesel production Soybeans have also been used since 2001 as fermenting stock in the manufacture of a brand of vodka Soybean and soybean products are a common part of the diet (https://en.wikipedia.org/wiki/Soybean, 2016)

The FDA granted the following health claim for soy: "25 grams of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease” (Henkel and John 2000) Soybean helped to reduce cholesterols and prevent and manage certain types of cancer, kidney disease, osteoporosis, diabetes, and obesity (Illinois Center for Soy Foods, 2007)

In addition, one of most important agronomic characteristic for soybean and other legume species was their ability to took nitrogen from the air and convert it to metabolizable ammonium N, a process known as nitrogen fixation It also helps to keep the production costs for soybean relatively lower compared to other cops The ability of soybean plant to fix N2 has played a very significant role in maintaining the nitrogen balance in biosphere and environmental cleanness Saving billion of dollar for provided fertilizer nitrogen (Liu, 1997)

1.2 The sucrose

Soybean seed are normally valued not only for their high oil contents and protein but also contain significant amount of carbohydrate, also known as saccharides with the general chemical formula Cn(H2O)m and their derivatives It including simple sugar (mono and disaccharide), oligosaccharide, and polysaccharide

Recent studies have shown that some beneficial effects of dietary oligosaccharides dietary for humans’ health and there are including:

Increasing population of indigenous bifid bacteria in the colon which, by their antagonistic effect, suppress the activity of putrefactive bacteria

Reducing toxic metabolites and detrimental enzyme Preventing pathogenic and autogenous diarrhea by the same mechanisms as described in the reduction of detrimental bacteria

Preventing constipation due to production of high levels of short-chain fatty acids by bifidobacteria

Protecting liver function due to reduction of toxic metabolites Reducing blood pressure Having anticancer effect Producing nutrients such as vitamins, also due to increased activity of Bifidobacterium (Liu, 1997)

Sucrose is often extracted and refined from either cane or beet sugar for human consumption Modern industrial sugar refinement processes often involve bleaching and crystallization, producing a white, odorless, crystalline powder with a sweet taste

of pure sucrose, devoid of vitamins and minerals This refined form of sucrose is commonly referred to as table sugar or just sugar

The two sugars are linked (via an alpha 1,2glycosidic bond) - effectively

an oxygen bridge - formed as a result of a condensation reaction

In the sucrose molecule there are 12 carbon atoms, and 2 ring-shaped structures, each containing an oxygen atom The glucose ring - initially on the left side - is a 6-sided structure (5 carbons and an oxygen), whereas the fructose ring - on the right side

- is a 5-sided structure (4 carbons and an oxygen) This structure is described as glucopyranosyl-(1→2)-β-D-fructofuranoside, by reference to pyran (a six-membered heterocyclic, ring compound, consisting of five carbon atoms and one oxygen), and furan (similar, with one less carbon atom) The -OH groups originally on C1 of glucose and C2 of fructose which give reducing properties to the original glucose and fructose molecules have been used up in forming the bond between the two sugar units,

α-D-so they cannot form reactive open-chain forms like the original sugars

Sucrose crystallizes in the monoclinic space group P21 with room-temperature

lattice parameters a = 1.08631 nm, b = 0.87044 nm, c = 0.77624 nm, β = 102.938° (Beevers at al., (1952), Hynes and Le (1991))

1.2.2 The function of sucrose

The sucrose holding important role in the body, food and nutrition, sugar has been used for many centuries to make our traditional homemade foods Besides its sweet taste, sugar contributes to the colour, flavour and texture of food

1.2.2.1 The role of sucrose in the body

Provide Fuel for the Central Nervous System

Nerve cells are very dependent upon glucose for their functioning When insufficient carbohydrates are consumed to meet the energy needs of the central nervous system, besides the occurrence of gluconeogenesis, another phenomenon occurs during a fast of three weeks or more: The cells of the central nervous system adapt their metabolic apparatus to use ketone bodies in place of glucose

Provide Fuel for the Muscular System

Carbohydrates (as well as sucrose) provide the major fuel for muscular exercise

Energy Storage

If the body already has enough energy to support its functions, the excess glucose is stored as glycogen (the majority of which is stored in the muscle and liver) The amount of glycogen in the body at any one time is equivalent to about 4,000 kilocalories—3,000 in muscle tissue and 1,000 in the liver Prolonged muscle use can deplete the glycogen energy reserve The liver, like muscle, can store glucose energy

as a glycogen, but in contrast to muscle tissue it will sacrifice its stored glucose energy

to other tissues in the body when blood glucose is low (Figure 4)

Building Macromolecules

Although most absorbed glucose is used to make energy, some glucose is converted to ribose and deoxyribose, which are essential building blocks of important macromolecules, such as RNA, DNA, and ATP Glucose is additionally utilized to make the molecule NADPH, which is important for protection against oxidative stress and is used in many other chemical reactions in the body (http://2012books.lardbucket.org/books/an-introduction-to-nutrition/s08-03-the-

functions-of-carbohydrates.html, 2012)

Besides, they also important role as participants make enzymes such as 6- phosphate dehydrogenase (G6DH or G6PDH) is a cytosolic that catalyzes the chemical reaction Clinically, an X-linked genetic deficiency of G6PD predisposes a

Glucose-person to non-immune hemolytic anemia (Cappellini at al., 2008)

The sucrose blinding protein (SBP), has been implicated as an important component of sucrose uptake system in plan SBP –mediated sucrose transport displays unique kinetic features and the protein is not similar to other transport

proteins (Carlos et al., 2002)

Supposedly Spare Proteins

In a situation where there is not enough glucose to meet the body’s needs, glucose is synthesized from amino acids The presence of adequate glucose basically spares the breakdown of proteins from being used to make glucose needed by the body (http://2012books.lardbucket.org/books/an-introduction-to-nutrition/s08-03-the-

functions-of-carbohydrates.html, 2012)

Figure 4: The structure of glycogen enables its rapid mobilization into free

glucose to power cells.

1.2.2.2 The role of sucrose in food product

Sugar plays a major role in the production of thousands of food products from cured meats through preserves and frozen fruits to confections

All green plants manufacture sugar through photosynthesis, the process by which plants transform sunlight and soil nutrients into their food and energy supply Sugar is first and foremost used because of its sweetening properties It has a clean sweet taste with no aftertaste and is the reference against which other sweeteners are compared Beyond its sweetening properties, sugar provides structure and texture to many traditional foods, such as bakery products and jam Sugar helps to create crispness and texture in biscuits, and is central to the browning process which gives bread and pastries their traditional golden color and characteristic flavor

Moreover, the Sugar Association suggested that beyond its contributions as a sweetener and flavor-enhancer, sugar has other: Interacts with molecules of protein or starch during baking and cooking process Acts as a tenderizer by absorbing water and inhibiting flour gluten development, as well as delaying starch gelatinization Incorporates air into shortening in the creaming process Caramelizes under heat, to provide cooked and baked foods with pleasing color and aroma Speeds the growth of yeast by providing nourishment Serves as a whipping aid to stabilize beaten egg foams Delays coagulation of egg proteins in custards Regulates the gelling of fruit jellies and preserves Helps to prevent spoilage of jellies and preserves Improves the appearance and tenderness of canned fruits Delays discoloration of the surface of frozen fresh fruits Enables a wide variety of candies through varying degrees of recrystallization Controls the reformation of crystals through inversion (breakdown to fructose and glucose) Enhances the smoothness and flavor of ice cream (Sugar Association, 1991)

An important property of sugar is that it can enhance tastes and aromas both above and below the sweetness threshold value Sucrose plays an important role,

nutritionally, and otherwise According to (Mathlouthi and Cedus, 1995) the typical application effects of sucrose to food was presented below:

as principal leavening, which contain 30- 37% sugars One recent development in this area is the developing market in the USA for fat-free, or lowered fat, cakes and sweet breads, with formulations containing higher levels of sugar and oligosaccharides to supply bulk substitute for the missing fat

Cookies and sweet biscuits

Sucrose contributes flavor, taste, color, texture and tenderness to cookies

Icings and frostings

Sugars are essential in these products for flavor, taste, texture - depending

on grain size of the sugar - and appearance The purity and consistent composition of sucrose are important to the simple water-sugar icings most commonly used on baked goods

Beverages

Sugar provides sweetness, flavor and mouthfeel in carbonated soft drinks, and

in simulated juice-type beverages Low microbial level, and low levels of turbidity and non-sugars are important to protect the flavor and appearance of the beverage

Jams, jellies and preserve

The preservation effect, resulting from lowered water activity and high osmotic pressure, is important here to maintain texture of fruit and to prevent microbial contamination The properties of sweetness and flavor are also significant

Confectionery

In chocolate products, sucrose is preferred because of its range of grain size, solubility, and dispersant ability in fat, stability bulk and texture In non-chocolate confectionery, such as hard, boiled sweets or candy, the high viscosity of sugar solutions, and the solubility of sugars which allow them to remain in supersaturated solution, and provide a stable syrup phase (Flanyak (1991) and Jeffrey (1993)) are of significance

Dairy products

Simple sugars give the necessary freezing point lowering to maintain texture and product quality in ice creams and frozen desserts Too Iowa freezing point means too little water may be frozen: an icy texture can develop (Smith, 1990) Sucrose provides good dispersion among fats in ice creams, and increases viscosity in lower fat products Sugars are used for sweetness, flavor, preservation (high osmotic pressure) and texture in sweetened condensed milk Flavor, sweetness and texture are added by sugars to yogurt products Again, sugars are not fat substitutes, but are finding use in lowered fat products to increase bulk, flavor and texture or tenderness

Ready-to-eat breakfast cereals

Sugars added to cereals contribute flavor, color (browning characteristics)

and sweetness, but also give important textural modifications Sugars are added to doughs at levels from 6 to 25% Sugars act as binders, and can act

to increase crispness, spread, and surface porosity in much the same way as

in baked cookies

Meats

Sugar is often added in small quantities to cured, dried or preserved meats, especially to pork products The purposes of the sugars are: as a preservative, to lower water activity, and as a flavor enhancer, to contrast with salt and extend the meat flavor

Frozen and tinned vegetables

Sugars are sometimes added in small (sub-percent) levels to cooked tinned and frozen vegetables to enhance flavor and help preserve color and texture A well-known example is the addition of sugar to baked beans: the flavor and preservative effects of added sugar are significant to anyone who has tasted plain boiled or baked navy or pea beans without sauce, and the regular baked beans in sauce The addition of sugar-containing sauce causes a maximum increase of 0.8 g carbohydrate (or 3.2 kcal) per 100 g of baked beans (USDA, 1991)

• Role of sucrose in aroma and flavor of foods:

Sucrose interacts with food ingredients and in processed foods in many different ways The major flavor function of sucrose is, certainly, to sweeten food, but sucrose also influences the flavor quality of foods in diverse other way There is our basic taste sensations are recognized in sensory studies - sweet, sour, salty and bitter

In general, bitter, acid and salty tastes are suppressed in the presence of sucrose

Studies have shown that color has a strong effect on the perception of flavor, especially of fruit-flavored beverages (Kotyla and Clydesdale, 1978) Sweetness perception can be strongly affected by the type of color, especially red color (Johnson and Clydesdale, 1982) showed that the perception of sweetness in a dark red solution was 2-10% higher than in a light red solution, even though the sucrose concentration

in the dark red solution was actually 1% lower than in the light red solution They also showed that perceived sweetness increased approximately 2-12% as red color intensity increased at the same time that a constant 4% sucrose concentration was maintained

(Johnson et al., 1983)

1.2.3 Trade and economic

One of the most widely traded commodities in the world throughout history, sugar accounts for around 2% of the global dry cargo market International sugar prices show great volatility, ranging from around 3 to over 60 cents per pound in the past 50 years (see Figure 5)

Consumption of sugar ranges from around 3 kilograms per person per annum in Ethiopia to around 40 kg/person/year in Belgium Consumption per capita rises with income per capita until it reaches a plateau of around 35 kg per person per year in middle income countries

Figure 5: World raw sugar price for Calendar years 1969-2014

(From source: https://upload.wikimedia.org/wikipedia/commons/a/ae/World_raw_

sugar_prices_since_1960.svg.)

Sugar plays a central role as an additive in food production and food consumption all over the world Demand for sucrose still is continuing on the world below (Figure 6):

The rising pace of global consumption has been sustained by drawing down stock levels in recent years Consequently, stocks are approaching what appear to be historically low levels As prices react to high demand and as the Brazilian

real struggles to find equilibrium against the dollar, market returns are needed to provide incentives for producers to catch up with demand (USDA, 2016)

Figure 6: The chart showed global sugar consumption to again outpace

production, 2009/2010-2016/2017

(From source: USDA, Sugar: World Markets and Trade, 2016)

1.2.4 Situation research

In recent years, many analytical methods have been used for determining soluble carbohydrate content in diverse sample matrix such as the thin layer chromatography

(TLC) (Kuriyama et al., 1990) and high performance liquid chromatography (Kuo,

Vanmiddlesworth and Wolf (1988); Lowell and Kuo (1989)) such as:

Several studies have been conducted in soybean to analyze sucrose content by HPLC High pressure liquid chromatography (HPLC) may be preferable for separation

of larger oligosaccharides (larger than verbascose, a pentamer) but resolution of monosaccharides and separation of different cyclitols and different galactosyl cyclitols

are sometimes problematic when using HPLC (Obendorf at al., 2011) Analysis of

soluble carbohydrates by gas chromatography (Horbowicz and Obendorf, 1994) requires that pure authentic compounds be used as reference standards Fortunately, many soluble carbohydrates found in soybean seeds are commercially available

(Kadlec et al., 2001)

The HPAEC-PAD detection method has been reported as sensitive, fast, and capable of separating individual soluble sugars in soybean seed (Giannoccaro, Wang mand Chen, 2008)

Total sugar and individual sugar content in seed were determined by the liquid chromatography method of Hymowitz (Hymowitz and Collins, 1974)

gas-Also, the application of rapid analytical techniques such as near-infrared reflectance spectroscopy (NIRS) has many advantages compared to standard and traditional techniques such as chromatographic methods NIRS analysis is performed with considerable reduction in analytical time and cost, without using hazardous chemicals

(Hollung et al., 2005) had attempted with NIRS to predict the contents of

nonstarch polysaccharides (NSP) and oligosaccharides in 38 soybean samples simultaneously However, that result was useful for determination of total NSP,

unsoluble NSP, total uronic acid, and raffinosecontent and impossible to predict other low-molecularweight carbohydrates in soybean seeds

Besides, the sucrose content of the soybean seeds was also determined by the

enzymatic method published by (Stitt et al., 1989) Sucrose quantification by the

GOD/invertase method which developed requires basically a spectrophotometer

adapted for reading ELISA plates and low-cost reagent (Arlindo at al., 2012)

Although methods mentioned above usually offer accuracy result, they are time-consuming, expensive, and labor-intensive, therefore, not advantageous for the selection of superior soybean varieties and the quality control soybean products

Thus, rapid and accurate methods for the evaluation of quality traits of soybean seeds are in high demands for the soybean breeding program So, we chose a fast and simple method for the analysis of sugar using the Megazyme Sucrose/D-Glucose Test Kit (by the GOPOD/invertase or called enzymetic method) employs high purity glucose oxidase, peroxidase and β-fructosidase (invertase) and can be used with confidence for the specific measurement of D-glucose and sucrose in plant and food

extracts It is an unexpensive alternative for sucrose quantification analyses in soybean

breeding programs and can be easily adapted to other species, allowing low cost scale analyses

large-1.2.5 Chemical Mutagenesis

The using chemical mutagenesis is one of the best technologies in soybean production Mutations can be artificially induced in plants by chemical mutagens to

produce new superior lines from traditional (Henffner at al., 2009) Unlike

transgenic breeding the resulting lines used to make cultivars can be considered Non-GMO’s which gives it an upper hand intoday’s marketplace (Kavithamani, 2010) One of the most common chemical mutagens is Ethyl Methane Sulfonate

(EMS) because it results in a higher frequency or irreversible mutations (Van et al.,

2000) In mutation breeding the chemical mutagen is used to alter the DNA of the plant to cause random genetic mutations Using chemical mutagenesis in the search

for elevated high sucrose profiles in the soybean seed is on the rise with the demand for soybean sugar continuing to increase

Ethyl methane sulfonate (EMS) is a mutagenic, teratogenic, and possibly carcinogenic organic compound with formula C3H8SO3 It produces random mutations in genetic material by nucleotide substitution; particularly

by guanine alkylation This typically produces only point mutations It can induce mutations at a rate of 5.10-4 to 5.10-2 per gene without substantial killing (https://en.wikipedia.org/wiki/Ethyl_methanesulfonate)

1.3 Research Objective

As mention above: Both consumer demand for soybean and sucrose continued and still is continuing quickly In addition to, sucrose (sugar) content in soybean are relatively low Especially, sucrose play an important role in food industry such as: confectionery, cream, freshwater, milk…Even if, it play important functions in the body Moreover, sucrose one of the sugars found, is easily digestibility whereas raffinose and stachyoest

Thus, enhancing and improving the natural sucrose content in soybeans is necessary to meet demand and reduce lower product prices The objective of this study was to screen and select high sucrose mutant soybean lines among lines derived from a mutation population The selected lines will be used to understand genetics of high sucrose in soybean seed and to improve soybean variety

II MARERIAL AND METHODS

2.1 Material and equipment

2.1.1 Plant Material

To improve quality sucrose from cultivar Pungsanamul (M1) which conducted

by 0.3 % EMS treatment of the seeds of a soybean wild cultivar Pungsannamul (Chae

et al., 2013) in the Korean environment, the seeds which were planted at Affiliated

Experiment and Practice fields of Kyungpook National University (Gunwi, 36˚07’ N, 128˚38’ E, Republic of Korea) in 2010 The seeds were harvested at maturity from each plant Obtaining a mutation population there were 3,774 lines (M2) Subsequently, the M2 air-dried then stored in basement under condition 4 ˚C Both 710 mutant lines

of those 3774 lines (M2) and the soybean cultivar Pungsannamul were utilized to determine sucrose concentration using Megazyme Kit (Enzymatic method) (see Figure7)

2.1.2 Equipment and chemical

• The list of equipments:

Oven set at 50-70°C PCR plate

Poly lid Shinil Electric Spec plate Spectrophotometer set at 510 nm

Standard testing sieve

96 deep well block

• Chemical:

50% Ethanol (see appendices)

Megazyme kit (see appendices)

Figure 7: Schema displayed procedure of plant material

2010 Pungsanamul (wild soybean)

2.2 Method

2.2.1 Carbohydrates extraction

In envelopes, 10-15 randomly selected seeds as a composite from each sample Then, those seeds were ground to form the powder by a Shinil Electric mixer (SFM-555SP, Korean) and screened through a 300 µm standard testing sieve (Figure 8)

Figure 8: Soybean powder were ground and filtered by standard testing sieve

Subsequently, seeds were kept in a paper envelope which were marked each concrete label

Carbohydrates extraction was conducted following the procedure

Weight of 0.0125 grams soybean flour into a 2mL Eppendorf microcentrifuge tubes The soybean carbohydrate was extracted by keeping the soybean power in 1 mL extraction solution (Ethyl Alcohol, distilled water [50:50, v/v]) The mixture was vortexed by vortex mixture and incubated at 70°C in an oven (Vision, Scientific, Co, LTD, Korean) for 30 minutes, shaking every 10 minutes The suspension was then centrifuged at 15000 rpm for 10 minutes (Figure 9)

B

Figure 9: Extracted the sugars from sample using ethanol 50% in oven (A) and

after centrifugal finish (B)

Using pipette 1mL was adjusted down 700µL volume to transfer 700 µL of clear supernatant into a new 96 deep well block (if not using right away, seal plate with non-slit silicone lid in the refrigerator at minus 4 °C) This extract was used for the sucrose determination by the enzymatic method, developed in this study

2.2.2 Sucrose quantification by the GOPOD/invertase method (enzymatic method)

In this experiment was used a method to quantify sucrose in soybean seeds with potential use in breeding programs, which enables large-scale, low-cost analyses to be carried out The Megazyme Sucrose/D-Glucose Test Kit (Figure 10) employs high purity glucose oxidase, peroxidase and β-fructosidase (invertase) and can be used with confidence for the specific measurement of D-glucose and sucrose in plant and food extracts Sucrose and D-glucose are two of the most commonly occurring sugars in plant and food products such as: sugar cane, beet, soybean…, and have dramatic

impacts on human nutrition

A

Figure 10: The Megazyme Sucrose/D-Glucose Test Kit The principle

At pH 4.6, sucrose is hydrolysed by the enzyme β-fructosidase to D-glucose and D-fructose (Equation 1)

Free D-glucose in the sample extract is determined by conversion to a red coloured quinoneimine dye compound through the action of glucose oxidase (Equation 2) and peroxidase (Equation 3) at pH 7.4, and employing p-hydroxybenzoic acid and 4-aminoantipyrine

The determination of D-glucose after inversion (total D-glucose) is carried out simultaneously according to the principle outlined above The sucrose content is calculated from the difference of the D-glucose concentrations before and after

enzymatic inversion

Equation 1

Equation 2

Equation 3

Preparation of reagent solutions/suspensions:

Bottle 3 (GOPOD Reagent Buffer) was diluted to 1 L with distilled water

Note: (see appendices)

The contents of bottle 4 dissolved in 20 mL of solution 3 and quantitatively transfer this to the bottle containing the remainder of solution 3 This bottle was covered with aluminium foil to protect the enclosed reagent from light This is Glucose Determination Reagent (GOPOD Reagent) Stable for ~ 3 months at 2-5°C or > 12

months at -20°C

This reagent is to be stored in the frozen state, it should be divided into aliquots

to be convenient employment (e.g 200 mL in polypropylene containers) Do not freeze/thaw more than once

When the reagent is freshly prepared it may be light yellow or light pink in colour It will develop a stronger pink colour over 2-3 months at 4°C The absorbance

of this solution should be less than 0.05 when read against distilled water

Sucrose analyses by the GOPOD/invertase method

Intake 100 µL of solution extraction above to a new clear PCR 96 well plate by pipette 100 µL volume contained 100 µL distilled water and mixing up by pipette Similarly, transfer 10 µL diluted solution above to a novel 96 well PCR plate with the invetase was prepared at a 10 µL of bottle 2 contain β-Fructosidase (invertase) solution (yeast; 5 mL) plus sodium azide as a preservative (0.02% w/v) Stable for > 2 years at

4°C) Note: after utilizing preservative in fridge right away

After that, the plate was then sealed and incubated in an oven (Vision, Scientific, Co, LTD, Korean) 50°C for 20 min After this period, the plate was removed from the oven and 150 µL out of 1L of GOPOD (Megazyme kit consist of Bottle 3: GOPOD Reagent Buffer and Bottle 4: GOPOD Reagent Enzymes Glucose oxidase plus peroxidase and 4-aminoantipyrinereagent were been mixed previously) was added, and the plate was sealed again by clear poly lid (spec plate lid) and placed

in an oven (Vision, Scientific, Co, LTD, Korean) 50°C for 20 min and mixing up by

pipette Note: Cover this bottle with aluminium foil to protect the enclosed reagent from light, after utilizing preservative in fridge right away at 4°Cl

After this time, the plate was removed from the oven (Vision, Scientific, Co,

LTD, Korean) transfer 125 µL to spec plate The absorbance at 510 nm was read in a spectrophotometer, equipped for reading spec plates (See Figure 11)

A B

C

Figure 11: A The carbohydrates extraction poured invertase and GOPOD in oven at

50°C

B That solution (A) was transferred to new spec plate

C The solution (B) were read by spectrophotometer at 510 nm

Determination standard curve

Standard D-glucose solutions were also added to each plate in separate wells at

concentrations 0, 62.5 mg/mL, 125 mg/mL, 187.5 mg/mL, 250 mg/mL corresponding

with 0%, 25%, 50%, 75%, 100%, respectively This allowed a calibration curve to be

constructed which was used to determine the sucrose concentration in each sample

Duplicate each standard solutions preparation:

100% = 0.1mL D-Glucose standard solution in bottle 5 (1mg/mL) + 0.3 mL of distilled water

75% = 75 µL of 100% + 25 µL distilled water

50% = 50 µL of 100% + 50 µL distilled water

25% = 75 µL of 100% + 75 µL distilled water

0% = 100 µL distilled water

Blank = 80 µL distilled water + 600 µL GOPOD

Add 20 µL of the standard and 170 µL of the blank and the plate was sealed

again by clear poly lid (spec plate lid) and placed in an oven (Vision, Scientific, Co,

LTD, Korean) 50°C for 20 min and mixing up by pipette Note: Cover this bottle with

aluminium foil to protect the enclosed reagent from light, after utilizing preservative in

fridge right away at 4°Cl

After this time, the plate was removed from the oven (Vision, Scientific, Co,

LTD, Korean) transfer 125 µL to spec plate The absorbance at 510 nm was read in a

spectrophotometer, equipped for reading spec plates

Bottle 5 was diluted by distilled water at different concentration 0, 62.5, 125,

187.5, 250 mg/mL then measured at 510nm The equation was built by above

concentration with form:

y = 0.013x+0.019 and R2=0.9889 (Figure 12)

Figure 12: The chart displaying standard curve of D-Glucose standard solution

mg

(g/100g)

Where: 342/180 = conversion from µg of D-glucose (as measured) to µg of sucrose