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 BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2012 – 2016 Thai Nguyen, 2016/08/20 n 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 BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2012 – 2016 Supervisors : Prof. Lee Jeong-Dong ______________ Ph.D Duong Van Cuong _____________ Ph.D Pham Bang Phuong ____________ Thai Nguyen, 2016/08/20 n Thai Nguyen University of Agriculture and Forestry Major Biotechnology Student name Duong Thi Bich Huong Student ID DTN1253150047 Thesis Title Selection high sucrose soybean lines from a mutation population derive from EMS treatment. Lee Jeong 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 2.2%, raffinose ranges from 0. Sucrose content in soybean seeds is desired to be high because it is a sweetness-imparting component, it helps extends acceptance for soy- 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.
Finishing the preliminary analysis, as a results we obtained 390 lines with sucrose content of larger than that of Pungsanamul. The remaining 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 (21.818 g/100g) were confirmed with extremely low concentrations. Pungsanamul was identified around 16. 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 Number of pages 63 Date of Submission 2016/08/20 i n 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. ii n 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. 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 iii n CONTENTS ACKNLOWLEDGMENTS .iv LIST OF FIGURES .v LIST OF TABLES .vi LIST OF ABBREVIATION. The function of sucrose.
The role of sucrose in the body. The role of sucrose in food product. Trade and economic. MARERIAL AND METHODS.1 Material and equipment .2 Equipment and chemical .2 Sucrose quantification by the GOPOD/invertase method (enzymatic method).
RESULTS AND DISCUSSION.1 The soybean seeds treated by EMS. Determination carbohydrates extraction .3 Preliminary evaluation of the sugar content .4 Selection of high sucrose content lines .46 iv n 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. 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. 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 : v n 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 vi n LIST OF ABBREVIATION ATP Adenosin triphosphate BU Bushel DN Deoxyribonucleic acid EMS Ethyl Methane Sulphonate ELISA Enzyme-Linked ImmunoSorbent Assay FAO Food and Agriculture Organization FDA Food and Drug Administration g Gram G6DH Glucose-6- phosphate dehydrogenase (or G6PDH) GO Glucose oxidase GOPOD Glucose oxidase peroxidase HPAE-PAD High-Performance Anion-Exchange Chromatography-Pulsed Amperometric Detection HPLC High pressure liquid chromatography mL Milliliter N Nitrogen NADPH Nicotinamide adenine dinucleotide phosphate NIRS Near-infrared reflectance spectroscopy NSP Nonstarch polysaccharides Rep Replication PCR Polymerase Chain Reaction RMP Revolutions Per Minute Pungsan Pungsanamul RNA Ribonucleic acid SBP Sucrose blinding protein TLC Thin layer chromatography µL Microliter USA United States Dollar USD The United States of America USDA United States Department of Agriculture °C Celsius degree °F: Fahrenheit degree % Percent vii n I. Soybean Cultivated soybean [G.) 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.
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 1 n 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.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 2.2%, raffinose in the range 0.9%, stachyose in the range 1.1% (Hymowitz et al. 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: 2 n 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. 8 million metric tons, respectively (USDA, 2016).75 USD/BU or 2.25 on Friday June 17 to 1134.50 in the previous trading session.8 USD/BU or 18.71 percent during the last 12 months from 976.50 USD/BU in June of 2015 (http://www.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.