University of Kentucky UKnowledge Theses and Dissertations--Plant and Soil Sciences Plant and Soil Sciences 2017 INCREASING RENEWABLE OIL CONTENT AND UTILITY William Richard Serson University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2017.243 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Serson, William Richard, "INCREASING RENEWABLE OIL CONTENT AND UTILITY" (2017). Theses and Dissertations--Plant and Soil Sciences. 89. https://uknowledge.uky.edu/pss_etds/89 This Doctoral Dissertation is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant and Soil Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. 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William Richard Serson, Student Dr. David Hildebrand, Major Professor Dr. Arthur Hunt, Director of Graduate Studies INCREASING RENEWABLE OIL CONTENT AND UTILITY ________________________________ DISSERTATION ________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky By William Richard Serson Lexington, Kentucky Director: Dr. David Hildebrand, Professor of Plant and Soil Sciences Lexington, Kentucky 2017 Copyright © William Richard Serson 2017 ABSTRACT OF DISSERTATION INCREASING RENEWABLE OIL CONTENT AND UTILITY Since the dawn of agriculture man has been genetically modifying crop plants to increase yield, quality and utility. In addition to selective breeding and hybridization we can utilize mutant populations and biotechnology to have greater control over crop plant modification than ever before. Increasing the production of plant oils such as soybean oil as a renewable resource for food and fuel is valuable. Successful breeding for higher oil levels in soybean, however, usually results in reduced protein, a second valuable seed component. We show that by manipulating a highly active acyl-CoA: diacylglycerol acyltransferase (DGAT) the hydrocarbon flux to oil in oilseeds can be increased without reducing the protein component. Compared to other plant DGATs, a DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in yeast, insect cells and soybean. Soybean lines expressing VgDGAT1A show a 4% increase in oil content without reductions in seed protein contents or yield per unit land area. Furthermore, we have screened a soybean fast neutrino population derived from M92-220 variety and found three high oil mutants that do not have reduced levels of protein. From the F2 plant populations we quantitatively pooled the high oil and low oil plants and performed comparative genomics hybridization (CGH). From the data it appears that two families have a 0.3 kb aberration in chromosome 14. We are performing further analysis to study this aberration and develop markers for molecular breeding. Mutagenic techniques are also useful for developing other traits such as early flowering varieties and adapting new high oil crops to a new region. Chia (Salvia hispanica) is an ancient crop that has experienced an agricultural resurgence in recent decades due to the high omega 3 fatty acid (ω-3) content of the seeds and good production potential. The area of cultivation has been expanded to Kentucky using mutagenized populations and the composition traits are similar to that of the original regions of cultivation in Central and South America. KEYWORDS: diacylglycerol acyltransferase, triacylglycerides, mutagenesis, comparative genomics hybridization, chia, soybean ____William R Serson June 28, 2017 INCREASING RENEWABLE OIL CONTENT AND UTILITY By William R. Serson David Hildebrand________ Director of Dissertation Arthur Hunt ___________ Director of Graduate Studies June 28, 2017___________ TABLE OF CONTENTS LIST OF TABLES ..................................................................................................................... v LIST OF FIGURES .................................................................................................................. vi Chapter 1 INTRODUCTION ..................................................................................................... 1 Soybean Oil Content and Composition .................................................................................. 1 Chia: Salvia hispanica .......................................................................................................... 10 Chapter 2 VgDGAT1A INCREASES RENEWABLE OIL CONTENT AND OLEIC ACID CONTENT IN SOYBEANS (Glycine max) ............................................................................ 20 Introduction .......................................................................................................................... 20 Materials and Methods ......................................................................................................... 22 cDNA cloning .................................................................................................................. 22 Expression in insect cells ................................................................................................. 23 Yeast microsome assays .................................................................................................. 23 Lipid analysis ................................................................................................................... 24 Seed-specific expression vector construction and soybean somatic embryo transformation .................................................................................................................. 26 Segregating Line Analysis ............................................................................................... 28 Results .................................................................................................................................. 28 In vitro assays of VgDGAT1A ........................................................................................ 28 Oil and protein content of transgenic soybeans expressing VgDGAT1A ....................... 29 Segregating Line Analysis ............................................................................................... 30 Fatty acid composition in high oil lines ........................................................................... 30 Discussion ............................................................................................................................ 31 Chapter 3 A LARGE COPY NUMBER VARATION ON CHROMOSOME 14 APPEARS TO INCREASE OIL CONTENT IN SOYBEANS ................................................................. 41 Introduction .......................................................................................................................... 41 Materials and Methods ......................................................................................................... 43 Genetic Material ............................................................................................................... 43 Backcrossing .................................................................................................................... 43 Tissue Sampling, Seed Composition and DNA Extraction ............................................. 43 NMR Methods ................................................................................................................. 44 CGH Analysis .................................................................................................................. 44 Results .................................................................................................................................. 44 Oil and Protein Content of Backcrosses .......................................................................... 44 CGH Analysis .................................................................................................................