Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Plant and Soil Sciences

First Advisor

Dr. David Hildebrand

Abstract

New soybean lines have been developed with significantly higher oil, protein + oil and higher meal protein. These soybeans contain a VgD1 gene (highly active acyl-CoA:diacylglycerol acyltransferase, DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in soybean. Soybean with active DGAT from Vernonia galamensis (VgDGAT1A) has active TAG biosynthesis relative to other DGATs including from soybeans and Arabidopsis. DGATs catalyze the final step of TAG synthesis: DAG (diacylglycerol) + acyl-CoA → TAG + CoASH (Coenzyme A is notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle). A thorough analysis of the major components in VgD1 lines, especially those of nutritional or anti-nutritional value including what else changed (decreased); and what remained at normal levels was conducted. A field study was conducted in Spindletop and Princeton KY, reviled no reduction in yield nor protein, and about 4 % (DW) more oil was obtained in Princeton and 2% (DW) in Spindeltop. No consistent reduction in the other seed composition.VgDGAT1A soybean lines indicated noticeably early maturation compared to the parental line. This is associated with higher expression of the flowering genes FT2 (FLOWERING LOCUS T2) and FT5 (FLOWERING LOCUS T5), for the high oil lines. A single recessive mutation in soybean (MIPS) myo-inositol 1-phosphate synthase, confers a seed phenotype of increase inorganic phosphate (Pi) crossed with high oil lines expressing a DGAT from Vernonia galamensis (VgDGAT1A) (VgD). The oil and protein were maintained compart to VgD. VgD X MIPS (VM), had 21.2, and 22 % oil in 2015, and 23.3 and 24.0 oil in 2016, and protein 46, 49 in 2015, and 37 and 39 % in 2016. Phosphate results suggesting the cross MV is still segregating for MIPS and more selection and planting are needed.

Measurement of seed phosphate levels is an established technique for screening for low phytate mutants but to date, it has not been performed non-destructively from single soybean seeds. A protocol was developed greatly reducing the sample size thereby reducing the cost and time and saving a generation in the selection of low phytate mutant seeds based on the high Pi phenotype.

Genotyping single seeds are useful in breeding and genetics while maintaining high germination rates. Nondestructive single-seed genomic DNA extraction protocols using 12 mg cotyledon tissue with a modified cetyl trimethyl ammonium bromide (CTAB) technique and a commercial seed DNA extraction kit using 1 mg cotyledon tissue were developed for dry soybean seeds and cross-verified with leaf DNA analysis.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.433

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