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The objective of this research was to isolate and characterize soybean genes involved in phytic acid metabolism for use in genetic engineering strategies to improve phosphorus utilization. A soybean phytase from germinated cotyledons was purified 28,000-fold to apparent homogeneity and was determined to be a glycosylated homodimer with 70 kD subunits. Soybean phytase preferred phytate as substrate (Km = 60 mM) and was capable of removing of all six phosphate groups from phytate. The pH and temperature optima for soybean phytase activity were 4.5 and 58*C, respectively. The N-terminus and four internal peptides from the purified soybean phytase were sequenced by Edman degradation. The amino acid sequence data were used to design degenerate oligonucleotide primers for PCR amplification of the soybean phytase coding sequence. A protein 547 amino acids in length was predicted from the 1641 bp coding sequence. The phytase protein showed significant similarity to plant purple acid phosphatases (PAPs) and contained the conserved metallo-phosphomonoesterase active site motif. The soybean phytase coding sequence was placed under the control of a constitutive 35S CaMV promoter in a soybean biolistic transformation vector and was introduced into "Williams 82" suspension culture cells by particle bombardment. Stably transformed cell suspension lines were recovered. DNA blot analysis demonstrated that the recombinant soybean phytase coding sequence had integrated into the genomes of two cell lines. Expression of the transgene was confirmed by RNA blot analysis. Phytase activity was three to four fold higher in these two lines compared to control non-transformed cultures. A soybean L-myo-insoitol-1-phosphate synthase (MIPS) cDNA was isolated from total RNA from developing seeds. The protein encoded by the soybean MIPS cDNA showed 87-91% homology to MIPS protein sequences from other plant species. RNA blot analysis of staged developing soybean seeds revealed that MIPS is transcribed early in the cotyledonary stage of development. Compared to other soybean tissues, MIPS expression levels were highest in developing seeds. DNA blot analysis demonstrated that multiple copies of the MIPS gene are present within the soybean genome. / Ph. D.

Soybean is an integral oilseed crop that is used all over the world. Despite this, soybean has been experiencing a decrease in seed quality seen by the decreasing trend in test weight, and is limited in its uses due to the antinutritional factor, trypsin inhibitor (TI). This project first worked to identify single nucleotide polymorphisms (SNPs) associated with high test weight through a genome-wide association study (GWAS) as well as determine the relationship between important seed composition traits and test weight. Additionally, it focused on identifying and knocking out KTI genes specifically expressed in soybean seed tissue through CRISPR/Cas9. Lastly, this project investigated further use of soybean in aquaculture by evaluating the performance of Rainbow trout fed diets with low-TI, 'VT Barrack' soy meal. Nine SNPs on chromosome 15 were found to be significantly associated with high test weight as and eight potential gene candidates were identified. Test weight was found to be significantly, and negatively related to seed oil content and had inconsistent correlations with protein and sugar content. The KTI genes KTI1, Glyma01g095000, and KTI3, Glyma08g341500, were found to be only expressed in seed tissues and multiple KTI knock-out soybean plants with decreased seed TI content and activity were developed. Lastly, we found that our low-TI soymeal could replace 30% of fish meal in aquafeeds with no negative effects on trout growth and health. / Doctor of Philosophy / Soybean is an essential and lucrative crop due to its countless uses. Despite its importance and flexibility, it contains antinutritional factors, such as trypsin inhibitors (TI), and has been experiencing a decline in test weight, the amount of soybean seed in kilograms per hectoliter, that can limit the profits that growers receive. This dissertation seeks to contribute to the development of improved soybean varieties in addition to evaluating current low-TI varieties' application in aquaculture as a substitute for fish meal. A genome-wide association study (GWAS) was performed to identify molecular markers associated with high test weight and the relationship between high test weight and important seed composition traits was determined. Gene editing was conducted to knock-out expression of Kunitz trypsin inhibitor (KTI) genes specifically expressed in seed tissue. A fish feeding trial was conducted to evaluate the performance of Rainbow trout-fed diets using our low-TI soybean cultivar, 'VT Barrack'. We were able to successfully identify multiple molecular markers associated with high test weight as well as identify multiple candidate genes. We were able to determine that high test weight has a strong, negative relationship with seed oil content. We identified two KTI genes for gene knockout and were able to successfully develop a low-TI soybean line that has limited TI in just seed tissue. Lastly, we found that our low-TI soymeal could replace 30% of fish meal in aquafeeds with no negative effects on trout growth and health.

Soybean [Glycine max (L.) Merr.] seed cost has increased dramatically with the introduction and adoption of herbicide-resistant cultivars, generating interest from growers to reduce seeding rates to the lowest possible level that does not affect yield. Research indicates that greater seeding rates are needed to maximize yield under low-yielding environments and less seed is needed in high-yielding environments, but this has not been confirmed with recent research in Virginia. The objectives of this research was to 1) determine the yield response of soybean cultivars with differing growth habits and maturities grown in full-season and double-crop systems to seeding rate under different yield environments; and 2) compare two seeding rates in large on-farm strip-plots to determine if the growth environment within the field affects the yield response to seeding rate. For objective 1, small-plot research was conducted on Piedmont and Coastal Plain sites across Virginia from 2017 thru 2020. Maturity group (MG) 4 or 5 cultivars were planted in 46-cm rows at the following seeding rates: full-season soybean – 74,130, 148,260, 222,390, 296,520, 370,650, and 444,780 seed ha-1; and double-crop soybean – 197,680, 296,520, 395,360, 494,200, 543,620, and 593,040 seed ha-1. One cultivar per MG was used in 2017 and 2018, but the experiments were expanded to include two cultivars, differing in canopy structure, within each MG in 2019 and 2020. On-farm research compared a high and low seeding rate with a 100,000 seed ha-1 difference based upon grower current practices. To determine growth influence on the yield response, normal difference vegetative index (NDVI) was measured at 2-week intervals from late-vegetative to late-reproductive stages in small-plot and on-farm experiments. Double-crop soybean required an average of 205,000 more seed ha-1 than full-season soybean. Although yield response varied with site and year, MG 4 cultivars usually yielded more than MG 5 at higher seeding rates, but less at lower seeding rate. No differences between cultivar canopy structure were present in full-season systems; differences were revealed in double-crop systems but were not consistent over sites. To obtain 95% of maximum yield, 170,000 to 390,000 seed ha-1 were required in full-season soybean and 470,000 to 550,000 seed ha-1 were required in double-crop soybean. While the NDVI response to seeding rate generally reflected the yield response at most site-years, relationship between yield and NDVI was weak. In on-farm experiments, higher seeding rates yielded more at 3 of 6 sites, but differences varied within the field. The yield-NDVI relationship was stronger due to greater variability within the field, but these differences due to seeding rate could not be discerned. Growing environment, primarily amount and distribution of rainfall, greatly influenced these results; therefore, more exact site-specific seed rate recommendations will be difficult in Virginia's environment. / Master of Science / Soybean is one of the world's most important seed legumes and contributes a major portion of global protein concentrate for livestock feeding and oil for human consumption therefore, it is essential to continually and sustainably increase yield while maintaining profitability for growers. Through adoption of precision agriculture technology by Southeastern farmers, site-specific management using variable rate application and/or automatic section control of inputs are able to be utilized when field variability is assessed. Additionally, on-farm experiments validate small plot research with larger field-scale evaluations with proper technology and design. In this study various seeding rates on soybean yield in both small-plot and on-farm experiments, as influenced by variable growth and yield differences within the field, was determined. On-farm research allowed better observation of yield response to seeding rate for each field, allowing the evaluation of production practices under realistic growing conditions. Ultimately, it is expected that more early-season growth in areas of the field with greater productivity will result in greater yields using less seed per hectare. The utilization of vegetative indices as well as remote sensing technology contributed largely to the analysis of yield and varying seeding rates. Accordingly, measurements were correlated with one another to determine if remote sensing techniques can be substituted for ground measurements in predicting yields.

Thesis (Ph.D)-University of KwaZulu-Natal, Pietermaritzburg, 2008. / Full text also available online. Scroll down for electronic link.