Soybean meal has been widely used in animal feed, including swine, poultry,
cattle, and even fish. However, soybean has several anti-nutritional factors such
as trypsin inhibitors, phytic acid, raffinose family of oligosaccharides, and
antigenic factors that prevent animals’ protein digestibility. Processing of
soybean meal requires precise control of moisture content and temperature in
order to denature those anti-nutritional factors. Those extra processing steps
add cost to soybean meal production. For example, roasting is applied on raw
soybean meal to inactive trypsin inhibitors, so the feed cost becomes higher due
to higher energy cost; and synthetic phytase is added to soybean meal to
increase the bioavailability of phosphorus. The most economical and reliable way
to improve animals’ protein digestibility is to feed them with soybean meals
containing low concentrations of anti-nutritional factors. However, no such
commercial soybean variety is available for growers and end users. This
proposed study will help to increase Virginia soybean growers’ feed market
share since VA farmers will first have access to the value-added varieties adapted
to Virginia. Our long-term goal is to improve seed quality attributes for meal
markets using new and improved soybean varieties that bolster the economic
capabilities of producers. Our overall objective is to develop soybean varieties
and germplasm with nutritional bundles such as high protein and low trypsin
inhibitors. In previous years (June 2019 to July 2023) we developed a low-TI
transgene-free line from Williams 82 using the gene-editing CRISPR-Cas9
technology. This transgene-free line (#5-26) has low Kunitz Trypsin inhibitor (KTI)
content and activity. The specific objectives of this proposal are to 1) discover
the relationship between the treatment of phytopathogens and expression levels
of TI genes, 2) conduct field evaluation of edited low-TI lines, 3) perform a short
shrimp feeding trial using raw meal of edited low-TI lines, and 4) develop
soybean breeding lines with nutritional bundles including low trypsin inhibitor
(TI) and other nutritional traits, and disease resistance package. Our expected
outcomes include new and improved breeding lines and cultivars with desirable
nutritional profiles that enable the improvement of protein digestibility using
both genome editing and traditional breeding methodologies. These outcomes
will have a positive impact on the soybean industry by adding value to current
soybeans, expanding market share, and enhancing U.S. soy value proposition in
key feed markets in the world.
We have initialized trypsin inhibitor research and breeding projects since 2014.
We have developed and advanced high protein, low trypsin inhibitor, low
phytate and low raffinose family of oligosaccharides soybean lines. We also have
developed a simple and accurate HPLC methods to detect and quantify KTI and
BBTI and a SNP marker that are used for marker-assisted selection for low KTI. In
2019, we incorporated the genome editing system CRISPR-Cas9, to edit KTI
genes(s) in order to have a super low or null TI lines for variety development.

The overall goal is to develop soybean varieties and germplasm with nutritional
bundles such as high protein and low trypsin inhibitors (TIs).
Specific Objectives are
i. TI involvement in pest resistance to discover the relationship between the
treatment of phytopathogens and expression levels of TI genes.
ii. Field evaluation of edited low-TI lines. The evaluation included field trials for
agronomic traits and pest evaluation, including nematodes.
iii. Perform a short shrimp feeding trial using raw and low-heated meals of edited
low-TI lines
iv. Keep developing low TI lines bundled with traits of interest to develop
advanced low trypsin inhibitor (LT), high protein, high oil, high sucrose, modified
fatty acids breeding lines with disease resistance package.

1) Advanced breeding lines
? Low TI transgene-free lines will be tested in VA for yield trials in 2023-2024.
? Populations with low TI and other beneficial traits will be developed.
2) Low-TI homozygous lines will be obtained as parental lines to cross with.
3) Publication(s) and presentation(s)
? A manuscript of low TI transgene-free line pest/disease resistance will be
submitted to Crop Science in 2024-2025.
? A manuscript of low TI transgene-free lines as dietary ingredients in shrimp
feed will be presented at a professional conference and submitted to the journal
Aquaculture in 2024-2025.
? The studies of field trials of low TI transgene-free line, TI measurement and
disease resistance, and genetic markers will be presented at an annual
professional conference in 2024-2025.
? Also, the studies described above will be presented at an annual soybean field
day in VA 2025.