Soybeans major economic value owes to its use as a protein source in animal feed mixtures, as well as high seed yield, relatively high biotic stress tolerance and its ease of agricultural rotation with cereal crops. Despite these boons, soybean meal cannot be directly used for animal feed mixtures due to the presence of various anti-nutritional compounds which reduce animal weight gain. There are two major proteinaceous anti-nutritional factors in soybean seeds which are named after the individuals who identified them: 1) Kunitz-trypsin inhibitors (KTi) which inactivate the animal digestive enzyme trypsin; and 2) Bowman-Birk inhibitors (BBi) which inactivates both trypsin and chymotrypsin animal digestive enzymes. Our group is proposing a three-year project which will culminate in development of adapted soybean germplasm which bears drastically reduced (ideally eliminated) major proteinaceous anti-nutritional Kunitz-trypsin inhibitors and Bowman-Birk inhibitors. We have recently developed soybean germplasm that has the lowest levels of trypsin inhibitor reported for conventional soybeans. However, it still retains significant levels of trypsin inhibitor activity, due to the presence of BBi proteins. Therefore, it is essential to remove both KTi and BBi for the purpose of developing soybean lines that would require extremely low or no processing. The proposed grant has the potential to serve farmers by increasing the seed value of soybean and potentially allowing use of raw of soybean meal in current markets (swine, poultry). Drastically lowering or eliminating both KTi and BBi may also open up new markets currently served by costly fishmeal.

Updated March 2, 2022:
Soybean meal cannot be directly used for animal feed mixtures due to the presence of various anti-nutritional compounds which reduce animal weight gain. There are two major proteinaceous anti-nutritional factors in soybean seeds which are named after the individuals who identified them: 1) Kunitz-trypsin inhibitors (KTi) which inactivate the animal digestive enzyme trypsin; and 2) Bowman-Birk inhibitors (BBi) which inactivates both trypsin and chymotrypsin animal digestive enzymes. Our group is attempting to drastically reduce (ideally eliminate) major proteinaceous anti-nutritional Kunitz-trypsin inhibitors and Bowman-Birk inhibitors. During this quarter the following activities were carried out:

1. Have identified several BBi genes that are specifically expressed in soybean seeds by RT-PCR.
2. Nucleotide and amino acid sequence analyses reveal significant sequence identity among the BBi genes.
3. Target sequences suitable for CRISPR-CAS9 editing that will result in the elimination of abundant BBi genes have been identified.
4. Currently working with Wisconsin Crop Innovation Center, Wisconsin to initiate soybean transformation.
5. USDA-ARS Technology Transfer Coordinator has requested some changes to be made to the Service Agreement Terms before the soybean transformation can be initiated. Currently, we are waiting for this agreement to be worked out.

Updated March 2, 2022:
Soybean meal cannot be directly used for animal feed mixtures due to the presence of various anti-nutritional compounds which reduce animal weight gain. Our group is attempting to drastically reduce (ideally eliminate) major proteinaceous anti-nutritional Kunitz-trypsin inhibitors and Bowman-Birk inhibitors. During this quarter the following activities were carried out:
Finally, USDA-ARS Technology Transfer Coordinator and Wisconsin Crop Innovation Center has worked out the Service Agreement Terms. However, due to COVID-19 pandemic, the Wisconsin Crop Innovation Center has not yet initiated the soybean transformation work. It is anticipated that the Wisconsin Crop Innovation Center will commence this project once the restrictions enforced due to COVID-19 pandemic is lifted. In the meantime, we are pursuing soybean transformation for the creation of CRISPR soybean lines at the University of Missouri Transformation Facility as well.
In addition to the CRISPR approach, my lab with funding from USDA-ARS is also pursuing RNAi approach for down-regulating the expression of Bowman-Birk inhibitors from soybean seeds. For this purpose, we have created RNAi constructs that either contain a 168 bp or 286 bp region of the Bowman-Birk inhibitor sequences cloned in an inverted repeat orientation and separated by the intron from pKannibal. These RNAi constructs are either under the control of the soybean ß-conglycinin a'-promoter or the native Bowman-Birk inhibitor promoter. These constructs also contain a gene expression cassette that includes the cauliflower mosaic virus 35S promoter, the bar-coding region and the 3' region of the nopaline synthase gene (nos). Using these constructs, we have already initiated soybean transformation work and are expecting to generate transgenic soybean plants in the next 9 months.

Updated January 24, 2021:
September 15, 2020
Soybean meal cannot be directly used for animal feed mixtures due to the presence of various anti-nutritional compounds which reduce animal weight gain. Our group is attempting to drastically reduce (ideally eliminate) major proteinaceous anti-nutritional Kunitz-trypsin inhibitors and Bowman-Birk inhibitors. As indicated in our previous quarterly report the Wisconsin Crop Innovation Center is handling the soybean transformation part of this project. My understanding is that they have initiated the CRISPR constructs that will be used for soybean transformation to knock out Bowman-Birk inhibitors from soybean seeds. However, the restrictions enforced due to COVID-19 pandemic will cause delays in obtaining the transgenic soybean lines from the Wisconsin Crop Innovation Center. However, we are also pursing soybean transformation at the University of Missouri plant Transformation facility. This work has commenced and is progressing smoothly. We expect to obtain transgenic soybean events in the next six months.

Updated March 2, 2022:
Soybean meal cannot be directly used for animal feed mixtures due to the presence of various anti-nutritional compounds which reduce animal weight gain. During the life of this project, we have successfully created two gene-editing constructs to knock out Bowman-Birk inhibitors from soybean seeds. Using these constructs, we have successfully generated transgenic soybean plants. These transgenic events were grown in the greenhouse and seeds harvested from these plants were subjected to immunoblot analysis using antibodies raised against soybean Bowman-Birk inhibitor. The results of our analysis clearly demonstrate that the accumulation of Bowman-Birk inhibitor in these transgenic plants is inhibited. Thus, we have successfully generated transgenic soybean lines that do not accumulate the major proteinaceous anti-nutritional Bowman-Birk inhibitor. Now, we are ideally positioned to take advantage of our BBi knock out soybean lines to develop soybean cultivars that will require little or no processing. Additional research will lead to the development of soybean cultivars that will allow the use of raw of soybean meal in current markets (swine, poultry).