This proposal seeks to use modern biotechnology tools and advances in plant transformation to develop a deeper understanding of the genes that control critical traits in soybean, and identify modifications of these genes for trait improvement.

There are two major types of modern crop biotechnology: (a) genetic transformation, which adds a gene from another species into the crop (e.g., the glyphosate resistance transgene) and (b) genome engineering, which can modify specific genes in soybean without the permanent introduction of DNA from other species (i.e., the resulting plants are non-transgenic).

With support from MSR&PC, our group has been at the forefront in developing and utilizing these approaches for soybean research and improvement. In recent years, the MSR&PC has provided the base funding to support the Stupar lab soybean transformation facility; this has been leveraged to support various projects that focus on traits of critical importance to Minnesota soybean producers. This year’s proposal continues efforts to maintain this facility and use genetic transformation and genome engineering methods to investigate soybean gene function and improve plant architecture and stress tolerance traits. Furthermore, this year’s project continues to merge work from previously funded projects respectively lead by PI Stupar and co-PI Muehlbauer.

Maintain an efficient soybean genetic transformation facility.
Obj 1: Provide transformation capacity to leveraged projects funded through the National Science Foundation, United States Department of Agriculture, and the North Central Soybean Research Program.

Use transformation and gene editing to assess gene functions and enhance traits of interest for Minnesota soybean growers.
Obj 1: Test functions of a key gene hypothesized to influence IDC tolerance using transgenic overexpression and targeted mutagenesis.
Obj 2: Generate novel architecture mutants by mutagenizing and characterizing candidate genes previously identified from Dr. Muehlbauer’s project.

Fine-map the branch orientation trait.
Obj 1: Provide a map and molecular markers that are useful to breeders for selecting an improved branch orientation for soybean.

The Stupar research group has recently developed a system that can either add a new gene to the genome (standard transformation) or reliably generate targeted DNA changes for specific genes (using a CRISPR/Cas9 system )(Michno et al. 2015; Liu et al. 2019). For the proposed project, both of these methods will be used to create new traits in soybean plants for genes involved in IDC tolerance and plant architecture. A list summarizing the target genes for this project is shown below:

1. IDC genes: We have used genetics and genomics to identify a leading candidate gene for IDC tolerance on soybean chromosome 5. We are testing the actions of this gene by changing its function in transient hairy root tissues. We will overexpress the gene and measure iron content in these cells. We will also mutate this gene in ‘Bert’ using gene editing techniques at UMN and phenotype the resulting plant in IDC conditions in subsequent generations. Identification of this gene will lead to new insight into IDC mechanisms in soybean and lead to new genetic and/or management strategies that target the functions of these pathways.

2. Architecture genes: We have made significant progress on using gene editing to mutate a candidate architecture gene known as Lps1. In this funding cycle, we will identify the mutants and evaluate their phenotype to see if they show the hypothesized changes in plant branch angle. Additionally, numerous genes have been identified in other plant species that regulate shoot architecture. Genes that exhibit a large effect and appear to be conserved across plants are the LATERAL SUPPRESSOR (LAS) and TEOSINTE BRANCHED1 (TB1) genes, and the MORE AXILLARY GROWTH (MAX) gene family. We plan to examine the function of these genes in soybean. This information will be used to identify a stronger architecture type that informs molecular breeding efforts for more robust soybean varieties.

To map the branch orientation trait, we will order advanced inbred lines that are polymorphic for this trait. We will use identify molecular markers that segregate with the respective orientation types (spiral or flat). We will identify nearly identical siblings that vary for these markers and establish near isogenic lines (NILs) from the progeny of the siblings, which will allow us to compare the effect of this trait on harvestable yield. We will fine-map this locus by tracking molecular markers that segregate with the respective spiral and flat branching orientations. From this, molecular markers that segregate with the trait will be identified for breeders to use.

Update:
This report covers progress from 5/1/2021 to 4/30/2022. Efforts have mainly focused on developing targeted mutations in soybean using the CRISPR/Cas9 transgenic system and/or stable transformed lines to test for gene function. However, funding was not received for this project between May 2021 and April 2022. Therefore, provided below is a brief synopsis of efforts during that time:

1. IDC candidate genes: We continued to characterize the IDC MATE transporter gene (see previous progress reports for more details) using both whole plant gene editing and hairy root transformation experiments. While there has been some progress in developing these materials, there is not a clear outcome to date with respect to validating this gene’s function in IDC resistance. Experiments are ongoing.

2. Architecture genes: We have continued to track the mutation profile of CRISPR/Cas9 plants in which the Lps1 (Glyma16g33430; loss of function mutation may generate short petiole phenotype) was mutated. We genotyped T2-generation plants growing in our greenhouse, and identified two types of inherited mutations in this population, including one allele carrying a deletion of 3 base pairs and another allele carrying a deletion of 27 base pairs (see previous progress reports for more details). These are stable mutations, and we have identified plants carrying respective alleles in the homozygous state while no longer carrying the transgene. The mutants were field-planted in 2021 and it was determined that they do not exhibit new architectural phenotypes. However, we hypothesize this is because they are in-frame deletions. For now, our efforts our focused on developing alleles with the specific 3-bp insertion that was observed in the original Lps1 mutant (genotype D76). We are using advanced gene editing methods to develop these alleles.

In addition to the above projects, the transformation facility has continued to support transformation and/or CRISPR mutation efforts for additional targets, using funding leveraged from the United States Dept of Agriculture and the United Soybean Board. The project has also contributed to collaborative efforts with other research teams.

Challenges encountered:

Delivery of funds for this project was delayed by about one year. Therefore, the project is on a no-cost extension until April 2022.

Information dissemination of data/information from this research during this reporting period:

In this reporting period, this project contributed work towards three presentations:
1) Stupar R. Crop genome editing: An Academic Perspective. American Seed Trade Association CSS & Seed Expo 2021, Chicago, IL, Dec 2021.
2) Stupar R. Searching for short, bald and golden: The modern pursuit of genes underlying soybean traits. University of Nebraska Agronomy & Horticulture Department seminar series, Virtual, Nov 2021.
3) Stupar R. Exploring soybean in the age of gene editing. BASF, Virtual, Nov 2021.

Technology Transfer:

There was no technology transfer in this reporting period.

Update:

This proposal provides a vision for improved soybean traits, tailored to the needs to Minnesota soybean farmers. Minnesota farmers will possess improved soybean varieties based on the novel traits developed by this project, with an emphasis on improved architecture and stress tolerance traits. This proposal aligns with Category III, particularly the following: “Soybean Farmers continually need new high yielding varieties with genetic traits that will reduce the impact of diseases, pests, and other yield limiting factors across the wide array of relative maturities across Minnesota” and “Soybean Farmers need molecular and functional genomics research to complement existing University of Minnesota public breeding efforts. Research should focus on identification of new resistance to yield limiting pests such as SCN, IDC, insects, and plant diseases and integration of identified genetics into existing breeding lines adapted across Minnesota relative maturities.” Results from this proposal will be used to integrate superior genes into soybean germplasm adapted to Minnesota.

Furthermore, breakthroughs from leveraged projects, including North Central, USDA and NSF sponsored efforts to addresses traits like seed composition and herbicide resistance, will also be of great value to genetic improvements for seed varieties grown in Minnesota.