Location: Plant Science Research
2025 Annual Report
Objectives
Objective 1: Conduct research to optimize gene editing reagents and transformation methods in soybean, alfalfa, and other legumes to increase functional efficiency and improve the screening methods that are used to identify successfully edited plants.
Goal 1A: Generate a collection of gene editing reagents comprised of various promoter, Cas9 enzyme, transcriptional/translational enhancers, and terminator combinations and screen their efficiency for mutagenesis of candidate root-trait related targets using a rapid hairy-root transformation assay.
Goal 1B: Improve transformation efficiencies of alfalfa and soybean by a 0.5- to 2-fold range and reduce time in tissue culture for plant regeneration in order to generate candidate root-trait related mutant plants.
Goal 1C: Develop tissue-culture-free-transgene-free gene editing.
Objective 2: Conduct research for functional validation of novel and previously identified genes that contribute to root architecture, nitrogen fixation, and nutrient acquisition in legume species, especially soybean and alfalfa.
Goal 2: Use reagents constructed and screened for efficiency in Objective 1 to generate mutant plants for the validation of candidate root-trait related genes in legumes.
Approach
Optimize gene editing reagents and the transformation methods of recalcitrant elite, wild, and un-adapted genotypes of soybean, alfalfa, and other legumes to generate mutant plants associated with root system architecture and other root traits. Improve the screening methods used to identify successfully edited plants. Using the generated mutants, validate previously identified root system architecture and other root trait related candidate genes. Demonstrate novel tissue culture-free gene editing of legume using phloem mobile elements.
Progress Report
Legume crops like soybean, alfalfa, and common bean play a vital role in sustainable food systems due to their nitrogen-fixing capabilities and high protein content. However, genetic improvement of these crops has lagged behind other crops largely due to technical barriers in transformation and gene editing. Our research addresses these limitations by developing efficient transformation and gene editing tools to accelerate crop improvement. This project achieved significant progress across all objectives, building on the previous year’s achievements. For Objective 1, we identified two promising gene components that enhance the accuracy and efficiency of CRISPR-based editing. These tools enable targeted gene disruption to study loss-of-function effects, subtle modifications that mimic natural variants and the precise insertion of beneficial alleles into specific genomic locations to improve gene function. We successfully modified elite genotypes of soybean and alfalfa and we're making strong progress in modifying common beans. Overcoming these challenges is critical as gene editing holds the potential to boost yield, strengthen resistance to pests and diseases, and improve drought tolerance in crops. Moreover, increasing the overall efficiency of gene editing is essential. The process remains complex and resource-intensive, especially in legumes, where genetic modification has historically been more difficult. For Sub-Objective 1C, previously generated mobile gRNAs targeting multiple loci in soybean and Medicago truncatula were tested. While seed were successfully recovered, no mutagenesis activity was detected. For Objective 2, we applied the optimized reagents Objective 1A and Objective 1B to generate gene edited soybean, Medicago truncatula, and alfalfa. We successfully regenerated multiple edited plants, confirmed several mutations and are in the process of validating others. These plants have been self-fertilized to verify the heritability in the next generation, and seeds are being bulked for future phenotyping assays focused on root development, pathogen resistance, seed oil to protein ratio, and drought stress tolerance. This work directly benefits plant breeders and geneticists, by providing validated gene targets and mutant lines that accelerate trait selection and crop improvement as well as farmers through the development of legume varieties with enhanced nutrient uptake, resilience to environmental stress, and pathogen resistance.
Accomplishments
1. Identification of barrel medic disease resistance genes for improving alfalfa cultivars. Plant pathogenic fungi cause most plant diseases, resulting in $200 billion in crop losses annually. The fungal disease, spring black stem (SBS) and leaf spot is the most devastating foliar disease of alfalfa, the premier forage crop used for dairy and beef livestock feed. Disease resistant cultivars are needed for combating this disease, but little resistance is found in alfalfa. SBS also occurs on barrel medic, an alfalfa relative, with disease resistance identified in the accession HM078. To increase understanding of SBS disease resistance, ARS scientists in Saint Paul, Minnesota, worked with University of Minnesota colleagues to assemble the DNA sequence for the HM078 genome and identified several possible genes for SBS disease resistance. A comparative gene expression study with HM078 and a disease susceptible accession identified additional genes that were uniquely expressed in HM078 in response to disease. Five potential disease resistance genes were tested by inoculating modified barrel medic plants with the SBS pathogen. Mutation of a gene that increases disease susceptibility reduced pathogen biomass by approximately 34% while over- expression of a second gene involved in cuticular wax synthesis reduced pathogen biomass by approximately 75%. This research further elucidates the legume immune system in response to pathogenic fungi, and results can be used by plant breeders to develop alfalfa cultivars with improved disease resistance, which will increase stand survival, yield, and quality of the forage.
Review Publications
Botkin, J., Curtin, S.J. 2024. Transcriptome analysis of resistant and susceptible Medicago truncatula genotypes in response to spring black stem and leaf spot disease. BMC Plant Biology. 24. Article 720. https://doi.org/10.1186/s12870-024-05444-3.
Botkin, J.R., Medina, C.A., Park, S., Poudel, K., Cha, M., Lee, Y., Prom, L.K., Curtin, S.J., Xu, Z., Ahn, E.J. 2024. Analyzing Medicago spp. seed morphology using GWAS and machine learning. Scientific Reports. https://doi.org/10.1038/s41598-024-67790-4.
Lim, S., Park, S., Baek, I., Botkin, J., Jang, J., Hong, S., Irish, B.M., Kim, M.S., Meinhardt, L.W., Curtin, S.J., Ahn, E.J. 2025. Integrative analysis of seed morphology, geographic origin, and genetic structure in Medicago with implications for breeding and conservation. BMC Plant Biology. https://doi.org/10.1186/s12870-025-06304-4.
Stupar, R.M., Locke, A.M., Allen, D.K., Stacey, M.G., Ma, J., Weiss, J., Nelson, R., Hudson, M.E., Joshi, T., Li, Z., Song, Q., Jedlicka, J., Macintosh, G.C., Grant, D., Parrott, W.A., Clemente, T.E., Graham, M.A., O'Rourke, J.A., Stacey, G., An, Y., Aponte-Rivera, J., Bhattacharyya, M.K., Baxter, I., Bilyeu, K.D., Campbell, J.D., Cannon, S.B., Clough, S.J., Mcgrinn, M., Curtin, S.J., Diers, B.W., Dorrance, A.E., Gillman, J.D., Graef, G.L., Hancock, N., Hudson, K.A., Hyten, D.L., Kachroo, A., Koebernick, J., Libault, M., Lorenz, A.J., Mahan, A.L., Massman, J.M., Meksem, K., Okamuro, J.K., Pedley, K.F., Rainey, K.M., Scaboo, A.M., Schmutz, J., Song, B., Steinbrenner, A.D., Stewart-Brown, B.D., Toth, K., Wang, D., Weaver, L., Zhang, B. 2024. Soybean genomics research community strategic plan: a vision for 2024-2028. The Plant Genome. https://doi.org/10.1002/tpg2.20516.