Location: Plant Science Research
2024 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
This project made significant progress on all objectives. In support of Objective 1, a suite of reagents used for targeted mutagenesis, adenine and cytosine base editing, prime editing and targeted knock-in by exonuclease mediated homology-directed repair (HDR) have been constructed. The testing of these reagents in hairy-root and whole plants is currently in progress. The transformation of three soybean genotypes has been carried out using a rapid embryonic axis (EA) protocol and the recovery of transgenic plants has been confirmed. A preliminary transformation efficiency of 10-20% has been observed for the Bert genotype, and successful shoot regeneration and rooting has been demonstrated. For Sub-Objective 1C, mobile gRNAs for multiple targets in soybean and Medicago truncatula were generated and tested using a hairy-root transformation protocol on a Cas9 expressing scions. Seeds have been recovered however no mutagenesis activity has been detected yet.
For Objective 2, previous phenotyping of 600 root samples from 260 M. truncatula accessions using laser ablation tomography (LAT) technology led to the identification of six promising candidate genes associated with various root architecture traits. Mutant plants have been generated for the six candidate genes and mutations have been sequenced to confirm disruption of gene function. T1 plants are being screened to confirm heritable transmission of target mutations into the next generation as well as the identification of T-DNA null segregants, (non-transgenic plants) so they can be transformed with transcriptional and translational reporter constructs for further investigation of candidate gene function.
Accomplishments
1. High efficiency DNA base editing in soybean. Gene editing is an emerging technology that enables precise changes to be made in plant genes to rapidly improve agronomic traits without the introduction of foreign genes or lengthy cross breeding. A novel type of gene editing reagent called a base editor causes specific point mutations in DNA without generating deleterious off-target changes. Legume species like soybean, alfalfa, and the model legume Medicago truncatula (barrel medic) have been difficult to base edit ever since the technology was first introduced. ARS scientists in Saint Paul, Minnesota optimized base editor reagents and demonstrated editing in soybean at high frequency. Using a natural yellow mutant soybean plant resulting from a single nucleotide difference in a chlorophyll gene, the ARS scientists used the reagent to correct the mutation and restore the green phenotype. These reagents will be powerful new tools for the legume research community to validate functions of genes by precisely targeting changes to DNA sequences and for making gene edits that improve crop characteristics.
Review Publications
Stupar, R.M., Curtin, S.J. 2024. Gene editing in soybean: Promise to products. In: Bansal, K.C., Lakra, W.S., Pathak, H. (eds). Transformation of Agri-Food Systems. Springer, Singapore. p. 287-296. https://doi.org/10.1007/978-981-99-8014-7_21.
Botkin, J., Farmer, A.D., Young, N.D., Curtin, S.J. 2024. Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance. BMC Genomics. 25. Article 204. https://doi.org/10.1186/s12864-024-10112-9.