2017
Non-transgenic soybeans with broad plant disease resistance and high protein
Contributor/Checkoff:
Category:
Sustainable Production
Keywords:
Crop protectionDiseaseField management
Lead Principal Investigator:
Ling Li, Iowa State University
Co-Principal Investigators:
Project Code:
450-21-08
Contributing Organization (Checkoff):
Institution Funded:
Brief Project Summary:

Soybean disease, nematode, insect pest and abiotic stress each year can cause major loss to soybean production under varying weather conditions. The high-protein soybean seed is of economic and nutritional value. A new molecular tool can generally increase plant defense to plant pathogen, with higher-protein trait, with normal morphology, development and yield similar to the control plants. A non-transgenic approach has been set up to generate non-regulatory soybean with potentially both high-protein and plant-pathogen-resistance traits. This research aims to understand soybean composition and defense, and application to avoid loss of yield from soybean disease, nematode, insect pest and abiotic stress, via a non-transgenic approach.

Key Benefactors:
farmers, agronomists, Extension agents, soybean breeders, seed companies

Information And Results
Project Deliverables

Timelines:
1. The transgenic GmGene2-OE Williams 82 with increased seed protein have been developed, has been tested for seed composition (protein and oil) in the field, and propagated for disease field experiments that require a lot of seeds. (Timeline: by Year 1)
2. Soybean disease (bacterial and SCN) and insect pest (aphids) tests on transgenic GmGene2-OE soybean mutants (Williams 82) in growth chamber/greenhouse. (Timeline: by Year 1)
3. The plasmid construct for non-transgenic approach via CRISPR/Cas9 will be generated and submitted for transformation. (Timeline: by Year 1)
4. Soybean disease (bacterial, viral, SCN) tests on transgenic GmGene2-OE Williams 82 and yield trials in the field in Ames. (Timeline: by Year 2)
5. Transcripts that are differentially accumulated in transgenic GmGene2-OE upon pathogen infection will be identified; our understanding of soybean defense, integrated with the carbon and nitrogen partitioning in the metabolic and regulatory network will be deepened by RNA-Seq analyses. (Timeline: by Year 2 and Year 3)
6. The soybean Williams 82 plants containing CRISPR/Cas9-edited soybean GmGene2 will be generated. Homozygous deletions, overexpressed GmGene2 and transgene-free CRISPR/Cas9-GmGene2promoter Williams 82 will be identified. (Timeline: by Year 2)
7. Application to USDA (United States Department of Agriculture) for non-regulatory CRISPR/Cas9-GmGene2promoter Williams 82 with homozygous deletions of repressor via CRISPR/Cas9 in promoter region, and free of transgene. (Timeline: by Year 2)
8. Soybean disease (bacterial, viral, SCN) tests on non-transgenic CRISPR/Cas9-GmGene2promoter Williams 82, with yield data in Ames. (Timeline: by Year 3)
9. At least two lines of non-transgenic CRISPR/Cas9-GmGene2promoter Williams 82 and control will be planted in Ames and two other different locations, for soybean disease tests (fungal, SDS), and yield trials. (Timeline: by Year 3, or when gets the USDA approval as non-regulatory materials)
10. Publications as research develops. (Timeline: by Year 2 and Year 3)
Key Performance Indicators:
1. Transgenic soybean germplasm with increased disease resistance (and increased protein/no effect on yield) will be available for seed companies and farmers by 2017.
2. Non-GMO soybean germplasm aiming for increased disease resistance (and increased protein/no effect on yield) will be available for seed companies and farmers by 2017/2018.
3. Transgenic approach to develop soybeans with increased disease resistance (and increased protein/no effect on yield will be available for seed companies by 2017.
4. Non-GMO approach to develop soybeans with increased disease resistance (and increased protein/no effect on yield) will be available for seed companies by 2018/2019.

Final Project Results

Update:
1. Seeds were planted in the field in spring 2016 to bulk more seeds for transgenic soybeans (in Williams 82 background) overexpressing Target Gene 1 or 2 for disease experiments, to screen more events overexpressing Target Gene 2 that were created but had missed the 2015 field growth season, and to confirm their seed composition. The bulked seeds have been harvested, cleaned, organized and analyzed for weight and composition.
Analyses of the composition of the mature soybean seeds overexpressing Target Gene 2 harvested from the field in 2016 screened more lines from more independent events with increased seed protein plus oil content, and decreased seed fibers. Seed yield per plant was not affected; generated more seeds that were enough for more pathogen experiments (for bacterial and viral infection experiments) in growth chamber and in the field (bacterial and SDS infection experiments).
2. Transgenic soybeans expressing Target Gene 1 or overexpressing Target Gene 2 with increased resistance to soybean pathogens and pests:
a. In growth chamber, in Williams 82 background, bacterial assay (bacterial infection: PsgR4) and viral assay (viral infection: BPMV) of soybeans overexpressing Target Gene 1 and for soybeans overexpressing Target Gene 2, using seeds harvested from the field in 2016. RNA-Sequencing experiments were also conducted for the bacterial assay and viral assay.
a1), Bacterial growth (bacterial infection: PsgR4) was similarly decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2 in growth chamber.
a2), Viral foci size was decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
a3), Soybean Cyst Nematode female count numbers were decreased in transgenic soybeans (Williams 82) overexpressing Target Gene 2 in greenhouse.
b. Field experiments were performed for SDS/bacterial infection assays in the field for lines overexpressing Target Gene 1 and for lines overexpressing Target Gene 2, using seeds generated from the field in 2016.
b1), Field bacterial assay was repeated twice in summer in 2017. But the weather was not suitable for bacterial experiment. The bacterial infection symptoms did not show up.
b2), Experiment in growth chamber to test SDS screening in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2 indicated these transgenic soybeans had decreased scores of foliar SDS symptoms. The field SDS experiment indicated less foliar disease symptoms in transgenic soybeans (Williams 82) overexpressing Target Gene 1 or 2.
3. Non-transgenic approach.
a. Promoter regions have been identified for Target Gene 2 that deletion of these promoter regions can overexpress Target Gene 2, which opens route for targeted mutagenesis via CRISPER/Cas9 technologies to increase the seed protein content of agronomic species, to reduce crop susceptibility to pathogens and pests.
b. Five constructs were generated and confirmed, and the confirmation of the transformation in soybean hairy roots was performed. They have been submitted to ISU PTF for soybean transformation to generate soybean plants. Plants generated from one construct have been moved to greenhouse. Transformation for other four constructs are going on.
4. Developing molecular tools of both Transgenic and Non-transgenic approaches, for soybeans with normal growth and development, increased soybean seed protein, reduced soybean susceptibility to pathogens and pests, and to increase sustainability. A company has licensed the patents of the technologies and will work on commercializing the high-protein soybean.
5. Manuscript for composition experiment results has been accepted for publication as a book chapter in a book that will be published by Springer. Manuscript for disease experiment results are being updated and will be submitted soon to peer-review journal for publication.

1. The transgenic soybeans (Williams 82) have been tested for seed composition (protein and oil content) in the field; identified that the transgenic mutants had increased seed protein.
2. Soybean disease of viral and bacterial infection and pest (soybean aphids, SCN, and SDS) tests on transgenic soybeans (Williams 82) in growth chamber, and bacterial and SDS tests in the field; identified that the transgenic mutants had increased broad disease resistance. RNA-Sequencing is undergoing to identify genes whose transcripts are associated with disease resistance.
3. The plasmid constructs for non-transgenic approach via CRISPR/Cas9 have been generated and submitted for transformation at ISU PTF. The transformation has made progress.
4. Developing molecular tools of both transgenic and non-transgenic approaches, for soybeans with normal growth and development, increased soybean seed protein, reduced soybean susceptibility to pathogens and pests, and to increase sustainability. A company has licensed the patents of the technologies and will work on commercializing the high-protein soybean.
5. Manuscript for composition experiment results has been accepted for publication as a book chapter in a book that will be published by Springer. Manuscript for disease experiment results are being updated with results from the field this summer and will be submitted soon to peer-review journal for publication.

The United Soybean Research Retention policy will display final reports with the project once completed but working files will be purged after three years. And financial information after seven years. All pertinent information is in the final report or if you want more information, please contact the project lead at your state soybean organization or principal investigator listed on the project.