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.