The proposed research will produce molecular research tools and a viable strategy for targeted gene stacking in transgenic soybean plants. The GE soybean will be free of selectable marker and recombinase genes. Novel soybean transformation vectors will be available for use with novel gene(s) of interest and high oleic and seed meal modified founder lines can be used for stacking additional improvement traits. These GE soybean lines will form the basis for future soybean biotechnology research enabling the production of novel stacked transgenic soybean cultivars. The studies will also provide data on the effectiveness of the individual recombinase genes in the soybean, which in turn can be applied to self-excision bio-containment protocols or for use in other engineering techniques in soybean. Finally the founder line/RMCE protocol is designed to include regions for Polymerase Chain Reaction (PCR) primers to enhance marker assisted breeding efforts and facilitate the introgression of the GE traits in elite cultivars via plant breeding.

This project was initiated preceding the incorporation and standardization of KPIs. The progress of objectives are as follows:

Achieving Objectives:
• Three single copy and two multi-copy founder lines were generated and crossed into Magellan, Williams, and Jack cultivars for greater utilization. The position of the T-DNA insert sites in each of the three lines with a single copy has been determined. This work involved using low-coverage Next Generation Sequencing (NGS) and custom designed software. The software was developed by USDA and is available to researchers.
• The initial test pEXCH vectors for biolistic and agro-bacterium transformation have been completed. The targeting (RCME) process has yielded 10 positive plants. Site specific targeting still needs to be validated.
• The Fad2-1 and Fad3 RNAi suppression transgenes and another gene designed to modify the beta carotene pathway have been cloned and testing is underway. The group is working with another USB funded project (1420-532-5604) to test disease resistance genes that were shown to be important using VIGS. This platform will allow optimal and consistent expression.

Not meeting Objectives:
• No issues at this time.