Updated March 26, 2020:
Key Activities/Accomplishments:
The major activities undertaken over this past year have been aimed at successfully overcoming challenges associated with the initial batch of soybean transformant plants that we obtained from Dr. Zhang at the Mizzou Plant Transformation Core. These initial transformant lines, which arrived significantly behind schedule, did not exhibit successfully BADC RNAi silencing. Over the course of this year, we:
1) Screened the initial BADC RNAi lines and determined that BADC silencing was not successful.
2) Assembled a new seed-specific BADC RNAi vector using a proven backbone supplied by Dr. Tom Clemente at the University of Nebraska (rather than the original backbone supplied by Dr. Zhang).
3) Assembled a seed-specific Ps a-CT over-expression vector also using Dr. Clemente’s backbone. Over-expression of Pisum sativum (garden pea) a-carboxyltransferase (a-CT) has successfully increased oil content in Arabidopsis and Camelina seeds (manuscript in preparation).
4) Obtained 25 BADC RNAi lines and 35 Ps a-CT over-expression lines transformed with these new vectors at the Iowa State Plant Transformation Facility. We are currently growing these plants in the greenhouse and expect them to produce seed for screening in the next couple of months.
5) Validated PCR primer sets and prepared an accelerated screening plan.

Key Performance Indicators (KPIs):
1) At least three independent transgenic soybean lines with 70-100% reduction in total BADC gene expression as compared to non-transgenic reference lines.
2) A significant and reproducible increase in soy seed oil content of at least 5% dry weight in at least three independent transgenic events as compared to non-transgenic reference lines.
3) No significant change in soy seed protein content in high oil transgenic lines compared to non-transgenic reference lines.
4) Identification of either government or industrial partners capable of transferring this trait into elite soy breeding lines through either selective breeding (gene mutation) or transgenic silencing.

Progress towards each KPI:
1) The eleven original transgenic lines that were transformed with our BADC RNAi cassette in Dr. Zhang’s expression vector did not exhibit successful BADC silencing. We therefore incorporated our BADC RNAi cassette into a proven soybean expression vector from Dr. Tom Clemente and we have obtained 25 lines transformed with this new vector, which we are about to screen.
2) No progress at present time, T1 seed will be ready for screening over the next few months.
3) No progress at present time, T1 seed will be ready for screening over the next few months.
4) We currently have a corporate partner on the pending worldwide patent and exclusive licensing options therein. They are moving this trait into canola and Camelina for tentative field trials in 2018.

Expected Outputs/Deliverables:
1) A binary vector RNA interference construct capable of reducing expression of all isoforms of BADC genes in any soybean variety through RNA interference. This construct will contain gene sequences specific to BADC isoforms 1 and 2 in tandem as part of a coupled RNAi gene silencing strategy.
2) Transgenic soybean lines with significantly (t-test, one-way ANOVA) higher levels of seed oil than nontransgenic reference lines.

Results on Deliverables:
1) Re-completed. As a result of failed RNAi silencing with the original pMUC binary vector backbone, we produced a new BADC RNAi vector based on a pPTN1138 backbone from Dr. Clemente at the University of Nebraska.
2) Ongoing. We have twenty-five putative transgenic soybean lines (all independent events) growing in the greenhouse, which we expect to produce seed for screening in the next couple of months.

We were repeatedly set back due to challenges in obtaining reliable soybean transformation services from the Mizzou Plant Transformation Facility. This has been a systematic issue and as a result the facility was recently placed under new management. Unfortunately, for our initial round of transformants, we relied on a vector from this same facility, and the resultant transformant plants did not exhibit successful BADC silencing. As a result, we had to design a new vector and proceed with an additional round of transformations. For this new round of transformations, we established a productive relationship with Diane Luth, the soybean transformation lead at the Iowa State Plant Transformation Facility, who was able to deliver an abundance of healthy plantlets ahead of schedule, in less than a quarter of the time that it previously took at the Mizzou facility. We intend to continue this relationship and do not anticipate further transformation difficulties for as long as this relationship continues.

What, if any, follow-up steps are required to capture benefits for all US soybean farmers?
If soybean oil levels are successfully increased without adversely affecting protein content, as has been observed in comparable Arabidopsis and Camelina transformants, this benefit would be available to US soybean farmers as soon as our licensing partners are able to distribute the technology.

We produced an additional soybean over-expression vector beyond what we proposed (a seed-specific a-CT over-expression vector) and generated 35 transgenic lines with this vector. Over-expression of a-CT successfully appears to increase oil content in Arabidopsis and Camelina seeds. We anticipate that if a-CT similarly increases oil content in soybean, we may be able to stack it with our BADC RNAi trait to further increase soybean oil content, providing additional benefits to US soybean farmers.