Project Details:

Genome editing and engineering pipeline

Parent Project: This is the first year of this project.
Checkoff Organization:Tennessee Soybean Promotion Board
Categories:Breeding & genetics
Organization Project Code:19-117-R
Project Year:2019
Lead Principal Investigator:C Neal Stewart Jr (University of Tennessee-Institute of Agriculture)
Co-Principal Investigators:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

The goals of this project are to expand on improvements made to our maturity group IV (MG IV) soybean transformation pipeline and continue to pursue genome edited MG IV soybean with an enhanced fatty acid oil profile. In addition, we will begin to pursue editing root genes from our sister proposal as well as overexpress a soybean trypsin inhibitor in leaves using a strong soybean leaf promoter for deer feeding deterrence. The direct transformation of MG IV germplasm will reduce unnecessary breeding steps and save time during cultivar development. In addition, new cultivars created using this these methods are not currently regulated by the USDA directly benefitting Tennessee farers by significantly reducing germplasm development time, The deer deterrent line may be counted as a “cisgenic” line by the USDA since it combines soybean DNA for trypsin inhibitor production in leaves. My team will continue to tailor CRISPR tools to improve Tennessee-adapted germplasm.

Project Objectives

Objective 1: Continue to establish a reliable Agrobacterium-mediated cot-node transformation method using Tennessee MG IV germplasm and evaluate tissue culture responsiveness of MG IV cultivars grown outside Tennessee. In previous work my team successfully established a routine cot-node transformation method in my lab using the antiquated MG III cultivar ‘Williams 82.’ Recently, we observed that three MG IV breeding lines from Vince Pantalone’s breeding program at UT (‘TN15-5007,’ ‘TN15-4009,’ and ‘TN17-4474’) that perform exceptionally well in our tissue culture system. The aim is to use these lines to develop a reliable and consistent MG IV cot-node transformation protocol with transformation efficiencies at or above those observed with ‘Williams 82.’ Tissue culture responsiveness will be assessed in a minimum of 10 additional cultivars grown outside of Tennessee.

Objective 2: Continue to produce and analyze soybean plants with genome edited fatty acid desaturase 2 (FAD 2) gene knockouts. My team will evaluate levels of monounsaturated (oleic acid) and reduced levels of polyunsaturated (linoleic acid) fats in seed oil of T0 and their progeny plants (T1) that are sequence confirmed as edited. Edited ‘Williams 82’ and ‘TN15-5007’ will be recovered, verified through molecular analysis, and selfed for T1 line production in the greenhouse in 2019. For these lines, in-depth analyses will be performed to quantify FAD2 gene disruption and fatty acid profiles in oil. In addition, productivity measures of seed and vegetative yield will be made and compared to non-genome edited controls.

Objective 3: Begin a new speed breeding program to produce engineered/edited deer resistant soybean starting with altered gene expression profiles of soybean trypsin inhibitor. All legume seeds produce trypsin inhibitors as well as other protease inhibitors that can hinder mammalian digestive enzymes. Thus, mature seeds must be treated by heat or chemicals to deactivate these protease inhibitor proteins that are found in soybean meal. Soybean, therefore, already has a built-in deterrent to feeding by mammals in the seed. We will transfer this mechanism to engineered leaves of soybean aimed at giving grazers such as deer a belly-ache upon ingestion. Since deer are adept leaners, we hypothesize they would learn to avoid eating soybean leaves. The strategy will be to combine soybean promoters characterized in earlier iterations of this grant (e.g., 2017) that enable high gene expression in leaves (at least 2 different promoters will be used) with a soybean trypsin inhibitor gene.

Project Deliverables

Objective 1: Performing these experiments will add to the genetic diversity of our MG IV transformation germplasm and increase the chances of generating an efficient and robust transformation and speed breeding pipeline for Tennessee-adapted germplasm.

Objective 2: Once high performing lines with high oleic and low linoleic oil are identified, they will be immediately made available to the UT soybean breeding team with the goal of ultimately releasing new cultivars to Tennessee soybean farmers. The results will be compared to high oleic lines produced via conventional breeding and transformation.

Objective 3: The goal will be to produce a cisgenic soybean line that may not be regulated by the USDA. In 2019, the team will build engineering vectors for a soybean cisgenic approach (new to UT) and begin the transformation experiments in the top-responding germplasm in the summer.

Progress of Work

Final Project Results

Benefit to Soybean Farmers

Performance Metrics

Project Years