PHYSARIA FENDLERI CROP IMPROVEMENT BY GENE EDITING AND BIOENGINEERING FOR A DOMESTIC HIGH YIELDING SOURCE OF INDUSTRIALLY VALUABLE FATTY ACIDS

Goals:Our long-term goals are: to elucidate the genetic components that control the accumulation of novel seed oil fatty acid compositions (and total amounts) that can be used for enhanced breeding or engineering of oilseed crops as feedstocks for the oleochemical industry.Within this proposal our goals are to: Identify mechanisms of efficient unusual fatty acid flux through the lipid metabolic network to enhance all oilseed crop bioengineering, characterize genes that can improve total oil and hydroxy fatty acid accumulation in Physaria fendleri through gene editing, and develop Physaria fendleri as a platform for the production of other valuable oil fatty acid compositions.OBJECTIVE 1: Functional genomics in P. fendleri to characterize the mechanisms of efficient hydroxy fatty acid flux through the lipid metabolic network into oil, and identify regulators of total oil accumulation (Years 1-3).Our newly developed VIGS based functional genomics approach in P. fendleri will be utilized to enhance our understanding of key unknowns in plant lipid metabolism and identify the molecular control of oil synthesis specifically in P. fenderli that could be targets for improving P. fendleri as a high yielding oilseed crop. The gene targets for our functional genomics approach will investigate mechanisms of fatty acid acyl editing, the selective production of phosphatidylcholine-derived diacylglycerol for triacylglycerol synthesis, and regulators of total oil synthesis and accumulation.OBJECTIVE 2. Targeted gene editing in for increased oil, HFA, and total seed yields (Years 1-2).Based on our preliminary RNA knockdown results we hypothesize a sdp1 knockout mutation will further enhance oil and HFA levels, and a srk knockout mutation will produce self-compatible flowers leading to an increase in total seed yield. Therefore, CRISPR/ cas9 technology will be used to generate mutations in SDP1 and SRK genes individually as well as together, to evaluate the individual and combined roles of each mutation on P. fendleri oil content and seed yield. Additionally, the self-compatible mutant will be a valuable background for producing homozygous transgenic P. fendleri (Objective 3), by reducing outcrossing and the manual labor required to force self-crossing during propagation of transgenic plants.?OBJECTIVE 3. Bioengineering P. fendleri as a platform crop for "castor oil" and other valuable unusual fatty acids (Years 2-3).We hypothesize that Physaria fendleri can be a highly useful platform species to engineering valuable unusual fatty acid compositions (over that of species such as camelina) due to its inherit ability to efficiently channel unusual fatty acids into seed oils. In Objective 3 we aim to produce two different seed oil compositions in P. fendleri to evaluate its value as an engineering platform crop through two phases: (1) gene editing to produce the platform backgrounds (fae1, and fae1/fah12); (2) transgene expression to produce designer oils for triacylglycerol containing three ricinoleic acids, or triacylglycerol containing two expoxy fatty acids. If the SRK gene editing in Objective 2 indicates no adverse effect on plant growth then we will cross the background lines with the srk mutant such that self-compatibility will aid in development of homozygous transgenic lines.