Dayakar Pareddy

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

Image Analysis of Transient Expression in Bombarded Soybean (Glycine max) Immature Embryos.

Particle bombardment of cells traditionally results in low frequencies of regenerated transgenic plants, which could be due to low (1-2%) cell viability in particle containing cells (Hunold, Bronner, & Hahne, 1994). Several methods for reducing damage to cells during bombardment have been developed, including the use of osmotic treatments (Vain, McMullen, & Finer, 1993) and the reduction of gold particle size (Randolph-Anderson, et al., 1995). It was determined that reducing the total number of gold particles per shot at the target tissue might reduce the damage received by each cell, thereby improving cell survival and regeneration. Immature embryos of soybean were bombarded using five different concentrations of gold particles to determine the effects of gold particles as measured by transient expression. Image analysis of bombarded immature embryos was performed using ImageJ to determine the number and location of foci with the bombardment target area and this information was used to optimize concentration of gold density for particle bombardment.

Zinc finger nuclease-mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non-homologous end joining

Summary Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.