Production of γ‐Linolenic Acid and Stearidonic Acid in Seeds of Marker‐Free Transgenic Soybean¹

Abstract

Through a single desaturation step, the Borago officinalis L. Δ6 desaturase can convert linoleic acid and α‐linolenic acid to γ‐linolenic acid (GLA) and stearidonic acid (STA), respectively. Both GLA and STA are of interest to the pharmaceutical and nutraceutical industries. Production of these fatty acids is costly. One potential strategy to reduce production cost would be to generate them in a major oilseed crop. To this end, a cDNA of the B. officinalis Δ6‐desaturase gene was cloned downstream of the embryo‐specific promoter β‐conglycinin. The resultant cassette was assembled into a two T‐DNA binary vector, in which the second T‐DNA element harbored a selectable marker cassette. The final plasmid was subsequently used to transform soybean [Glycine max (L.) Merr.]. The simultaneous delivery of two T‐DNA elements was used as a strategy to derive soybean progeny transgenic for the Δ6 desaturase T‐DNA and free of the marker gene T‐DNA. Twenty‐nine transgenic soybean lines were recovered that harbored both T‐DNA elements, of which 17 produced GLA and STA in the seed storage lipids. Average GLA levels ranged from 3.4 up to 28.7%, while STA levels varied from just under 0.6 to 4.2% in the T1 generation. Among the 17 lines that produced GLA and STA, four lines were identified that were free of the selectable marker T‐DNA element.

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

“…A borage 6 desaturase expression cassette harboring a cDNA clone of the gene under the control of the soybean seed-speciWc -conglycinin promoter was previously described (Sato et al 2004). A cDNA clone of the Arabidopsis 15 desaturase (Fad3) fused to the soybean -conglycinin promoter was obtained from DuPont Company.…”

“…PCR was used to engineer EcoRV sites just out side the left (LB) and right (RB) border elements of pPTN377. The resultant product was subcloned to the binary vector pPTN200 (Sato et al 2004) to make the Wnal two T-DNA binary plasmid pPTN382 (Fig. 2).…”

“…The NTL 4 / pTiEHA105 transconjugant carrying the two binary plasmids pGPTV-bar and pPTN7373 was used in soybean transformation experiments conducted with soybean genotypes, Thorne (Ohio State University) and NE3001 (University of Nebraska). The EHA101 transconjugant carrying the two T-DNA binary vector pPTN382 was used in soybean transformation experiments conducted with soybean genotypes Thorne, NE3001 and the marker-free transgenic event 420-5 (Sato et al 2004). Event 420-5 is derived from genotype NE3001.…”

“…Hence, the 12 desaturase activity is required to shift the fatty acid pool to LA, thereby permitting the build-up of substrate for GLA production, while the ectopic expression of the 15 desaturase will in turn inXuence the level of ALA to allow for increased STA accumulation. In soybean, expression of the borage 6 desaturase will divert fatty acid biosynthesis for the production of GLA and STA averaging approximately 25 and 4%, respectively (Sato et al 2004). For a number of reasons soybean is an ideal target for the production of these novel fatty acids.…”

“…Field studies on a marker-free transgenic soybean event designated 420-5 (Sato et al 2004) that harbors the borage 6 desaturase gene are currently being conducted across multiple environments. This event has consistently produced GLA and STA at levels ranging from 24 to 30 and 2 to 4%, respectively, depending on the environment in which seeds were harvested.…”