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Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9–11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Seed-specific modification of crop plants is a common goal in plant metabolic engineering. Ability for early assessment and validation of seed-specific constructs for metabolic engineering in crop plants is desirable. We describe a method in which the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2) transcription factor is constitutively expressed to generate somatic embryos that can generate a seed-like fatty acid profile from cotyledonary petiole of Brassica napus. We also demonstrate that the super-transformation of 35S-LEC2 construct in B. napus lines that are already stably-transformed with seed-specific constructs producing arachidonic acid (ARA) or ricinoleic acid (RA) in mature seed results in the generation of somatic embryos that actively expressed these seed-specific transgenes and produced ARA and RA similar to transgenic T2 seeds. Stable transformation of seed-specific construct and analysis of T1 seed in B. napus requires around 7–8 months. We have used this method to functionally assess single (RA) or multi-gene (ARA) seed specific construct in somatic embryos within 6–7 weeks of transformation and have found the results to be reasonably indicative of mature (T2) seed fatty acid profiles. A similar strategy can also be extended to induce somatic embryogenesis in oil seed crops for the testing and validation seed specific traits quickly before stable transformation.

One thousand five hundred rapeseed accessions were collected from gene banks in several countries and surveyed for their seed traits such as oil content, protein content, fatty acid composition and seed weight. Seed chemicals were analyzed by near-infrared reflectance spectroscopy (NIRS). An extensive genetic variation existed among accessions for all seed traits examined. The oil content ranged from 24.7 to 56.9%, and the protein content ranged from 6.7 to 36.6%. A hundred seed weight ranged from 0.13 to 0.70 g. The fatty acid compositions of germplasms also varied from 2.6 to 9.2% of C16:0 (palmitic acid), 0.3 to 70.2% of C18:1 (oleic), 10.3 to 28.9% of C18:2 (linoleic), 4.8 to 15.3% of C18:3 (linolenic), 0 to 10.5% of C20:1(eicosenoic) and 0 to 59.6% of C22:1 (erucic). The oil content had strong negative correlation with protein content (r = −0.848). However, it did not have any close correlations with other traits. When the genetic stability of seed traits was examined by comparison between original seeds and self-pollinated seeds (S1 seeds), higher positive and significant correlation coefficients between original seeds and S1 seeds were found in all seed traits.

Background Omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) such as eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) are critical for human health and development. Numerous studies have indicated that deficiencies in these fatty acids can increase the risk or severity of cardiovascular, inflammatory and other diseases or disorders. EPA and DHA are predominantly sourced from marine fish although the primary producers are microalgae. Much work has been done to engineer a sustainable land-based source of EPA and DHA to reduce pressure on fish stocks in meeting future demand, with previous studies describing the production of fish oil-like levels of DHA in the model plant species, Arabidopsis thaliana. Principal Findings In this study we describe the production of fish oil-like levels (>12%) of DHA in the oilseed crop species Camelina sativa achieving a high ω3/ω6 ratio. The construct previously transformed in Arabidopsis as well as two modified construct versions designed to increase DHA production were used. DHA was found to be stable to at least the T5 generation and the EPA and DHA were found to be predominantly at the sn-1,3 positions of triacylglycerols. Transgenic and parental lines did not have different germination or seedling establishment rates. Conclusions DHA can be produced at fish oil-like levels in industrially-relevant oilseed crop species using multi-gene construct designs which are stable over multiple generations. This study has implications for the future of sustainable EPA and DHA production from land-based sources.