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.

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 T 5 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.

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.

Competing interests: This study was partly funded by Nuseed Pty Ltd and the Australian Grains Research and Development Corporation (GRDC). Some of the data provided in this study may have been included in patent applications. CSIRO is in a commercial arrangement with GRDC and Nuseed to develop DHA land plants, although there is no product name as yet. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Funding: This research was funded by CSIRO Food Futures Flagship, Nuseed Pty Ltd and the Australian Grains Research and Development Corporation. The funders had no role in study design, data collection and analysis, or preparation of the manuscript. The authors were granted permission to publish the manuscript by the funders.

Copyright: © 2014 Petrie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Camelina sativa, also known as Gold of Pleasure and False Flax, is an ancient cultivated oilseed crop [13] , [14] with naturally high levels of the C18 ω3 ALA in seed oil. Commercial cultivation of the crop slowed significantly with the introduction of oilseed rape but has recently garnered significant attention as an underutilised species. Some C. sativa varieties have high oil content (in excess of 40%) and have strong agronomic performance on marginal lands [15] and approvals have recently been granted for C. sativa meal or oil use in food and animal feed applications in USA and Canada. In this article we describe the production of fish oil-like levels of DHA (12%) in C. sativa by the introduction of a transgenic Δ6-desaturase pathway ( Figure 1 ) consisting of both yeast and microalgal genes to convert native OA (oleic acid, 18∶ω9), LA and ALA substrates to the beneficial ω3 LC-PUFA EPA and DHA.

The omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) EPA (eicosapentaenoic acid, 20∶5ω3) and DHA (docosahexaenoic acid, 22∶6ω3) are recognized for their strong health benefits. Developing an oilseed source of these fatty acids is desirable since these provide far stronger health benefits than the shorter chain terrestrial plant precursors of these fatty acids, α-linolenic acid (ALA, 18∶3ω3) and stearidonic acid (SDA, 18∶4ω3) [1] . Moreover, the conversion of these precursor fatty acids to long-chain variants occurs at surprisingly low levels in humans [2] . Importantly, studies have also found that high concentrations of ω6 fatty acids such as LA (linoleic acid, 18∶2ω6), GLA (γ-linolenic acid, 18∶3ω6) and ARA (arachidonic acid, 20∶4ω6) can decrease the bioconversion efficiency of C18 ω3 fatty acids to long-chain PUFA [2] . The production of ω3 LC-PUFA in land plants as been a long-standing goal of bioengineers. The first demonstrations of LC-PUFA production were published in 2004 and showed EPA and ARA production in leaf [3] and seed [4] . The first demonstrations of DHA production in seed were published soon after [5] , [6] . Subsequent work resulted in increasing levels of production, particularly for EPA [7] – [9] . The first report of production of fish oil-like levels of DHA in seed was published in 2012 [10] . Progress has been extensively reviewed in recent years [11] , [12] .

A hexane-extracted oil (>99% TAG, confirmed by TLC-FID; results not shown) was obtained by sequential extraction of crushed seeds and prior to analysis was stored at -18°C. Samples were warmed to room temperature 1 h prior to NMR sample preparation. 100 mg oil was dissolved in deuteriochloroform containing 25 mM Tris(acetylacetonate) chromium(III) as a relaxation agent (0.6 mL). The solutions were transferred to 5 mm O.D. NMR tubes (New Era NE-UL5-7) and sealed with PTFE lids. Solutions for NMR spectroscopy were stored at 4°C until they were inserted into the magnet. Quantitative 13 C NMR spectra were acquired on a Bruker BioSpin Av500 NMR spectrometer equipped with a 5 mm 1 H- 13 C/ 15 N triple-resonance inverse probe operating at 125.8 MHz for 13 C. The data were acquired and processed in Bruker BioSpin TopSpin v3.2. The samples were maintained at 25°C during acquisition. 128 k data points were collected over a spectral width of 26.3 kHz summed over 46 k scans. Inverse-gated, bilevel adiabatic 1 H-decoupling was employed with an acquisition time of 2.49 s and a recycle delay of 2.5 s. Data were processed to 128 k data points using a Gaussian multiplication with a Gaussian position factor of 0.12 and a line broadening of −0.15 Hz prior to Fourier transformation; a 5th-order polynomial baseline correction was applied to each spectrum. Spectra were referenced to the peak arising from C1 of 22∶6ω3 in the sn-2 position of TAG at 172.13 ppm [19] and the signals assigned using the published assignments [20] . The raw data were processed in a similar fashion in triplicate and the mean and standard deviations calculated.

RNA was extracted from developing seed taken from three plants of a high DHA line transformed with either GA7, mod-F or mod-G using RNeasy Plant Mini kit (Qiagen, Hilden, Germany). Total RNA (1 µg) was converted to cDNA using First-Strand cDNA synthesis mix (OriGene Inc., Australia). Diluted cDNA (0.2×) was used for quantitative real-time PCR by Bio-Rad CFX Real-Time System (BIO-RAD, USA) using iQSYBR Green Supermix (BIO-RAD). Reactions were carried with initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 10 sec, 58°C for 30 sec and 68°C for 30 sec. Target gene expression was normalized to the endogenous C. sativa HMG gene in the Bio-Rad CFX Manager software.

DHA levels in T 5 seeds from 15 randomly selected homozygous plants grown in three glasshouses were analysed by One Way Analysis of Variance (ANOVA) using Sigmaplot (v12) by the Holm-Sidak method [18] .

Total lipid was extracted from seeds using chloroform: methanol: 0.1 M KCl (2∶1∶1 v/v/v as described in [17] . Neutral lipid classes and polar lipid were fractionated from the total lipid on a TLC plate (Silica gel 60, MERCK) using a mixture of hexane: diethylether: acetic acid (70∶30∶1, v/v). The lipid bands were visualized under UV after spraying the plate with 0.001% primuline dissolved in acetone:water (80∶20 v/v). The individual lipid bands were identified on the basis of authentic lipid standards, which were run parallel to the seed lipid samples in the TLC, collected into separate glass vials and their fatty acid methyl esters (FAME) were prepared together with known amount of heptadecanoic acid as internal standard. FAMEs were analysed by GC as previously described [10] and individual lipids were quantified on the basis of the amounts of FAMEs produced from the known amount of internal standards used.

Constructs were transformed in Agrobacterium tumefaciens strain AGL1 and cultured at 28°C on a rotary shaker to appropriate growth phase. C. sativa (“Celine”) was transformed by a floral dip method adapted from Liu et al. [16] . Briefly, the freshly opened flower buds were dipped in A. tumefaciens solution for 15 s, wrapped in plastic film and left overnight in the dark at 24°C after which the plastic was removed.

The box denotes the unchanged region. Abbreviations are: TER, terminator or polyadenylation region; PRO, promoter; NOS, Agrobacterium tumefaciens nopaline synthase terminator; FP1, Brassica napus truncated napin promoter; FAE1, Arabidopsis thaliana FAE1 promoter; Lectin, Glycine max lectin terminator; Cnl1 and Cnl2 denotes the Linum usitatissimum conlinin1 or conlinin2 promoter or terminator. MAR denotes the Rb7 matrix attachment region from Nicotiana tabacum and gene identities are given in the text and the Materials and Methods section.

Figure 2. DHA pathway construct maps described in this study: A) GA7 parental construct; B) mod-F variant with an additional Δ6-desaturase, elongase coding regions switched and the original FP1 promoter replaced by Cnl2; C) mod-G with the Δ5-elongase and Δ6-desaturase coding regions switched.

Constructs mod-F and mod-G were made using a combination of DNA synthesis and restriction enzyme-based cloning starting with the previously described GA7 parent vector [10] , Figure 2 . Mod-F was made by replacing the SbfI-flanked Δ5-elongase expression cassette near the left border with a Δ6-elongase expression cassette. The original Δ6-desaturase expression cassette and the adjacent Δ6-elongase promoter and coding region were then excised with AscI + PmeI and replaced with the Cnl2:: Δ6-desaturase::NOS expression cassette plus the replacement Δ5-elongase promoter and coding region. The second Δ6-desaturase expression cassette was then added at the PmeI site as a PmeI-SwaI blunt-ended fragment to generate mod-F. Construct mod-G was generated by first replacing the original Δ6-desaturase expression cassette and the adjacent Δ6-elongase promoter and coding region (flanked by AscI + PmeI) with the new Δ5-elongase expression cassette and adjacent Δ6-elongase coding region. The original Δ5-elongase expression cassette adjacent to the left border (flanked by SbfI) was then replaced with the Δ6-desaturase expression cassette.

Results and Discussion

Construct design and manufacture Use of construct pJP3416_GA7 (GA7) to generate DHA-containing seeds in A. thaliana has previously been described [10]. Two GA7 construct variants, referred to here as mod-F and mod-G, were designed to improve the efficiency of the Δ6-desaturase and Δ6-elongase steps. The core GA7 sequence was left intact (Figure 2a) with only the terminal regions that contained the genes of interest modified. Specifically, the GA7 terminal FP1/NOS promoter/terminator pair was replaced with the Linum usitatissimum conlinin2 (Cnl2) promoter/terminator pair in both new variants. The mod-F changes consisted of the switching of the two elongase coding regions as well as the addition of a second Micromonas pusilla Δ6-desaturase coding region with different codon usage to the original GA7 version (Figure 2b). In addition to the conlinin2 cassette changes described above, the Δ6-desaturase and Δ5-elongase coding regions were also switched in mod-G (Figure 2c). Conversion of ALA to SDA (Δ6-desaturation) had previously been identified as a bottleneck in DHA production in A. thaliana and in this study we tested both the effect of using different promoters (A. thaliana FAE1 and L. usitatissimum Cnl2) and the addition of a second expression cassette with different gene codon usage to avoid gene silencing. The changes made in mod-G also tested whether the expression cassette adjacent the right border was intrinsically compromised [21] by switching the Δ6-desaturation and Δ5-elongase cassettes. Similarly, the Δ5-elongase had previously been shown to have very high activity whilst the Δ6-elongase had lower activity. The changes in mod-F included switching the two elongase coding regions to test whether this was due to the expression cassette rather than the gene.

Camelina sativa transformation Constructs were sequence confirmed and transformed in Agrobacterium strain AGL1 before floral dip of C. sativa. After the floral dip, plants were grown to maturity, seed harvested, and germinated in soil trays. Established seedlings (7–10 days) were sprayed with 0.1% BASTA herbicide (250 g/L glufosinate ammonium; Bayer Crop Science Pty Ltd, VIC Australia) to kill plants not expressing the selectable marker gene. The original GA7 construct was used to establish the C. sativa transformation protocol in the lab and was transformed before the mod-F and mod-G constructs.