Abstract
The rapid assessment of metabolic engineering strategies in plants is aided by crops that provide simple, high throughput transformation systems, a sequenced genome, and the ability to evaluate the resulting plants in field trials. Camelina sativa provides all of these attributes in a robust oilseed platform. The ability to perform field evaluation of Camelina is a useful, and in some studies essential benefit that allows researchers to evaluate how traits perform outside the strictly controlled conditions of a greenhouse. In the field the plants are subjected to higher light intensities, seasonal diurnal variations in temperature and light, competition for nutrients, and watering regimes dictated by natural weather patterns, all which may affect trait performance. There are difficulties associated with the use of Camelina. The current genetic resources available for Camelina pale in comparison to those developed for the model plant Arabidopsis thaliana; however, the sequence similarity of the Arabidopsis and Camelina genomes often allows the use of Arabidopsis as a reference when additional information is needed. Camelina’s genome, an allohexaploid, is more complex than other model crops, but the diploid inheritance of its three subgenomes is straightforward. The need to navigate three copies of each gene in genome editing or mutagenesis experiments adds some complexity but also provides advantages for gene dosage experiments. The ability to quickly engineer Camelina with novel traits, advance generations, and bulk up homozygous lines for small-scale field tests in less than a year, in our opinion, far outweighs the complexities associated with the crop.
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Notes
Canadian Food Inspection Agency, The Biology of Camelina sativa (L.) Crantz (Camelina). http://www.inspection.gc.ca/plants/plants-with-novel-traits/applicants/directive-94-08/biology-documents/camelina-sativa-l-/eng/1330971423348/1330971509470?wbdisable=true.
USDA-APHIS response to Yield10 Bioscience “Am I Regulated?” letter of inquiry for CRISPR/Cas9 genome editing of a gene in Camelina. https://www.aphis.usda.gov/biotechnology/downloads/reg_loi/17-166-01_air_response_signed.pdf.
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Acknowledgements
This work was supported in part by the US Department of Energy’s Bioenergy Technologies Office (BETO) under Award Number DE-EE0007003, and the Advanced Research Projects Agency—Energy (ARPA-E), US Department of Energy, under Award Number DE-FOA-0000470.
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All authors are employees of either Metabolix Oilseeds, Inc. or Yield10 Bioscience, Inc. Metabolix Oilseeds is a wholly owned subsidiary of Yield10 Bioscience, Inc.
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Communicated by Neal Stewart.
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Online Resource 1
T1 seeds of Camelina sativa cv. 10CS0043 co-transformed with expression cassettes for genes encoding the visual markers DsRed and GFP. a. Transformation construct pMBXS700 containing expression cassettes for the DsRed2B, a derivative of DsRed (Malik et al. 2015), and nptII genes, both under the control of the CaMV35S promoter. Expression of the nptII gene provides plants resistance to kanamycin. b. Transformation construct pMBXO12 containing an expression cassette for SYN-GFP-S65T, a variant of GFP with a mutation in the chromophore at position 65 (Chiu et al. 1996), under the control of the soybean oleosin promoter (Rowley and Herman 1997), and an expression cassette for Bar under the control of the CaMV35S promoter. The expression of the Bar gene provides the plant resistance to the herbicide bialophos. Both vectors pMBXS700 and pMBXO12 are pCAMBIA based binary vectors (Centre for Application of Molecular Biology to International Agriculture, Canberra, Australia). T1 seeds from co-transformation of pMBXS700 and pMBXO12 visualized with a Nikon AZ100 microscope as seen under (c) white light, (d) a filter for DsRed visualization [TRITC-HQ(RHOD)2 filter module (HQ545/30X, Q570LP, HQ610/75M)], and (e) a filter for GFP visualization (eGFP filter, excitation bandpass 470/40, emission bandpass 525/50). Wild-type seeds (WT, DsRed-, GFP-) are shown as a negative control. Seeds in (c), (d), and (e) are a pool of pre-selected T1 and wild-type seeds to illustrate visualization of the two markers and do not represent the efficiency of co-transformation (PPTX 225 KB)
299_2018_2308_MOESM2_ESM.pptx
Online Resource 2 A reverse glyoxylate shunt (rGS) pathway designed for increased carbon fixation in seed plastids of Camelina (Malik et al. 2016). Genes encoding enzymes for this pathway are engineered into binary vectors pMBXS919 and pMBXS1023 (Online Resource 3 and 4a). Variants of this pathway contain an additional gene encoding malate synthase (aceB, binary vectors pMBXS919 and pMBXS1022, Online Resource 4a) or contain the additional gene encoding malate synthase (aceB) but do not contain the gene encoding pyruvate oxidoreductase (por, binary vectors pMBXS919 and pMBXS1024, Online Resource 4a) (PPTX 204 KB)
Online Resource 3
Seed-specific expression of plastid targeted rGS pathway shown in Online Resource 2. Transformation constructs pMBXS919 and pMBXS1023 were co-transformed into Camelina sativa cv. 10CS0043. a. Plasmid map of pMBXS919 with seed-specific expression cassettes for MDH5 (NADP specific malate dehydrogenase from C. reinhardtii), fumC (fumarate hydratase class II from Escherichia coli), FRDg, (fumarate reductase from Trypanosoma brucei), acnA (aconitase from Escherichia coli), aclA-1, (subunit of ATP-citrate lyase from A. thaliana), and aclB-2 (subunit of ATP-citrate lyase from A. thaliana). A DNA sequence encoding a plastid signal peptide was fused to the N-terminus of each gene to direct the encoded protein to the plastid. The plastid signal peptide consisted of DNA encoding the signal peptide from the ribulose-1,5-bisphosphatase carboxylase (Rubisco) small subunit from Pisum sativum, including the first 24 amino acids of the mature protein (Cashmore 1983). A three amino acid linker containing an Xba I restriction site allowed direct fusion of the desired transgene to the plastid signal peptide (Kourtz et al. 2005). Each plastid targeting signal modified gene was placed between the soybean oleosin seed-specific promoter (pOle) (Rowley and Herman 1997) and corresponding soybean 3’-termination sequence (tOle) to form the seed-specific expression cassettes. An expression cassette for the bar gene, driven by the CaMV35S promoter, imparts transgenic plants resistance to the herbicide bialophos. Seed-specific expression cassettes were cloned into pCAMBIA based vectors using conventional cloning techniques. T-DNA LB and T-DNA RB denote the left and right border, respectively, of the T-DNA. b. Plasmid map of pMBXS1023 with seed-specific expression cassettes for the genes por (pyruvate oxidoreductase from Desulfovibrio africanus), pyc (pyruvate carboxylase from Bacillus subtilis), mcl (malyl CoA lyase from Rhodobacter sphaeroides), and iclA (isocitrate lyase from Cupriavidus necator). Co-expression of M. capsulatus sucC and sucD in recombinant E. coli was recently shown to provide malate thiokinase activity ADDIN EN.CITE (Mainguet et al. 2013). Expression cassettes for a synthetic gene encoding green fluorescent protein (SYN-GFP-S65T), with a mutation in the chromophore at position 65 (Chiu et al. 1996), were driven by either the CaMV35S or soybean oleosin promoters to allow detection of transgenic seeds by fluorescent microscopy. To identify co-transformed lines, SYN-GFP-S65T expressing seeds were visualized by fluorescent microscopy using a Nikon AZ100 microscope with an eGFP filter (Excitation bandpass 470/40, Emission Bandpass 525/50) and planted in soil. The presence of the bar gene on the T-DNA of the pMBXS919 construct allowed selection of co-transformants by spraying a solution of 400 mg/L of the herbicide Liberty (active ingredient 15% glufosinate-ammonium) on plantlets obtained from GFP expressing seeds. c. PCR confirmation of all genes for rGS pathway in a T1 plant (top panel, line LU03) co-transformed with plasmids pMBXS919 and pMBXS1023. Genomic DNA was extracted from leaf tissue for line LU03 and the wild-type control. The primers used for screening were designed to produce an amplicon of ~ 300 bp from each gene. d. RT-PCR confirmation of expression of all genes for rGS pathway in T3 developing seeds of line LU03. A two-step RT- PCR procedure was performed in 20 DAF (days after flowering) developing seeds of line LU03 according to previously described protocols (Malik et al. 2015) (PPTX 233 KB)
Online Resource 4
Yield parameters from seeds of Camelina sativa cv. 10CS0043 co-transformed with various rGS pathway plasmids compared to seeds of control line JS11. a. Plasmids used in study. Abbreviations for genes and the enzymes that they encode are listed in Online Resource 3. b. Seed yield per plant (g) and seed oil content (% oil). Oil content is expressed as the percent of seed weight. Red dotted horizontal lines show the average seed yield and average % oil content of control line JS11. c. Total seed oil produced per plant. Red dotted horizontal line shows the average total oil produced per plant in seeds of control lines JS11. d. Seed weight/100 seeds of high oil producing plants from c. (n=3 100 seed weight measurements, *P ≤ 0.001). 100 seed weight of plant 15-0417 from control event JS11 is 106.64 + 2.71. e. Seed yield and oil content increases in best plants. Control for all data is line JS11 obtained from transformation of Camelina sativa cv. 10CS0043 with plasmid pMBXO12 expressing the bar and GFP genes. Plasmid maps of pMBXS919 and pMBXS1023 are shown in Online Resource 3. Floral dip in planta transformation to generate T1 seeds is described in Online Resource 3. T1 plants were propagated in soil in a greenhouse (22/18◦C day/night and a photoperiod of 16h under supplemental light ≥500µmolm-2s-1) to produce T2 seeds. Events LX03, LG08, and LU03 were chosen for yield analysis. Eight plants of each event (T2 plants for events LX03, LG08, and LU03; T3 plants for control Event JS11) were grown in a greenhouse in a randomized complete block design at 22/18◦C day/night and a photoperiod of 16h under supplemental light ≥500µmolm-2s-1. Total seed yield per plant was determined by harvesting all of the mature seeds from a plant and drying them in an oven with mechanical convection set at 22°C for two days. The weight of the entire harvested seed was recorded. Total seed oil content was determined as previously described (Malik et al. 2015) (PPTX 384 KB)
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Malik, M.R., Tang, J., Sharma, N. et al. Camelina sativa, an oilseed at the nexus between model system and commercial crop. Plant Cell Rep 37, 1367–1381 (2018). https://doi.org/10.1007/s00299-018-2308-3
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DOI: https://doi.org/10.1007/s00299-018-2308-3