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Plant Cell Reports

, Volume 37, Issue 10, pp 1367–1381 | Cite as

Camelina sativa, an oilseed at the nexus between model system and commercial crop

  • Meghna R. Malik
  • Jihong Tang
  • Nirmala Sharma
  • Claire Burkitt
  • Yuanyuan Ji
  • Marie Mykytyshyn
  • Karen Bohmert-Tatarev
  • Oliver Peoples
  • Kristi D. Snell
Review

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.

Keywords

Camelina sativa Metabolic engineering Model crop Gene editing Doubled haploid Field trials 

Introduction

Camelina sativa (L.) Crantz, an annual plant that is part of the Brassicaceae family, has recently found widespread use as a model crop for testing metabolic engineering strategies as evidenced by the significant number of recent journal articles that have been published on the subject (Table 1). Like Arabidopsis thaliana, the ease of Camelina transformation using floral dip methods (Lu and Kang 2008) and its short lifecycle of approximately 85–100 days, depending on genotype and growth conditions (Moser 2010), are ideal for high throughput transformation systems making the plant a good platform for testing novel metabolic engineering strategies (Fig. 1). The ability to start evaluation of homozygous engineered Camelina plants in field tests as soon as a year after the original transformation procedure (Fig. 1), using conventional planting and harvesting equipment, is a key advantage of this crop over Arabidopsis. This allows the researcher to evaluate trait performance in real world conditions where the plants are subjected to the benefits, as well as the stresses, of higher light, variable weather, and where they must compete with other plants for available nutrient and water resources.

Table 1

Select examples of metabolic engineering performed in Camelina sativa

Target trait or product

Cultivara

Description

References

Biopolymers

 PHB

10CS0043

Seed plastid specific production of the biopolymer polyhydroxybutyrate

Malik et al. (2015)

Carbon fixation cycles

 Reverse glyoxylate shunt pathway

10CS0043

Expression of 12–13 genes to produce a reverse glyoxylate shunt pathway in seed plastids

Malik et al. (2016) Online Resources 2, 3 & 4

Fatty acids and oils

 Acetyl-triacylglycerols (acetyl-TAG)

Celine

Expression of a diacylglycerol acetyltransferase (DAcT) and RNAi suppression of DGAT1 increased acetyl-TAG levels in seed oil

Liu et al. (2015)

 Hydroxyl fatty acids (HFAs)

Suneson

High levels of HFAs produced in seeds through co-expression of a Lesquerella fatty acid elongase and the castor fatty acid hydroxylase

Snapp et al. (2014)

 Linoleic and α-linolenic acid composition of oil

Cam 139

Silencing DGAT and overexpression of PDAT led to an increase in linoleic and a decrease in α-linolenic acids in seed oil

Marmon et al. (2017)

 Medium chain saturated fatty acids

Calena

Seed-specific expression of a 12:0-acyl-carrier thioesterase gene combined with RNAi suppression of KASII genes yielded enhanced accumulation of medium chain saturated fatty acids in seeds

Hu et al. (2017)

 

Suneson

Seed-specific expression of various acyl-carrier-thioesterase (FATB) genes resulted in accumulation of medium chain fatty acids of various lengths

Kim et al. (2015)

 Oleic acid

Suneson

High levels of oleic acid produced in seeds through antisense repression of FAD2 genes in seeds

Kang et al. (2011)

 

Suneson

High oleic acid lines produced by downregulating FAD2 and FAE1

Nguyen et al. (2013)

 Very long chain fatty acids (VFLAs)

Suneson

Nervonic acid produced in seeds through seed-specific expression of ketoacyl-CoA synthase

Huai et al. (2015)

 ω-3 long chain polyunsaturated fatty acids

Celine used in Petrie et al. (2014), other references do not describe cultivar

ω-3 long chain polyunsaturated fatty acids produced in seeds

Petrie et al. (2014); Ruiz-Lopez et al. (2015); Ruiz -Lopez et al. (2014); Usher et al. (2017)

 ω-7 monounsaturated fatty acids

Suneson

High levels of ω-7 monounsaturated fatty acids produced in seeds

Nguyen et al. (2015)

Increased seed yield and/or seed oil content

 Seed yield and oil content

Suneson

Co-expression of DGAT1 and cytosolic GPD1 genes under the control of seed-specific promoters yielded significant increases in seed mass and oil content

Chhikara et al. (2017)

 

Came

Seed-specific expression of WRI1 gave increased seed mass, seed size, and oil content

An and Suh (2015)

 Seed oil content

Suneson

Constitutive expression of patatin-related phospholipase AIIIδ (pPLAIIIδ) significantly increased seed oil content while decreasing cellulose content and seed production; seed-specific expression of pPLAIIIδ increased oil content while maintaining normal seed production

Li et al. (2015)

 

Came

Seed-specific DGAT1 expression increased oil content

Kim et al. (2016)

 Seed yield

Cultivar not described

Constitutive expression of purple acid phosphatase 2 produced more seeds with increased seed size

Zhang et al. (2012)

 

Suneson

Increased seed size, number, and mass through seed-specific or constitutive expression of an Arabidopsis GTP-binding protein (Gγ, AGG3)

Roy Choudhury et al. (2014)

Photorespiration

 Photorespiratory bypass

Calena

Increased plant growth and yield through constitutive overexpression of chloroplast targeted bacterial photorespiratory bypass pathway

Dalal et al. (2015)

Terpenes

 Isoprene

Calena

Increased isoprene production through constitutive expression of an isoprene synthase gene resulted in shorter plants with smaller leaves

Rossi et al. (2017)

 Limonene

Calena

Higher limonene production in siliques and seeds using seed and valve specific promoters to express a limonene synthase gene

Borghi and Xie (2016)

 Limonene and δ-cadinene

Cultivar not described

Compared cytosolic and plastid pathways to produce limonene and cadinene; plastid-based expression produced higher amounts of product

Augustin et al. (2015)

 Increased β-amyrin and campesterol production

Calena

Constitutive expression of a plastid targeted, synthetic insect-plant geranyl pyrophosphate synthase gene enhanced plant growth and promoted early flowering; increased levels of β-amyrin and campesterol observed

Xi et al. (2016)

Waxes

 Wax esters

Ligena

Plants engineered to express fatty acid acyl-CoA reductase and wax synthase in seeds produced high levels of wax esters

Iven et al. (2016)

 

Cultivar not described

Pathways engineered to tailor composition of wax esters produced in seeds

Ruiz-Lopez et al. (2017)

DGAT, diacylglycerol acyltransferase; PDAT, phospholipid:diacylglycerol acyltransferase; FAD2, fatty acid desaturase 2; FAE1, fatty acid elongase 1; GPD1, glycerol-3-phosphate dehydrogenase 1; KASII, β-ketoacyl-acyl-carrier protein synthase II

aCamelina cultivars used in the metabolic engineering studies are described in Table 2

Fig. 1

The ease of transformation and short life cycle of Camelina allows the quick generation of homozygous seed that can be used in small-scale field trials to test a trait of interest. Wild-type plants with buds that are just starting to open are transformed by Agrobacterium-mediated floral dip procedures. Inclusion of a visual marker such as DsRed allows the identification of T1 transgenic seeds from untransformed seeds under a fluorescence microscope or by shining light of the correct wave length for excitation of the visual marker and viewing with an appropriate filter (Malik et al. 2015). T1 lines are grown in the greenhouse, PCR verified, and T2 seed is collected. Lines are grown for several additional generations to obtain homozygous lines and for seed bulk up purposes (typically two additional generations). Bottom pictures are from small scale Yield10 Bioscience and Metabolix Oilseeds field trials of yield traits

Table 2

Cultivars of Camelina mentioned in this review

Genotype

Variety

Origin

References

Blaine Creek

Spring

Developed by Duane Johnson and released through Montana State University

Eynck and Falk (2013); Jewett (2015)

Calena

Spring

Vienna, Austria; developed by Johann Vollmann

Eynck and Falk (2013); Vollmann et al. (2005)

Calinka

Spring

Vienna, Austria

Vollmann et al. (2005)

Came

Spring

Institut fur Pflanzengenetik und Kulturpflanzenforschung, Genebank-Aussenstelle Malchow, Germany; maintained at USDA-ARS North Central Regional PI Station, Ames, Iowa as PI 650140

https://training.ars-grin.gov/gringlobal/AccessionDetail.aspx?id=1522423

Cam 139

Spring

Maintained at Genebank, Leibniz Institute of Plant Genetics and Crop Plant Research, Germany

https://pgrdeu.genres.de/exsitu/steckbrief/ex_id/1875501/sdb_id/1/genus/Q2FtZWxpbmE%3D/species/c2F0aXZh/spauthor/KEwuKSBDcmFudHo%3D/subtaxa/c3Vic3AuIHNhdGl2YQ%3D%3D/subtauthor/KERjLikgTi5XLlppbmdlcg%3D%3D/navleaf/genbankpa

Celine

Spring

Developed by Limagrain, France

Eynck and Falk (2013)

DH55

Spring

Doubled haploid line used to generate genome sequence

Kagale et al. (2014)

Ligena

Spring

Developed by DSV Deutsche Saatveredelung, Germany

Eynck and Falk (2013)

Suneson

Spring

Developed by Duane Johnson and released through Montana State University

Eynck and Falk (2013); Jewett (2015)

10CS0043

Spring

Isolated in breeding program at Agriculture and Agri-Food Canada (AAFC)

Kevin Falk (personal communication)

Joelle

Winter

Origin unclear, available from North Dakota State University Research and Extension Center

Gesch and Cermak (2011)

Camelina has also received considerable interest as an industrial platform crop for the production of biodiesel, jet fuel, novel industrial lipids, oleochemicals, specialty oils, and polymers (Bansal and Durrett 2016; Berti et al. 2016; Iskandarov et al. 2014; Sainger et al. 2017; Snell and Peoples 2013). Camelina seeds contain high levels of oil, often between 32 and 49% of the seed weight depending on the genotype, growth conditions, and fertilizer (Vollmann and Eynck 2015), and the crop has the ability to thrive on marginal land with low inputs (Eynck et al. 2013). Adoption of the crop has however been slow, and increasing the crop’s yield has been suggested as a key driver to reduce feedstock production costs and increase crop revenue (Zering 2015).

For traits whose commercialization would likely occur as a specialty crop, such as oils containing omega fatty acids or biopolymers, Camelina can span basic research through commercialization by providing an easily transformable robust research model system that can transition to a field testing program and commercial line development if warranted. Our laboratories have been working with Camelina since 2010, first in efforts to produce the bacterial biopolymer polyhydroxybutyrate (PHB) as a value-added coproduct in seeds (Malik et al. 2015; Snell and Peoples 2013), and more recently to test novel yield genes and pathways. We have found the Camelina platform to be robust and amenable to the engineering of complex multi-gene pathways as well as the manipulation of genes through multiplex genome editing. This review will draw on our experience with the crop and will highlight the benefits and limitations of Camelina as a platform crop for plant metabolic engineering, as well as highlight the extensive work that has been done with Camelina to modify endogenous or engineer novel metabolic pathways.

The Camelina platform

Transformation

Camelina sativa includes both spring and winter varieties (Berti et al. 2016), with winter cultivars often requiring a vernalization step to flower. To date, all of the published transformation and metabolic engineering work with Camelina has utilized spring varieties which will be the focus of this review. There are multiple transformation methods available for Camelina, with Agrobacterium-mediated floral dip in planta transformation (Lu and Kang 2008) highly favored by most researchers due to its simplicity. This method, originally demonstrated with Camelina sativa cv. Celine (Table 2) (Lu and Kang 2008), is very similar to the routinely used floral dip transformation procedure of Arabidopsis (Clough and Bent 1998). The floral dip method was later demonstrated to be useful for transforming a variety of different cultivars of Camelina, even without a vacuum-infiltration step, achieving a transformation efficiency of up to 0.8% (Liu et al. 2012). In our hands, the transformation efficiency of Camelina ranges from 1–4 transformed seeds generated per dipped plant, a number that can vary with the health and genotype of the plant used for dipping as well as the transgene expression construct. Of the transformed events that we typically obtain, approximately 7–12% are single copy lines as determined by standard Southern blotting procedures. The pairing of floral dip transformation with the use of visual markers for identification of transformed seeds (Table 3; Fig. 1, Online Resource 1), makes the generation of large numbers of transformed lines routine. Several researchers have also transformed or co-transformed one or more multi-gene expression constructs expressing five or more genes (Table 4), including in our laboratory where we have transformed a novel carbon fixation pathway that utilizes 12–13 transgene expression cassettes and three visual and/or selectable marker expression cassettes [(Malik et al. 2016), Table 4, Online Resources 2, 3, and 4].

Table 3

Examples of visual markers that have been used successfully to identify Camelina transformants

Visual marker

Marker description

Examples of use in Camelina

DsRed

Red fluorescent protein from the Discosoma genus of coral (Matz et al. 1999)

Lu and Kang (2008); Malik et al. (2015); Ruiz-Lopez et al. (2014); co-transformation with GFP marker shown in Online Resource 1

mCherry

Marker generated by mutation of DsRed (Shaner et al. 2004)

Dalal et al. (2015)

GFP (green fluorescent protein)

Green fluorescent protein from Aequorea victoria (Chalfie et al. 1994)

Yield10, unpublished results with sGFP(S65T) (Chiu et al. 1996) including co-transformation with DsRed marker (Online Resource 1)

Table 4

Examples of complex pathways engineered into Camelina sativa that contain more than five expression cassettes

Target

Plasmids transformed

Expression cassettes

References

Transgenes

Markersb

Total

Seed-specific, plastid targeted, carbon fixation pathway

2a

6

1

16

Malik et al. (2016); Online Resources 2, 3, & 4

7

2

ω-3 Long chain polyunsaturated fatty acids

1

7

1

8

Ruiz-Lopez et al. (2014)

1

8

1

9

Petrie et al. (2014)

1

6

1

7

Ruiz-Lopez et al. (2015)

1

7

1

8

Usher et al. (2015)

1

7

1

8

Usher et al. (2017)

aPlasmids pMBXS919 and pMBXS1022 co-transformed into same plant (Online Resource 4)

bSelectable and visual markers

A tissue culture-based transformation system has also been described (Kuvshinov et al. 2011) that involves Agrobacterium-mediated transformation of excised Camelina leaves and was demonstrated with the Calena and Calinka cultivars (Table 2). In our laboratory, we have also developed polyethylene glycol (PEG) mediated transformation of Camelina protoplasts using the cultivar Suneson (Table 2) (Metabolix Oilseeds/Yield10 Bioscience, unpublished results). In our hands, regeneration of shoots from Camelina callus cultures and their subsequent rooting is however inefficient which significantly reduces the number of plants that can be obtained from tissue culture-based transformation methods.

Genome

Camelina was proposed to have an allohexaploid genome by Hutcheon et al. in gene isolation studies that focused on fatty acid biosynthetic genes (Hutcheon et al. 2010). During this work, the researchers identified three copies of their genes of interest, as well as other genes that are typically single copy within the genomes of diploid species. The Camelina genome has been reported to be approximately 750 Mb (Hutcheon et al. 2010)–785 Mb (Kagale et al. 2014) in size with a chromosome count of n = 20 (Gehringer et al. 2006). The genome sequence of a doubled haploid line of Camelina, DH55 (Table 2), was first published in 2014 (Kagale et al. 2014) and is publically available (Table 5). The transcriptome of Camelina has been reported from several independent labs (Liang et al. 2013; Mudalkar et al. 2014; Nguyen et al. 2013) including studies focused on the seed transcriptome (Abdullah et al. 2016; Wang et al. 2015). A Camelina developmental transcriptome atlas has also been published based on comprehensive genome-wide expression profiling of various tissues covering major developmental stages during the plant’s life cycle (Kagale et al. 2016).

Table 5

Internet databases and bioinformatics tools useful for Camelina metabolic engineering

Database or tool (latest release version)

Descriptiona

Websites/references

Camelinadb.ca (V2)

Provides C. sativa cv. DH55 genome sequence and associated gene models

Kagale et al. (2014) http://www.camelinadb.ca/

GenBank

NIH genetic sequence database providing access to C. sativa cv. DH55 and cv. CAME genome sequences and gene models

For cv. DH55: NCBI Camelina sativa Annotation Release 100 https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Camelina_sativa/100/, Genome assembly accession GCF_000633955.1

For cv. CAME: Genome assembly accession https://www.ncbi.nlm.nih.gov/assembly/GCA_000496875.1/

Camelina developmental transcriptome atlas

The eFP browser provides easy access and enables visualization of the expression levels of C. sativa genes and allows basic queries by the annotated gene names

Kagale et al. (2016) http://bar.utoronto.ca/efp_camelina/cgi-bin/efpWeb.cgi

Camelina seed transcriptome

The seed transcriptome includes transcripts from developing Camelina seeds at 15–20 days after pollination (DAP); the data assembly is available as a BLAST database

Nguyen et al. (2013) http://www.camelinagenome.org/

CRISPOR (v4.3)

Helps design, evaluate and clone guide sequences for CRISPR/ Cas9 variants and wild-type Cpf1 based gene editing in C. sativa and other plant species and organisms

Haeussler et al. (2016) http://crispor.tefor.net/

Cas-OFFinder

algorithm that searches for potential off-target sites of Cas9 and Cpf1 in C. sativa and 77 other plant species; also can be used for other organisms

Bae et al. (2014) http://www.rgenome.net/cas-offinder/

aCamelina cultivars in this table are described in Table 2

Both the genome sequence and the transcriptome (Nguyen et al. 2013) have been found to be closely related to Arabidopsis thaliana. Almost 70% of the annotated genes in the Camelina genome (62,277) were found to be syntenically orthologous to A. thaliana genes (Kagale et al. 2014). It has been suggested that Camelina sativa may have formed from hybridization of three ancestors of A. thaliana with lower chromosome numbers (Kagale et al. 2014, 2016). Despite the complexity of the genome, Camelina displays diploid inheritance (Lu and Kang 2008), as is common for most allopolyploids (Hutcheon et al. 2010). Diploid inheritance simplifies the advancement of single copy transgenic lines to a homozygous state but, as described below, the allohexaploid genome, with little evidence of gene fractionation or loss with polyploidization (Kagale et al. 2016), can complicate gene editing and other gene specific modification procedures where all six copies of a gene may need to be modified to achieve a desired effect.

The current Camelina reference genome annotation (NCBI Camelina sativa Annotation Release 100, Table 5) has annotated 95,318 gene models, including 81,485 unique protein coding genes. Approximately, 95% of the genes have been located on the 20 assembled pseudochromosomes, with the remainder on unanchored scaffolds (Kagale et al. 2014). The size of the assembled genome (608.54 Mb) anchored to the 20 chromosomes in the three C. sativa subgenomes accounted for up to 77.5% of the estimated genome’s size of 785 Mb (Kagale et al. 2014). The incomplete genome annotation and assembly of the whole genome to the chromosome level can make manipulation of some genes difficult. Refining of current genome annotations with more genomic, transcriptomic, proteomic and mapping data from experiments conducted in Camelina would allow more accurate annotations of the gene models increasing the utility of the genomic databases for metabolic engineering projects. Only several dozen proteins from Camelina have been used for the current annotation (NCBI Camelina sativa Annotation Release 100, released in 2014) with the predominant input of protein information for annotations originating from related oilseed plant species such as A. thaliana, Arabidopsis lyrata, and Brassica rapa. Regular updates of the annotations based on newly published data are also needed—the latest available update is from 2016.1 A list of available internet databases and bioinformatics tools that are useful for working with the Camelina genome are listed in Table 5.

Germplasm

Collections of Camelina germplasm are maintained at the European Catalogue of Plant Germplasm Collection, The Plant Gene Resources of Canada, and the USDA-National Plant Germplasm System (Sainger et al. 2017; Vollmann and Eynck 2015). Several breeding programs have developed cultivars that have been used in metabolic engineering studies and the cultivar that appears most commonly published in these studies is Suneson (Table 1). Camelina has not undergone extensive breeding and there is scope to further improve the crop and develop more homogenous populations through tissue culture methods producing doubled haploids (Baenziger 1996) to reduce the notable plant to plant variability in existing cultivars that can make it difficult to analyze the performance of yield and oil traits. Procedures for creating doubled haploid lines of Camelina using microspore embryogenesis have been described (Ferrie and Bethune 2011). In fact, the sequenced genome of Camelina is from the doubled haploid (DH) line DH55 (Table 2) (Kagale et al. 2014) that was chosen due to the homozygous nature of DH lines. Our laboratories have generated doubled haploids of spring genotypes 10CS0043, Blaine Creek, and Suneson, and of a winter cultivar Joelle (Table 2), using a microspore embryogenesis procedure adapted from previously described procedures (Ferrie and Bethune 2011) with minor modifications. This work was initiated to create homozygous lines for analyzing yield traits (Metabolix Oilseeds and Yield10 Bioscience, unpublished results). Figure 2 shows the different stages of development of doubled haploid lines from microspores of 10CS0043 in work performed at Metabolix Oilseeds. We have recently evaluated a select number of these doubled haploid 10CS0043 lines in small plot field trials to identify homogeneous lines with good agronomic performance that have comparable or better yields as the parent cultivar (Metabolix Oilseeds and Yield10 Bioscience, unpublished results). Other researchers have also created doubled haploid lines from anther cultures of F1 hybrids obtained from a diverse panel of Camelina genotypes and screened the doubled haploid lines for freezing tolerance (Soorni et al. 2017). Besides creating uniform germplasm, doubled haploid lines can also be used to obtain homozygous and uniform lines of an engineered trait in a single generation (Dwivedi et al. 2015).

Fig. 2

Stages of development of doubled haploid lines generated from microspore cultures of Camelina sativa cv. 10CS0043. a Parent/microspore mother cells (2n) undergo meiosis/reduction division to form microspores in floral buds. b Light microscopy image of microspores (n) at mid-uninucleate stage isolated from ≤ 2 mm floral buds. Light microscopy was performed with a Motic inverted microscope using a 20X objective lens. c Multicellular embryos (2n) developed from embryogenic microspores in liquid cultures in the dark. Autonomous chromosome doubling was commonly observed such that chemically induced chromosome doubling, through the use of colchicine, was not required. d Greening of microspore-derived embryos (2n) in liquid cultures under light. e Doubled haploid plantlets (2n) in peat pellets after regeneration of shoots from embryos in d and formation of roots

Metabolic engineering

Many researchers have taken advantages of the benefits of using Camelina as a platform for metabolic engineering in studies to produce novel molecules, such as biopolymers (Malik et al. 2015; Snell and Peoples 2013), wax esters (Iven et al. 2016; Ruiz-Lopez et al. 2017), hydroxyl fatty acids (Snapp et al. 2014), acetyl-triacylglycerols (Liu et al. 2015), and terpenes (Augustin et al. 2015). Studies have also been performed to increase the level of endogenous molecules such as triacylglycerols (Li et al. 2015), to change the fatty acid profile of oils to a composition more suitable for a particular use (Ruiz -Lopez et al. 2014), and to increase yield through novel carbon fixation pathways [(Malik et al. 2016), Online Resources 2, 3, and 4] or through the use of a photorespiratory bypass (Dalal et al. 2015). Published metabolic engineering strategies range from overexpression of one target gene to expression of 7–13 genes. These and other examples are briefly described in Tables 1 and 4. At Yield10 and Metabolix Oilseeds, we are using Camelina as a platform to test a range of novel metabolic engineering strategies to increase crop yield. Promising strategies are engineered in parallel activities into commodity oilseed crops such as canola or soybean where the timelines are significantly longer to obtain field data. Using the rapid field testing of Camelina to guide which canola and soybean strategies to scale up for subsequent field testing has proven invaluable in our programs.

At Yield10 and Metabolix Oilseeds, we have also used the Camelina platform in pilot studies to evaluate the performance of complex carbon fixation pathways. In one of our studies, we have engineered genes encoding reverse glyoxylate shunt (rGS) pathways for operation in seed plastids that is designed to fix carbon in the form of carbon dioxide and/or bicarbonate and produce acetyl-CoA (Online Resource 2, 3, and 4), the precursor for fatty acid biosynthesis. Although this complex pathway involves the co-expression of 12–13 transgenes (Online Resource 4) and would be difficult to commercialize both from a technical and regulatory perspective (Skraly et al. 2018), it is a useful system to study. There are significant gene expression stability challenges associated with large multi-gene expression constructs, especially when designed with multiple copies of the same regulatory sequences to achieve coordinate expression of large metabolic pathways. However, given the critical need to double food production by the year 2050 to address global food security (Ray et al. 2013), it is crucial to evaluate such systems to determine the limits of how far it is possible to drive increases in seed yield using advanced metabolic engineering. Such highly engineered plants will also provide invaluable gene expression and metabolic data to enable the development of simpler strategies using genome editing and, where necessary, genes from non-plant sources. We choose to place 12–13 separate expression cassettes for the pathway genes on two different binary vectors (Online Resource 3 and 4) and to drive the expression of each transgene encoding a plastid targeted protein from the seed-specific promoter from the soybean oleosin isoform A gene (Rowley and Herman 1997). Although repetitive genetic elements, such as the promoters and terminators used in our transgene expression cassettes, can cause gene silencing (Aboulela et al. 2017), we chose to repeat the promoter sequences since coordinate expression of all 12–13 transgenes in the pathway is required. The identification of 12–13 different seed-specific promoters with the same expression profile to achieve the coordinate expression of transgenes required for functioning of the novel pathway would be difficult (Aboulela et al. 2017). The binary vectors were introduced into Camelina using co-transformation, transgenic plants containing both vectors were identified, and plants were propagated for several generations. T2 plants were grown in a randomized complete block design in a greenhouse for testing T3 seed yield and oil content (Online Resource 4). Large increases in seed yield in engineered plants were observed, with the best plants from line LU03 and LX03 (Online Resource 4e) reaching 216 and 228% of the control, respectively. Seed oil content also increased such that the total oil produced per plant in the best plants from line LU03 and LX03 reached 281% (line LU03) and 244% (line LX03) of the control. The weight of individual seeds, as measured by 100 seed weight, increased reaching 128% (line LU03) and 133% (line LX03) of the control in the best plants (Online Resource 4e). These observed increases were not, however, consistent across all replicate plants of the line tested, likely due to the duplication of promoter and terminator sequences in the constructs that could promote gene silencing. As expected, significant silencing was observed in later experiments with these constructs. Despite the issues faced with working with these large constructs, our experiments suggest that the hurdle of achieving large increases in seed and oil yield can be overcome using advanced metabolic engineering. The challenge is to find a minimum gene set capable of reaching these yields that can be stably engineered into plants.

Genome editing

The ability to make routine, targeted mutations or other edits to the plant genome with technologies such as CRISPR/Cas (clustered regulatory interspaced short palindromic repeat/ CRISPR-associated protein), TALENs (transcriptional activator-like effector nucleases), or zinc finger nucleases has only recently provided plant metabolic engineers with a key tool that has been available to bacterial metabolic engineers for several decades. Researchers have preferentially used the CRISPR/Cas technology in Camelina (Table 6), likely due to its relative simplicity compared to other methods. All that is required to achieve a CRISPR/Cas edit is a Cas enzyme, or other CRISPR nuclease (Murugan et al. 2017), and a single guide RNA (sgRNA) as reviewed extensively by others (Belhaj et al. 2015; Khandagale and Nadaf 2016). As with any polyploid plant, the presence of multiple copies of a gene can complicate editing. Comparative analyses of Camelina genome sequence data revealed that the three subgenomes of the allohexaploid are highly undifferentiated (Kagale et al. 2014) such that there are six copies (three homoeologs) for most genes in Camelina, compared to two for similar genes in diploid relative plants, and these copies may often have high sequence identity. While the allohexaploid nature of the Camelina genome makes sgRNA design and the editing itself more complex, there are multiple tools that can be used to help design the sgRNAs. A few of these tools are listed in Table 5. The allopolyploid nature of the genome can also provide some advantages. The diploid inheritance of Camelina, since each gene only pairs to its own homolog within its subgenome, makes it possible to edit the gene copies on only one or two of the subgenomes, if unique sgRNA’s can be designed. Alternatively, all three gene copies can be edited either through the use of one sgRNA that targets a region of homology to all three genes, or through the use of more than one sgRNA. Most, if not all of the genes targeted in CRISPR-Cas based editing experiments of Camelina published to date have a copy in each of the three subgenomes (Table 6). This ability to perform selected editing of some or all homoeologous copies in the three subgenomes can be useful for gene dosage experiments (Morineau et al. 2017) and genetic engineering of agronomic traits controlled by additive and recessive genes.

Table 6

CRISPR-Cas9 mediated editing in Camelina

Target gene(s)

Trait/phenotype

Promoter for Cas9/Cas9 codon usage

Promoter for gRNA expression

Cultivar/transformation method/marker

Type of edits reported

References

3 FAE1 genes

Decreased VLCFAs C20-C24 from 22 to 2%

Egg cell-specific promoter (EC1.1)/ Cas9 codon optimized for Zea mays

AtU6

Suneson/floral dip/DsRed

Predominantly small indels and substitutions of 1–5 bp; larger deletion of up to 14 bp detected; homozygous edited lines were obtained in a single generation

Ozseyhan et al. (2018)

3 DGAT1 and 3 PDAT1 genes

Reduced oil content and altered fatty acid composition

CaMV35S/Cas9 codon optimized for Zea mays

AtU6

Suneson/floral dip/ hpt conferring hygromycin resistance

Predominantly small indels of 1–7 bp; larger deletion of 16 bp and insertion of 31 bp detected in T1 plants

Aznar-Moreno and Durrett (2017)

3 FAD2 genes

Reduced PUFAs and increased oleic acid to 62% in oil

PcUbi4-2/Cas9 codon optimized for A. thaliana

CsU6 promoter gave edits; CsU3 did not

Celine/floral dip/DsRed

Predominantly small indels; a larger deletion of 40 bp detected; homozygous edits isolated in the T3 generation

Morineau et al. (2017)

3 FAD2 genes

Increased oleic acid and reduced PUFAs content of oil

CaMV35S/Cas9 codon optimized for Chlamydomonas reinhardtii

AtU6

Suneson/floral dip/DsRed

Predominantly small indels, a larger deletion of 15 bps detected; Cas9 negative plants contained homozygous edits in 2 of the 18 FAD2 homoelogous copies

Jiang et al. (2017)

AtU6, Arabidopsis thaliana U6 promoter; CaMV35S, 35S promoter from the Cauliflower mosaic virus; CsU3, Camelina sativa U3 promoter; CsU6, Camelina sativa U6 promoter; DGAT1, diacylglycerol acyltransferase 1; FAD2, fatty acid desaturase 2; FAE1, fatty acid elongase 1; hpt, gene encoding hygromycin phosphotransferase; PDAT1, phospholipid: diacylglycerol acyltransferase 1; PcUbi4-2, constitutive Ubiquitin 4 − 2 promoter from Petroselinum crispum; PUFA, polyunsaturated fatty acids; VLCFAs, very long chain fatty acids; Camelina cultivars are described in the Table 2

CRISPR genome editing of Camelina has primarily been done via Agrobacterium-mediated transformation of a binary vector into Camelina (Table 6). In our laboratories, we have also successfully edited Camelina using PEG-mediated transformation of protoplasts and observed high efficiency editing in callus cultures (Yield10 Bioscience and Metabolix Oilseeds, unpublished results). However, the difficulty in obtaining shoots and plantlets from Camelina tissue culture makes this procedure difficult to use if a larger number of plants is desired. Agrobacterium-mediated transformation and selection with a visual marker, such as DsRed, is our preferred method for genome editing in Camelina. This method allows for easy screening of seeds to identify transformants (Fig. 1) that contain the T-DNA insert with genes encoding the CRISPR/Cas editing machinery. After the desired edit is identified, removal of the T-DNA insert through segregation can easily be detected by monitoring the loss of the visual marker. This results in a plant containing only the desired edit. The thorough removal of the T-DNA from edited lines has implications for regulation of the resulting edited lines as described below.

Field trials and regulatory

One of the primary benefits of the Camelina model system compared to Arabidopsis thaliana is the ability to perform field trials with a crop with good agronomics using conventional planting and harvesting equipment. These field trials provide data on performance of engineered traits under real world agricultural conditions where plants must compete for limited resources and resist pests. Camelina is a predominantly self-fertilizing plant (Francis and Warwick 2009) with a low outcrossing rate [0.09–0.28%, (Walsh et al. 2012)] to other Camelina plants. Importantly, Camelina does not cross with Brassica napus (canola) (FitzJohn et al. 2007), a major agricultural crop that is extensively cultivated on the Canadian Prairies and the North Western United States where the climate is ideal for field trials of spring Camelina. A “Biology Document” on Camelina sativa has been compiled by the Canadian Food Inspection Agency (CFIA), the regulatory body in Canada with jurisdiction over plants with novel traits including transgenic plants, and this document lists what is known about inter-species/genus hybridization of Camelina sativa with other closely related relatives.2

Several researchers have published the results of their Camelina field trials, including for plants engineered to produce omega-3 long chain unsaturated fatty acids (Usher et al. 2015, 2017) and high-oleic acetyl-triacylglycerols (Liu et al. 2015). However, the results of most field trials remain unpublished. Since many countries require a permit prior to performance of transgenic trials, the number of trials and the types of traits tested can be estimated from public records from the regulatory agencies. These records however do not typically list detailed information on the genes manipulated, since this information is usually designated as confidential in the field trial applications. In the US, field trials of transgenic traits are regulated by the US Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service’s (APHIS) regulation 7 CFR part 340 if the plants are believed to pose a plant health risk. Plants transformed using procedures where Agrobacterium tumefaciens, a plant pest, is the DNA delivery vehicle, or plants containing sequences such as promoters, genes, or terminators, from a plant pest are typically regulated by USDA-APHIS and require permits for field release. Through January 2018, 25 field trial permits were listed as having been granted by USDA-APHIS to researchers working in Camelina for field testing of their traits (Online Resource 5), with sixteen of these permits granted to researchers at academic or government institutions suggesting the testing of traits developed in basic research projects.

Canada’s regulatory agency, the Canadian Food Inspection Agency (CFIA), regulates plants with novel traits, or PNTs, and not by the method by which the modified plants are produced. A trait is considered to be novel by CFIA when it is new to cultivated populations of the plant species in Canada and has the potential to have an environmental effect.3 Novel traits can be developed through techniques including genetic engineering, gene editing, mutagenesis, cell fusion, as well as traditional breeding.3 From 2009–2017, confined field trials of PNT Camelina for the purpose of evaluating plants modified for 86 traits associated with yield increase or modified oil composition were reported to be performed in Canada (Online Resource 6). Our laboratories have performed confined Camelina field tests in Western Canada to examine the performance of yield traits with two sites planted during both the 2016 and 2017 growing seasons. The results from these trials have been used by our organization to help decide the priority of traits to be transformed into commodity oilseed crops such as canola and soybean.

In the US, transgenic plants that do not pose a plant health risk, such as plants that were not engineered with Agrobacterium tumefaciens and do not contain plant pest sequences, may not be regulated by USDA-APHIS. Many genome edited lines, where DNA encoding the CRISPR, TALENS, or zinc finger editing machinery has been removed and the final plant only contains the edit, also fall in this category. For these plants, USDA-APHIS has an “Am I Regulated?” process that researchers can go through to determine if their plant variety is regulated. The researcher submits a letter to USDA-APHIS describing the genetic components and procedures used to develop the new plant, and, in the case of genome edited plants, a description of the final edited sequence and whether any inserted DNA remains in the final plant. The researcher can also state why he or she believes it should not be regulated by the agency. USDA-APHIS will respond with whether the plant falls under their jurisdiction and whether it is regulated by the agency. It is, however, important to note that these plants may still be regulated by the US Food and Drug Administration (FDA) or the US Environmental Protection Agency (EPA), depending on the crop and its intended use. As of the end of January 2018, one Camelina line has gone through the “Am I Regulated?” process and has been deemed by USDA-APHIS to not be regulated by the agency4 (Waltz 2018). This submission was made by Yield10 Bioscience and described a genome edited line in which a single base edit was made to all six copies of a gene to increase seed oil content. The non-regulated designation will significantly decrease the burden and cost of our field testing as well as importation and interstate movement of seed of this line.

Conclusions

Camelina sativa is a robust platform crop for investigating both simple and complex metabolic engineering strategies in studies that can be readily transitioned from lab, to greenhouse, to field. Strategies that increase the value of the seed, by producing higher seed yield, oil content, or a value-added coproduct, have the potential to be commercialized directly in Camelina as a specialty crop generating revenue potential. These opportunities are likely to be realized by small agricultural companies due to the small market size of a specialty crop. The high interest in the use of Camelina as a model plant system for metabolic engineering is evident by the number of journal articles published in the last several years (Table 1). Further improvements can be made to the Camelina platform by developing more homogenous germplasm, through conventional breeding methods or by creating doubled haploids, and improving and expanding the genomic resources and bioinformatics tools for the crop. Pairing the versatility of the Camelina plant platform with predictive models for metabolic target discovery (Skraly et al. 2018) has, in our hands, created a robust system for discovering and testing gene targets to significantly enhance crop yield.

Author contribution statement

All authors contributed to the compilation of information and data described in the manuscript. All authors read and approved the final manuscript.

Footnotes

Notes

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.

Compliance with ethical standards

Conflict of interest

All authors are employees of either Metabolix Oilseeds, Inc. or Yield10 Bioscience, Inc. Metabolix Oilseeds is a wholly owned subsidiary of Yield10 Bioscience, Inc.

Supplementary material

299_2018_2308_MOESM1_ESM.pptx (224 kb)
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 (205 kb)
 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)
299_2018_2308_MOESM3_ESM.pptx (234 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)
299_2018_2308_MOESM4_ESM.pptx (383 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 (22/18C 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 22/18C 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)
299_2018_2308_MOESM5_ESM.docx (20 kb)
Supplementary material 5 (DOCX 20 KB)
299_2018_2308_MOESM6_ESM.docx (18 kb)
Supplementary material 6 (DOCX 18 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Meghna R. Malik
    • 1
  • Jihong Tang
    • 2
  • Nirmala Sharma
    • 1
  • Claire Burkitt
    • 1
  • Yuanyuan Ji
    • 1
  • Marie Mykytyshyn
    • 1
  • Karen Bohmert-Tatarev
    • 2
  • Oliver Peoples
    • 2
  • Kristi D. Snell
    • 2
  1. 1.Metabolix Oilseeds, Inc.SaskatoonCanada
  2. 2.Yield10 Bioscience, Inc.WoburnUSA

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