WO2024102881A1 - Plantes de camelina transgéniques génétiquement modifiées pour être tolérantes au glufosinate sans présenter une diminution du rendement en graines - Google Patents

Plantes de camelina transgéniques génétiquement modifiées pour être tolérantes au glufosinate sans présenter une diminution du rendement en graines Download PDF

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WO2024102881A1
WO2024102881A1 PCT/US2023/079186 US2023079186W WO2024102881A1 WO 2024102881 A1 WO2024102881 A1 WO 2024102881A1 US 2023079186 W US2023079186 W US 2023079186W WO 2024102881 A1 WO2024102881 A1 WO 2024102881A1
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plant
camelina
gene
line
camelina plant
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Jihong Tang
Meghna MALIK
Nirmala Sharma
Lauren RAKOCHY
Kristi D. Snell
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Yield10 Bioscience, Inc.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8277—Phosphinotricin
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01006—Acetolactate synthase (2.2.1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • the invention is generally directed to transgenic Camelina plants genetically modified to be tolerant to glufosinate without exhibiting a decrease in seed yield, the transgenic Camelina plants comprising an insertion of a heterologous gene encoding phosphinothricin acetyltransferase within the genome of the transgenic Camelina plants.
  • Camelina sativa (also termed Camelina) is a flowering plant in the family Brassicaceae. There are two types of Camelina, spring and winter, named for their respective growth habit. Both are promising crops that produce high levels of seed oil (Vollmann and Eynck, 2015; Berti et al.2016.; Malik et al.2018). The spring growth habit is dominant in Camelina. Winter lines are better adapted as cover crops in colder, temperate climates. Winter lines require vernalization, or exposure to cold, with temperatures below 8 °C for two to three weeks at seedling to rosette stage to induce bolting and flowering, whereas spring lines do not (Anderson et. al.2018).
  • Camelina plants are highly sensitive to herbicides including glufosinate, glyphosate, sulfonylureas, and imidazolinones.
  • glufosinate is a naturally occurring broad- spectrum herbicide produced by various Streptomyces soil bacteria including Streptomyces hygroscopicus and Streptomyces viridochromogenes.
  • Glufosinate is a non-selective contact herbicide that, when applied to plants, inhibits glutamine synthase such that high levels of ammonia accumulate and inhibition of the photorespiratory pathway and photosynthesis occur, killing the plant (Takano and Dayan, 2020).
  • phosphinothricin N-acetyltransferase a protein that catalyzes the conversion of L-phosphinothricin (L-PPT) to the non-phytotoxic form, N- acetyl-L- phosphinothricin, by acetylation.
  • Plants engineered with a genetic construct for expression of the bar gene are thus tolerant to post-emergent applications of glufosinate containing herbicides (Thompson et al., 1987; De Block et al., 1987; Wehrmann et al., 1996).
  • Herbicide tolerant plants are designed to prevent yield losses arising from pest or weed infestation, not to increase yields (Bond et al., 2006). Under field conditions, which do not involve pest or weed infestation, a yield drag may occur (Bond et al., 2006).
  • SUMMARY OF THE INVENTION A transgenic Camelina plant genetically modified to be tolerant to glufosinate without exhibiting a decrease in seed yield is disclosed.
  • the transgenic Camelina plant comprises an insertion of a heterologous gene encoding phosphinothricin acetyltransferase within the genome of the transgenic Camelina plant.
  • the transgenic Camelina plant was obtained by transforming a parental-line Camelina plant with the heterologous gene, conducting self-pollination of the transformed parental-line Camelina plant to obtain T1 seed of the transformed parental-line Camelina plant, obtaining a T1 generation Camelina plant from the T1 seed, and conducting one or more rounds of self-pollination of the T1 generation Camelina plant or progeny thereof to obtain the transgenic Camelina plant.
  • the transgenic Camelina plant is homozygous for the insertion.
  • the transgenic Camelina plant exhibits no decrease in seed yield when treated with glufosinate in comparison to the parental-line Camelina plant that has not been treated with glufosinate.
  • the transgenic Camelina plant has not undergone cross- pollination.
  • the heterologous gene encoding phosphinothricin acetyltransferase encodes one or more of (i) phosphinothricin acetyltransferase of Streptomyces hygroscopicus, (ii) a phosphinothricin acetyltransferase that is at least 80% identical to phosphinothricin acetyltransferase of Streptomyces hygroscopicus, (iii) phosphinothricin acetyltransferase of Streptomyces viridochromogenes, or (iv) a phosphinothricin acetyltransferase that is at least 80% identical to phosphinothricin acetyltransferase
  • the parental-line Camelina plant was obtained from Camelina germplasm 10CS0043, directly or indirectly.
  • the parental-line Camelina plant is a doubled haploid Camelina plant.
  • the parental-line Camelina plant is spring Camelina line DH12, winter Camelina line WDH2, or winter Camelina line WDH3.
  • the parental-line Camelina plant exhibits an increase in seed yield relative to a progenitor plant from which the parental-line Camelina plant was derived, the parental-line Camelina plant comprising: (a) a first homeolog of the SUGAR- DEPENDENT1 (SDP1) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a wild-type allele; and (b) a second homeolog of the SDP1 gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein: (i) the wild- type allele encodes an active SDP1 triacylglycerol lipase and is identical to an allele of the first homeolog of the SDP1 gene from the progenitor plant; and (ii) the mutant allele does not encode an active SDP1 triacylglycerol lipase and includes one or more additions
  • the parental-line Camelina plant expresses about 20% to 80% of SDP1 triacylglycerol lipase activity in seeds relative to the progenitor plant. Also in some of these embodiments, the increase in seed yield is at least 9%. Also in some of these embodiments, the parental-line Camelina plant further comprises a third homeolog of the SDP1 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the SDP1 gene is homozygous for a mutant allele.
  • the parental-line Camelina plant further comprises a first homeolog of the SUGAR-DEPENDENT1-LIKE (SDP1-L) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein the mutant allele of the first homeolog of the SDP1- L gene does not encode an active SDP1-L triacylglycerol lipase and includes one or more additions, deletions, or substitutions of one or more nucleotides relative to an allele of the first homeolog of the SDP1-L gene from the progenitor plant.
  • SDP1-L SUGAR-DEPENDENT1-LIKE
  • the parental-line Camelina plant further comprises a second homeolog of the SDP1-L gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the second homeolog of the SDP1-L gene is homozygous for a mutant allele.
  • the parental-line Camelina plant further comprises a third homeolog of the SDP1-L gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the SDP1-L gene is homozygous for a mutant allele.
  • the parental-line Camelina plant further comprises a first homeolog of the TRANSPARENT TESTA2 (TT2) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein the mutant allele of the first homeolog of the TT2 gene does not encode an active TT2 transcription factor and includes one or more additions, deletions, or substitutions of one or more nucleotides relative to an allele of the first homeolog of the TT2 gene from the progenitor plant.
  • TT2 TRANSPARENT TESTA2
  • the parental-line Camelina plant further comprises a second homeolog of the TT2 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the second homeolog of the TT2 gene is homozygous for a mutant allele.
  • the parental-line Camelina plant further comprises a third homeolog of the TT2 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the TT2 gene is homozygous for a mutant allele.
  • the parental-line Camelina plant is Camelina line E3902.
  • the transgenic Camelina plant exhibits a decrease in mucilage in comparison to the parental-line Camelina plant.
  • the transgenic Camelina plant exhibits an increase in seed yield when treated with glufosinate in comparison to the parental-line Camelina plant that has not been treated with glufosinate.
  • the transgenic Camelina plant is tolerant to glufosinate applied at 0.53 pounds or more of active ingredient per acre (0.60 kg or more of active ingredient per hectare).
  • the transgenic Camelina plant is tolerant to glufosinate applied at 0.53 pounds of active ingredient per acre (0.60 kg of active ingredient per hectare) to 2.12 pounds of active ingredient per acre (2.40 kg of active ingredient per hectare).
  • the transgenic Camelina plant is further genetically modified to be tolerant to Group 2 herbicides, the transgenic Camelina plant further comprising a gene encoding acetohydroxy acid synthase mutated to provide tolerance to Group 2 herbicides within the genome of the transgenic Camelina plant.
  • the acetohydroxy acid synthase comprises a mutated Arabidopsis acetohydroxy acid synthase comprising one or more of the following mutations: (i) P197S, (ii) P197S and W574L, or (iii) W574L and S653N, with numbering of the mutations based on positions of amino acids in wild-type Arabidopsis acetohydroxy acid synthase.
  • the gene encoding acetohydroxy acid synthase comprises a wild-type promoter of Arabidopsis acetohydroxy acid synthase.
  • the transgenic Camelina plant is tolerant to one or more of the Group 2 herbicides Imazamox or chlorsulfuron.
  • the transgenic Camelina plant is further genetically modified to express barnase targeted to anthers of the transgenic Camelina plant, thereby leading to male sterility of the transgenic Camelina plant.
  • the transgenic Camelina plant is further genetically modified to express barstar targeted to anthers of the transgenic Camelina plant, thereby leading to a male fertility restorer line.
  • the transgenic male sterile Camelina plant is further genetically modified to express at least one more herbicide resistance gene than a male fertility restorer line, for simplified removal of male fertility restorer line.
  • Example embodiments include the following: [0024] Embodiment 1: A transgenic Camelina plant genetically modified to be tolerant to glufosinate without exhibiting a decrease in seed yield, the transgenic Camelina plant comprising an insertion of a heterologous gene encoding phosphinothricin acetyltransferase within the genome of the transgenic Camelina plant, wherein: the transgenic Camelina plant was obtained by transforming a parental-line Camelina plant with the heterologous gene, conducting self-pollination of the transformed parental-line Camelina plant to obtain T1 seed of the transformed parental-line Camelina plant, obtaining a T1 generation Camelina plant from the T1 seed, and conducting one or more rounds of self-pollination of the T
  • Embodiment 2 The transgenic Camelina plant according to embodiment 1, wherein the transgenic Camelina plant has not undergone cross-pollination.
  • Embodiment 3 The transgenic Camelina plant according to embodiment 1 or embodiment 2, wherein the heterologous gene encoding phosphinothricin acetyltransferase encodes one or more of (i) phosphinothricin acetyltransferase of Streptomyces hygroscopicus, (ii) a phosphinothricin acetyltransferase that is at least 80% identical to phosphinothricin acetyltransferase of Streptomyces hygroscopicus, (iii) phosphinothricin acetyltransferase of Streptomyces viridochromogenes, or (iv) a phosphinothricin acety
  • Embodiment 4 The transgenic Camelina plant according to any one of embodiments 1 to 3, wherein the parental-line Camelina plant was obtained from Camelina germplasm 10CS0043, directly or indirectly.
  • Embodiment 5 The transgenic Camelina plant according to any one of embodiments 1 to 4, wherein the parental-line Camelina plant is a doubled haploid Camelina plant.
  • Embodiment 6 The transgenic Camelina plant according to embodiment 5, wherein the parental-line Camelina plant is spring Camelina line DH12, winter Camelina line WDH2, or winter Camelina line WDH3.
  • Embodiment 7 The transgenic Camelina plant according to any one of embodiments 1 to 4, wherein the parental-line Camelina plant exhibits an increase in seed yield relative to a progenitor plant from which the parental-line Camelina plant was derived, the parental-line Camelina plant comprising: (a) a first homeolog of the SUGAR- DEPENDENT1 (SDP1) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a wild-type allele; and (b) a second homeolog of the SDP1 gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein: (i) the wild- type allele encodes an active SDP1 triacylglycerol lipase and is identical to an allele of the first homeolog of the SDP1 gene from the progenitor plant; and (ii) the mutant allele does not encode an active SDP1 triacy
  • Embodiment 8 The transgenic Camelina plant according to embodiment 7, wherein the parental-line Camelina plant expresses about 20% to 80% of SDP1 triacylglycerol lipase activity in seeds relative to the progenitor plant.
  • Embodiment 9 The transgenic Camelina plant according to embodiment 7 or embodiment 8, wherein the increase in seed yield is at least 9%.
  • Embodiment 10 The transgenic Camelina plant according to any one of embodiment 7 to 9, wherein the parental-line Camelina plant further comprises a third homeolog of the SDP1 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the SDP1 gene is homozygous for a mutant allele.
  • Embodiment 11 The transgenic Camelina plant according to any one of embodiments 7 to 10, wherein the parental-line Camelina plant further comprises a first homeolog of the SUGAR-DEPENDENT1-LIKE (SDP1-L) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein the mutant allele of the first homeolog of the SDP1-L gene does not encode an active SDP1-L triacylglycerol lipase and includes one or more additions, deletions, or substitutions of one or more nucleotides relative to an allele of the first homeolog of the SDP1-L gene from the progenitor plant.
  • SDP1-L SUGAR-DEPENDENT1-LIKE
  • Embodiment 12 The transgenic Camelina plant according to embodiment 11, wherein the parental-line Camelina plant further comprises a second homeolog of the SDP1- L gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the second homeolog of the SDP1-L gene is homozygous for a mutant allele.
  • Embodiment 13 The transgenic Camelina plant according to embodiment 12, wherein the parental-line Camelina plant further comprises a third homeolog of the SDP1-L gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the SDP1-L gene is homozygous for a mutant allele.
  • Embodiment 14 The transgenic Camelina plant according to any one of embodiments 7 to 13, wherein the parental-line Camelina plant further comprises a first homeolog of the TRANSPARENT TESTA2 (TT2) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein the mutant allele of the first homeolog of the TT2 gene does not encode an active TT2 transcription factor and includes one or more additions, deletions, or substitutions of one or more nucleotides relative to an allele of the first homeolog of the TT2 gene from the progenitor plant.
  • TT2 TRANSPARENT TESTA2
  • Embodiment 15 The transgenic Camelina plant according to embodiment 14, wherein the parental-line Camelina plant further comprises a second homeolog of the TT2 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the second homeolog of the TT2 gene is homozygous for a mutant allele.
  • Embodiment 16 The transgenic Camelina plant according to embodiment 15, wherein the parental-line Camelina plant further comprises a third homeolog of the TT2 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the TT2 gene is homozygous for a mutant allele.
  • Embodiment 17 The transgenic Camelina plant according to any one of embodiments 7 to 16, wherein the parental-line Camelina plant is Camelina line E3902.
  • Embodiment 18 The transgenic Camelina plant according to any one of embodiments 7 to 17, wherein the transgenic Camelina plant exhibits a decrease in mucilage in comparison to the parental-line Camelina plant.
  • Embodiment 19 The transgenic Camelina plant according to any one of embodiments 1 to 18, wherein the transgenic Camelina plant exhibits an increase in seed yield when treated with glufosinate in comparison to the parental-line Camelina plant that has not been treated with glufosinate.
  • Embodiment 20 The transgenic Camelina plant according to any one of embodiments 1 to 19, wherein the transgenic Camelina plant is tolerant to glufosinate applied at 0.53 pounds or more of active ingredient per acre (0.60 kg or more of active ingredient per hectare).
  • Embodiment 21 The transgenic Camelina plant according to embodiment 20, wherein the transgenic Camelina plant is tolerant to glufosinate applied at 0.53 pounds of active ingredient per acre (0.60 kg of active ingredient per hectare) to 2.12 pounds of active ingredient per acre (2.40 kg of active ingredient per hectare).
  • Embodiment 22 The transgenic Camelina plant according to any one of embodiments 1 to 21, further being genetically modified to be tolerant to Group 2 herbicides, the transgenic Camelina plant further comprising a gene encoding acetohydroxy acid synthase mutated to provide tolerance to Group 2 herbicides within the genome of the transgenic Camelina plant.
  • Embodiment 23 The transgenic Camelina plant according to embodiment 22, wherein the acetohydroxy acid synthase comprises a mutated Arabidopsis acetohydroxy acid synthase comprising one or more of the following mutations: (i) P197S, (ii) P197S and W574L, or (iii) W574L and S653N, with numbering of the mutations based on positions of amino acids in wild-type Arabidopsis acetohydroxy acid synthase.
  • Embodiment 24 The transgenic Camelina plant according to embodiment 22 or embodiment 23, wherein the gene encoding acetohydroxy acid synthase comprises a wild- type promoter of Arabidopsis acetohydroxy acid synthase.
  • Embodiment 25 The transgenic Camelina plant according to any one of embodiments 22 to 24, wherein the transgenic Camelina plant is tolerant to one or more of the Group 2 herbicides Imazamox or chlorsulfuron.
  • Embodiment 26 The transgenic Camelina plant according to any one of embodiments 1 to 25, further being genetically modified to express barnase targeted to anthers of the transgenic Camelina plant, thereby leading to male sterility of the transgenic Camelina plant.
  • Embodiment 27 The transgenic Camelina plant according to any one of embodiments 1 to 25, wherein the transgenic Camelina plant is further genetically modified to express barstar targeted to anthers of the transgenic Camelina plant, thereby leading to a male fertility restorer line.
  • Embodiment 28 The transgenic male sterile Camelina plant according to embodiment 26, wherein the transgenic male sterile Camelina plant is further genetically modified to express at least one more herbicide resistance gene than a male fertility restorer line, for simplified removal of male fertility restorer line.
  • FIG.1 shows a plasmid map of transformation vector pMBXS1341 (SEQ ID NO: 1) for Agrobacterium-mediated transformation of dicots, including Camelina, to develop lines with tolerance to glufosinate herbicide.
  • the pMBXS1341 vector contains an expression cassette of the bar gene in the T-DNA region, composed of the PssuAt promoter from the ribulose-1,5-biphosphate carboxylase small subunit gene of Arabidopsis thaliana (AT1G67090, GenBank accession no.
  • AEE34594.1 (Krebbers et al., 1988), operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.2 shows greenhouse spray results of control Camelina E3902 lines with applications of commercial glufosinate sprays. Plants were sprayed with solutions of 2X or 4X of commercial glufosinate solution A or B containing surfactant(s) as part of the commercial formulation, or with only active ingredient glufosinate with no surfactant.1X commercial sprays contained 150 g/L glufosinate with surfactant(s) to deliver the recommended 1X commercial rate used for in-crop canola applications (0.53 lbs active ingredient per acre, corresponding to 0.60 kg active ingredient per hectare). Herbicide was applied with a mist sprayer. Picture was taken 96 hours after spraying.
  • FIG.3 shows example greenhouse spray results for glufosinate tolerant lines with applications of commercial glufosinate sprays.
  • Event SR174 is a glufosinate tolerant line from transformations of vector pMBXS1341 (FIG.1). Plants were sprayed with solutions of 2X or 4X of commercial glufosinate solution A or B containing surfactants as part of their formulation, or with only active ingredient glufosinate with no surfactant.1X commercial sprays contained 150 g/L glufosinate with surfactant(s) to deliver the recommended 1X commercial rate used for in-crop canola applications (0.53 lbs active ingredient per acre, corresponding to 0.60 kg active ingredient per hectare).
  • FIG.4 shows a drone image of a field trial at a US site showing herbicide tolerance of glufosinate lines. Events of E3902/pMBXS1341 and DH12/pMBXS1341 along with their respective controls, were grown in the field and sprayed with glufosinate. The first spray occurred at the 2-5 leaf stage, the second spray prior to bolting.
  • Untreated controls were sprayed with no glufosinate (UTC), other plots were sprayed with solutions of 1X or 2X of commercial glufosinate solution B.1X commercial sprays contained 150 g/L glufosinate with surfactants to deliver the recommended 1X commercial rate used for in-crop canola applications (0.53 lbs active ingredient per acre, corresponding to 0.60 kg active ingredient per hectare). Each event was replicated in the field trial four times. Image was taken one week after the second spray application of glufosinate.
  • FIG.5 shows seed yields of E3902/pMBXS1341 events and controls harvested from the field trial described in FIG.4.
  • FIG.6 shows seed yields of DH12/pMBXS1341 events and controls harvested from the field trial described in FIG.4.
  • FIG.7 shows a plasmid map of transformation vectors (A) pMBXO128 (SEQ ID NO: 2) and (B) pMBXO133 (SEQ ID NO: 3) for Agrobacterium-mediated transformation of dicots, including Camelina, to develop lines with stacked tolerance to glufosinate herbicide and Group 2 herbicide tolerance.
  • the pMBXO128 vector contains the following expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): (i) An expression cassette of the codon-optimized GmAHAS- P197A/W574L gene, composed of the P-GmSAMS promoter containing the 5′ untranslated regions and intron from the S-adenosyl-L-methionine synthetase (SAMS) gene (GenBank Gene ID: 100820392) of soybean (Falco and Li, 2003)/US publication 2003-0226166); operably linked to a modified version of the acetolactate synthetase (AHAS) gene (GenBank Gene ID: 100782250) from soybean with two amino acid changes (P197A and W574L; amino acid or codon position numberings are in reference to A.
  • SAMS S-adenosyl-L-methionine synthetase
  • thaliana AHAS GenBank Accession No. NP_190425.1
  • additional nucleotides derived from the SAMS gene
  • codon optimized for Arabidopsis thaliana codon usage operably linked to the T-GmAHAS terminator sequence containing the 3 ⁇ untranslated region of the native soybean AHAS gene (Guida Jr. et al., 2011/ US patent no. 7951995 B2).
  • An expression cassette of the bar gene composed of the PssuAt promoter from the ribulose-1,5-biphosphate carboxylase small subunit gene of Arabidopsis thaliana (AT1G67090, GenBank accession no.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • the pMBXO133 vector contains the following expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): (i) An expression cassette of the gm-hra gene, composed of the P-GmSAMS promoter containing the 5′ untranslated regions and intron from the S-adenosyl-L-methionine synthetase (SAMS) gene (GenBank Gene ID: 100820392) of soybean (Falco and Li, 2003)/US publication 2003-0226166) operably linked to a modified version of the acetolactate synthetase (AHAS) gene (GenBank Gene ID: 100782250) from soybean with two amino acid changes (P197A and W574L) within the coding sequence and 15 additional nucleotides (derived from the SAMS gene) added to the 5′ end, operably linked to the T- GmAHAS terminator sequence containing the 3 ⁇ untranslated region of the native soybean
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.8 shows greenhouse spray results of Camelina line E3902 transformed with pMBXO133 (SEQ ID NO: 3). No tolerance to imazamox or chlorsulfuron was observed. Picture taken 96 hours after spraying. Rates of herbicide application used for screening – 30 mg/L of Imazamox; 2.5 mg/L of chlorsulfuron.
  • FIG.9 shows graphs of the expression of the bar and AHAS genes in DH12/pMBXO128. RNA was extracted from several DH12/pMBXO128 (SEQ ID NO: 2) lines with different copy numbers for transcript quantitation by RTPCR. All lines showed good expression of both the bar and AHAS transgenes.
  • FIG.10 shows a plasmid map of transformation vector pMBXS1391 (SEQ ID NO: 4) for Agrobacterium-mediated transformation of dicots, including Camelina, to develop lines with tolerance to glufosinate and Group 2 herbicides.
  • the pMBXS1391 vector contains the following two expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): (i) An expression cassette of the AtAHAS- P197S/W574L gene, composed of the 1,450-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-P197S/W574L coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (P197S and W574L) conferring tolerance to sulfonylurea and imidazolinone herbicides (Sathasivan et al., 1990; Tan et al., 2005), operably linked to the T- AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.11 shows greenhouse spray results of Camelina line E3902 transformed with pMBXS1391 (SEQ ID NO: 4). Good tolerance to both imazamox and chlorsulfuron were observed in most events with similar health and phenotypes for treated and untreated events. Picture taken 96 hours after spraying. Rates of herbicide application used for screening were 30 mg/L of Imazamox and 2.5 mg/L of chlorsulfuron.
  • FIG.12 shows a plasmid map of transformation vector pMBXS1393 (SEQ ID NO: 5) for Agrobacterium-mediated transformation of dicots, including Camelina, to develop lines with tolerance to glufosinate and Group 2 herbicides.
  • the pMBXS1393 vector contains the following two expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): (i) An expression cassette of the AtAHAS- P197S/W574L gene, composed of the P-e35S promoter sequence from the cauliflower mosaic virus (CaMV) 35S transcript with the duplicated enhancer region (Kay et al., 1987, Odell et al., 1985), operably linked to the AtAHAS-P197S/W574L coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (P197S and W574L) conferring tolerance to sulfonylurea and imidazolinone herbicides (Sathasivan et al., 1990; Tan et al., 2005), operably linked to the T-35S terminator sequence including the 3’ untranslated region of the 35S transcript of
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.13 shows a plasmid map of transformation vector pMBXS1397 (SEQ ID NO: 6) for Agrobacterium-mediated transformation of dicots, including Camelina, to develop lines with tolerance to glufosinate and Group 2 herbicides.
  • the pMBXS1397 vector contains the following two expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): (i) An expression cassette of the AtAHAS- P197S/W574L gene, composed of the 275-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-P197S/W574L coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (P197S and W574L) conferring tolerance to sulfonylurea and imidazolinone herbicides (Sathasivan et al., 1990; Tan et al., 2005), operably linked to the T- AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.14 shows a plasmid map of transformation vector pMBXS1395 (SEQ ID NO: 7) for Agrobacterium-mediated transformation of dicots, including Camelina, to develop lines with tolerance to glufosinate and Group 2 herbicides.
  • the pMBXS1395 vector contains the following two expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): (i) An expression cassette of the AtAHAS- W574L/S653N gene, composed of the 1,450-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-W574L/S653N coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (W574L and S653N) conferring tolerance to sulfonylurea and imidazolinone herbicides (Sathasivan et al., 1990; Tan et al., 2005), operably linked to the T- AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.15 shows a plasmid map of transformation vector pMBXS1399 (SEQ ID NO: 8) for Agrobacterium-mediated transformation of dicots, including Camelina, to develop lines with tolerance to glufosinate and Group 2 herbicides.
  • the pMBXS1399 vector contains the following two expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): (i) An expression cassette of the AtAHAS- W574L/S653N gene, composed of the 275-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-W574L/S653N coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (W574L and S653N) conferring tolerance to sulfonylurea and imidazolinone herbicides (Sathasivan et al., 1990; Tan et al., 2005), operably linked to the T- AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.16 shows a plasmid map of transformation vector pMBXO138 (SEQ ID NO: transformation of dicots, including Camelina, to develop lines with tolerance to glufosinate and Group 2 herbicides.
  • the pMBXO138 vector contains the following expression cassettes in the T-DNA region from the right border (T-DNA RB) to the left border (T-DNA LB): An expression cassette of the AtAHAS-P197S gene, composed of the P-e35S promoter sequence from the cauliflower mosaic virus (CaMV) 35S transcript with the duplicated enhancer region (Kay et al., 1987, Odell et al., 1985), operably linked to the AtAHAS-P197S coding sequence for a mutated version of the Arabidopsis thaliana acetohydroxyacid synthase large subunit (AT3G48560, GenBank Gene ID: 824015) (Swolick, D.
  • AtAHAS-P197S gene composed of the P-e
  • AtAHAS-W574L gene composed of the 1,450-bp P- AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding the acetohydroxyacid synthase large subunit (AT3G48560, GenBank Gene ID: 824015, GenBank Protein Accession No.
  • NP_190425.1 operably linked to the AtAHAS-W574L coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with the W574L mutation conferring tolerance to sulfonylurea and imidazolinone herbicides (Tan et al., 2005), operably linked to the T-AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.17 shows differences in the measured size of the mucilage layer of edited E3902 lines compared to control WT43 lines upon staining with ruthenium red.
  • A Microscope image of ruthenium red stained Camelina seeds showing the outer mucilage layer. The thickness of mucilage at the length of the seed was calculated with the formula x – y. The thickness of mucilage at the width of the seed was calculated with the formula a – b. Measurements were performed with Image J software.
  • FIG.18 shows the crop injury score after planting Camelina lines with stacked herbicide tolerance on soil containing group 2 herbicides.
  • A Crop injury score, or a rating of observed injury on plants (crop yellowing and growth retardation), was determined for plants grown on soil that was pretreated with imidazolinone (IMI) herbicides. The crop injury score was estimated visually. Rating is 1 to 100 where 1 is the lowest level of injury and 100 is the highest level of injury. IMI was incorporated into soil 13 days before planting. Stand counts (plants per plot) was determined 10 days after planting.
  • Crop injury data was taken 14 days after planting (27 days after incorporation of IMI into soil).
  • IMI used in the plot is a mixture of Imazamox (Commercial product Davai 80SL, 80g/L) and Imazethapyr (Commercial product Phantom, 240g active ingredient (ai)/L). This mixture was applied at the following rates to soil before planting: 1X application, 17.3 g mixture/acre (corresponding to 43.3 g/hectare); 0.5X application, 8.65 g mixture/acre (corresponding to 21.6 g/hectare); 0X application, 0 g mixture/acre (corresponding to 0 g/hectare).1X application is equivalent to the commercially recommended field application rate.
  • Crop injury score or a rating of observed injury on plants (crop yellowing and growth retardation), was determined for plants grown on soil that was pretreated with sulfonyl urea (SU) herbicides. The crop injury score was estimated visually. Rating is 1 to 100 where 1 is the lowest level of injury and 100 is the highest level of injury. SU was incorporated into soil 13 days before planting. Stand counts (plants per plot) was determined 10 days after planting. Crop injury data was taken 14 days after planting Camelina seeds (27 days after incorporation of SU into soil).
  • SU used in the plot is a mixture of 25% thifensulfuron methyl plus 25% tribenuron methyl (Barricade SG). This mixture was applied at the following rates to soil before planting: 1X application, 12 g/acre (corresponding to 30 g/hectare); 0.5X application, 6 g/acre (corresponding to 15 g/hectare); 0X application, 0 g/acre (corresponding to 0 g/hectare). Prior to all applications, a surfactant (Agral 90) was added to the herbicide such that the surfactant was applied at 0.2 L per 100 L. Numbers shown above graph bars in (A) and (B) are the crop injury for each line at the 1X rate.
  • FIG.19 shows the crop injury score of Camelina lines with stacked herbicide tolerance after spray of glufosinate.
  • A Crop injury score, or a rating of observed injury on plants (bronzing or speckling on leaves), was determined in plot where plants from FIG.18 (A) (IMI soil incorporation experiment) were treated with an over-the-top spray of glufosinate 18 days after planting. Injury was estimated visually nine days after glufosinate application. Rating is 1 to 100 where 1 is the lowest level of injury and 100 is the highest level of injury.
  • FIG.20 shows drone images of plots in field trial of stacked herbicide tolerant events with glufosinate and group 2 tolerance.
  • C Drone shot of entire field trial where stacked herbicide tolerant lines were grown in soil pre-treated with SU (left panel) or IMI (right panel) herbicide before planting as described in FIG.18. Plots were planted with herbicide tolerant lines and plants growing in plots were subsequently treated with glufosinate spray as described in FIG.19 (glufosinate). Drone images were captured 20 days after glufosinate spray.
  • Plot labeling in (A) and (B) indicate the following events: E1, event XL196; E2, event XC20; E3, event XC190; E4, event XG18; E5, event XG164; E6, event XL08; Control, E3902 control line without herbicide tolerance.
  • Plot labeling in (C) points out control events WA-HT1 (W) and E3902 (E). Descriptions for 0x, 0.5x and 1x applications of SU or IMI herbicides are in FIG. 18. Description of glufosinate application is in FIG.19. [0072] FIG.21 describes a hybrid seed production strategy using barnase/barstar methodology that requires mechanical removal of the restorer line after flowering but prior to seed formation.
  • T-DNA insert contains expression cassettes for barnase (Hartley et al., 1988), a strong ribonuclease, whose expression can be targeted to the anther with an anther specific promoter. This leads to male sterility in the plant.
  • the T-DNA insert also contains an expression cassette for bar, providing glufosinate herbicide tolerance.
  • B T-DNA inserts after propagation of MS line with Maintainer line (M). To propagate MS line, MS plants are pollinated with fertile plants with the same elite genotype used for the original transformation of the male sterile construct (Maintainer line, M).
  • MS line is a single copy T-DNA insert, harvested seeds are a mixture of 50% heterozygous transgenic seed containing the MS T- DNA and 50% M line used to pollinate the MS line. The plants without the MS T-DNA are removed in field growth described in (D).
  • C Genetic components of T-DNA inserts of homozygous restorer (R) line that includes an expression cassette for barstar, a ribonuclease inhibitor that can restore male fertility (Szeluga et al., 2023). T-DNA also contains an expression cassette for bar providing glufosinate tolerance.
  • R homozygous restorer
  • the MS line and the R line can be grown in alternating rows in the field or alternatively, mixed and broadcast seeded. Alternating rows are shown in (D).
  • FIG.22 describes a hybrid seed production strategy using barnase/barstar that removes the restorer line with herbicide spray after flowering but prior to seed formation.
  • A Genetic components of the T-DNA insert of the male sterile line (MS). T-DNA insert contains expression cassettes for barnase (Hartley et al., 1988), a strong ribonuclease, whose expression can be targeted to the anther with an anther specific promoter. This leads to male sterility in the plant.
  • the T-DNA insert also contains an expression cassette for bar, providing glufosinate herbicide tolerance, and an expression cassette for a mutated AHAS gene, conferring tolerance to group 2 herbicides such as sulfonyl ureas or imidazolinones.
  • B T- DNA inserts after propagation of MS line with Maintainer line (M). To propagate MS line, MS plants are pollinated with fertile plants with the same elite genotype used for the original transformation of the male sterile construct (Maintainer line, M). MS line is a single copy T- DNA insert, harvested seeds are a mixture of 50% heterozygous transgenic seed containing the MS T-DNA and 50% of the M line used to pollinate the MS line.
  • the plants without the MS T-DNA are removed in field growth (see D).
  • C Genetic components of restorer (R) line.
  • the restorer line contains an expression cassette for barstar, a ribonuclease inhibitor that can restore male fertility (Szeluga et al., 2023). It also contains an expression cassette for bar providing glufosinate tolerance.
  • D Production of hybrid seed.
  • the MS line and the R line can be grown in alternating rows in the field or alternatively, mixed and broadcast seeded. Alternating rows are shown in (D). Before flowering, the field is sprayed with a glufosinate herbicide allowing any plant expressing the bar gene to continue to grow.
  • FIG.23 describes plant transformation construct pMBXS1440 for Agrobacterium- mediated transformation to produce male sterile lines containing expression cassettes for glufosinate (bar) and Group 2 (AHAS W574/S653N) herbicide tolerance.
  • Plasmid pMBXS1440 contains the following expression cassettes in the T-DNA region from the right border to the left border (SEQ ID NO: 10).
  • AtAHAS-W574L/S653N gene composed of the 275-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-W574L/S653N coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (W574L and S653N) conferring tolerance to sulfonylurea and imidazolinone herbicides (Sathasivan et al., 1990; Tan et al., 2005), operably linked to the T-AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid) (Botterman et al., 1991), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.24 describes plant transformation construct pMBXS1446 for Agrobacterium- mediated transformation to produce male sterile lines containing expression cassettes for glufosinate (bar) and Group 2 (AHAS P197S/W574L) herbicide tolerance.
  • the plasmid construct pMBXS1446 has the following expression cassettes in the T-DNA region from the right border to the left border (SEQ ID NO: 11).
  • AtAHAS- P197S/W574L gene composed of the 1,450-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-P197S/W574L coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (P197S and W574L) conferring tolerance to sulfonylurea and imidazolinone herbicides (Tan et al., 2005), operably linked to the T-AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid) (Botterman et al., 1991), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.25 describes plant transformation construct pMBXS1450 for Agrobacterium- mediated transformation to produce male sterile lines containing expression cassettes for glufosinate (bar) and Group 2 (AHAS P197S/W574L) herbicide tolerance.
  • Plasmid pMBXS1450 contains the following expression cassettes in the T-DNA region from the right border to the left border, in addition to an insertion of a barstar gene in the vector backbone: (i) An expression cassette of the barstar gene (Bayer Crop Science, 2016, USDA Petition No.16_23501p), composed of the pNOS promoter sequence of the nopaline synthase gene in Agrobacterium tumefaciens (Depicker et al., 1982), operably linked to the barstar coding sequence from Bacillus amyloliquefaciens (GenBank Accession No.
  • X15545.1 operably linked to the 3’ barstar terminator sequence from downstream of the Bacillus amyloliquefaciens barstar coding sequence (Hartley, 1988) and the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • a barnase gene expression cassette composed of the Pta29 promoter of the anther-specific gene TA29 of Nicotiana tabacum, GenBank accession No.
  • X52283.1 (Seurinck et al., 1990), operably linked to the barnase gene encoding the mature extracellular ribonuclease of Bacillus amyloliquefaciens (Bayer Crop Science, 2016) where the gene has been mutated so that the first three amino acids are methionine, valine, proline (MVP), operably linked to the 3’ barnase terminator sequence containing the 3’ untranslated region downstream of the barnase coding sequence of Bacillus amyloliquefaciens (Hartley, 1988) and also the 3’ nos terminator sequence containing the 3’ untranslated region of the nopaline synthase gene from pTiT37 plasmid (Depicker et al., 1982).
  • MVP methionine, valine, proline
  • AtAHAS- P197S/W574L gene composed of the 1,450-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-P197S/W574L coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (P197S and W574L) conferring tolerance to sulfonylurea and imidazolinone herbicides (Tan et al., 2005), operably linked to the T-AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid) (Botterman et al., 1991), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.26 describes plant transformation construct pMBXS1480 for Agrobacterium- mediated transformation to develop fertility restorer lines containing glufosinate herbicide tolerance.
  • Plasmid pMBXS1480 contains the following two expression cassettes in the T- DNA region from the right border to the left border (SEQ ID NO: 13): (i) An expression cassette of the barstar gene (Bayer Crop Science, 2016, USDA Petition No.16_23501p), composed of the Pta29 promoter of the anther-specific gene TA29 of Nicotiana tabacum, GenBank accession No. X52283.1 (Seurinck et al., 1990), operably linked to the barstar coding sequence from Bacillus amyloliquefaciens (GenBank Accession No.
  • AEE34594.1 (Krebbers et al., 1988) operably linked to the bar gene encoding the phosphinothricin acetyltransferase (PAT) protein from Streptomyces hygroscopicus (Thompson et al., 1987), in which the initiation codon was changed from GTG to ATG for plant expression and the second codon from AGC (serine) to GAC (aspartic acid) (Botterman et al., 1991), operably linked to the 3'g7 terminator sequence containing the 3 ⁇ untranslated region of the TL-DNA gene 7 of the Agrobacterium tumefaciens octopine Ti plasmid (Dhaese et al., 1983).
  • PAT phosphinothricin acetyltransferase
  • FIG.27 describes plant transformation construct pMBXS1480 for Agrobacterium- mediated transformation to develop fertility restorer lines containing group 2 herbicide tolerance.
  • Plasmid pMBXS1481 contains the following two expression cassettes in the T- DNA region from the right border to the left border (SEQ ID NO: 14).
  • An expression cassette of the barstar gene (Bayer CropScience, 2016, USDA Petition No.16_23501p), composed of the Pta29 promoter of the anther-specific gene TA29 of Nicotiana tabacum, GenBank accession No.
  • X52283.1 (Seurinck et al., 1990), operably linked to the barstar coding sequence from Bacillus amyloliquefaciens (GenBank Accession No. X15545.1), operably linked to the 3’ barstar terminator sequence from downstream of the Bacillus amyloliquefaciens barstar coding sequence (Hartley, 1988) and also the 3’ nos terminator sequence containing the 3’ untranslated region of the nopaline synthase gene from pTiT37 plasmid (Depicker et al., 1982).
  • AtAHAS- P197S/W574L gene composed of the 1,450-bp P-AtAHAS promoter sequence from upstream of the Arabidopsis thaliana gene encoding acetohydroxyacid synthase large subunit (GenBank Accession No.
  • NP_190425.1 operably linked to the AtAHAS-P197S/W574L coding sequence for a mutated Arabidopsis thaliana acetohydroxyacid synthase large subunit with two point mutations (P197S and W574L) conferring tolerance to sulfonylurea and imidazolinone herbicides (Tan et al., 2005), operably linked to the T-AtAHAS terminator sequence containing the 3’ untranslated region of the Arabidopsis thaliana acetohydroxyacid synthase large subunit gene.
  • FIG.28 illustrates targets for CRISPR/Cas9 gene edits to significantly increase oil content and/or seed yield and their function in specific parts of plant metabolism.
  • FIG.29A-D shows a multiple sequence alignment of the Arabidopsis thaliana SDP1 and SDP1-like proteins with seven Camelina orthologs according to CLUSTAL O (1.2.4). Sequence descriptions and SEQ ID numbers are shown in TABLE 6 and TABLE 7.
  • ARABIDOPSIS_SDP1 SEQ ID NO: 31
  • Camelina SDP1_CH_8 SEQ ID NO: 33
  • Camelina SDP1_CH_13 SEQ ID NO: 34
  • Camelina SDP1_CH_20 SEQ ID NO: 35
  • ARABIDOPSIS_SDP1-LIKE SEQ ID NO: 32
  • Camelina SDP1-like_CH_9 SEQ ID NO: 40
  • Camelina SDP1-LIKE_CH_4 SEQ ID NO: 36
  • Camelina SDP1-LIKE_CH_6_ISO_X1 SEQ ID NO: 37
  • Camelina SDP1- LIKE_CH_6_ISO_X2 SEQ ID NO: 39
  • FIG.30 illustrates the genetic elements transformed into plants to achieve Cas9 mediated genome editing.
  • A Separate cassettes for expression of a DNA molecule encoding a single guide RNA (sgRNA) and a gene encoding the Cas9 enzyme.
  • the expression cassette for the sgRNA is composed of DNA encoding a guide target sequence, targeted to the gene of interest in the Camelina genome, fused to DNA encoding a guide RNA scaffold.
  • the DNA encoding the guide portion of the sgRNA is often identical to the “guide target sequence” of the genomic DNA to be cut, however several mismatches, depending on their position, can be tolerated and still promote double stranded DNA cleavage.
  • B An sgRNA and Cas9 enzyme are produced.
  • FIG.31 illustrates plasmid maps of binary vectors for Cas9 mediated genome editing of (A) sdp1 gene in Camelina sativa and (B) sdp1-like gene in Camelina sativa.
  • A Binary construct pMBXS1107 (SEQ ID NO: 19) for Cas9 mediated genome editing of the coding sequence of the sdp1 genes using guide target sequence SDP1 #71 (TABLE 8, SEQ ID NO: 18).
  • U6-26p a DNA fragment encoding the polymerase III promoter from the Arabidopsis U6-26 small nuclear RNA gene
  • Guide SDP1 #71 SEQ ID NO: 18
  • DNA encoding a 20 bp guide target sequence SEQ ID NO: 18
  • gRNA Sc DNA fragment encoding a Guide RNA scaffold encoding a crRNA- tracrRNA hybrid engineered from the Streptococcus pyogenes CRISPR locus (the DNA encoding the guide target sequence and gRNASc, when expressed together, form a functional sgRNA sequence)
  • U6-26t a DNA fragment encoding the terminator from the Arabidopsis U6-26 snRNA gene
  • 35S:C4PPDK promoter Chou et al., 1996, Curr.
  • 2X Flag a fragment encoding a FLAG polypeptide protein tag (Li et al., 2013, Nature Biotechnology, 31, 688) created by artificial design (Hopp et al., 1988, Bio/Technology, 6, 1204); NLS-5’, a nuclear localization sequence encoding the peptide MAPKKKRKVGIHGVPAA (SEQ ID NO: 41) (WO 2016114972) attached to the 5’ end of Cas9; pcoCas9-5’, DNA fragment encoding the 5' part of a Cas9 (CRISPR associated protein 9) from Streptococcus pyogenes codon-optimized for expression in plants (pcoCas9, Li et al., 2013, Nature Biotechnology, 31, 688); IV2, a DNA sequence encoding the second intron (IV2) of the nuclear photosynthetic gene ST-LS1 from Solanum tuberosum (Vancanneyt et al., 1990, Molecular and
  • An expression cassette for the DsRed protein driven by the 2X CaMV 35S promoter provides a visual selection of transgenic seeds.
  • B Binary construct pMBXS1126 (SEQ ID NO: 20) for Cas9 mediated genome editing of the coding sequence of the Camelina sativa sdp1-like genes using guide target sequence SDP1-like #4 (TABLE 13, SEQ ID NO: 24).
  • Promoter U6-26p Guide SDP1-like #4 (SEQ ID NO: 24), DNA encoding a 20 bp guide target sequence to the sdp1-like genes; gRNA Sc; U6-26t; 35S:C4PPDK promoter; 2XFlag, NLS-5’ nuclear localization sequence; pcoCas9-5’ encoding the 5' part of the Cas9 protein; the IV2 intron sequence; pcoCas9-3’ encoding the 3' part of Cas9; NLS-3’ nuclear localization sequence; nos termination sequence.
  • FIG.32 shows the expression profiles of the three different homeologs of SDP1 on Chromosomes 8, 13, and 20 according to the Camelina eFP Browser (website: //bar.utoronto.ca/efp_camelina/cgi-bin/efpWeb.cgi).
  • the expression signal is in units of FPKM, fragments per kilobase of transcript per million mapped reads.
  • FIG.33 illustrates the binary construct pMBXS1140 (SEQ ID NO: 30) designed for Cas9 mediated genome editing of the coding sequences of the sdp1, sdp1-like, and tt2 genes in Camelina sativa WT43.
  • Promoter U6-26p; Guide TT2#106/107 (SEQ ID NO: 29), DNA encoding a 20 bp guide target sequence to the tt2 genes; gRNA Sc; U6-26t; Promoter U6-26p; Guide SDP1-like #4 (SEQ ID NO: 24), DNA encoding a 20 bp guide target sequence to the sdp1-like genes; gRNA Sc; U6-26t; Promoter U6-26p; Guide SDP1 #77 (SEQ ID NO: 28), DNA encoding a 20 bp guide target sequence to the sdp1 genes; gRNA Sc; U6-26t; 35S:C4PPDK promoter; 2XFlag, NLS-5’ nuclear localization sequence; pcoCas9-5’ encoding the 5' part of the Cas9 protein; the IV2 intron sequence; pcoCas9-3’ encoding the 3' part
  • FIG.34 illustrates the seed coat phenotype of T3 seeds harvested from T2 multiplex edited lines targeting the sdp1, sdp1-like, and tt2 genes. A loss of pigmentation in the seed coat is observed in lines with 100% editing within the tt2 gene (lines 17-1013, 17-1011 and 17-1014; TABLE 18) compared to lines with partial tt2 editing (lines 17-1012 and 17-1042; TABLE 18) and WT43.
  • FIG.35 illustrates the development of stable, fertile homozygous lines with INDELS in the sdp1, sdp1-like, and tt2 gene targets.
  • INDELS is an abbreviation for insertions or deletions.
  • FIG.36 shows phenotypes of winter Camelina WDH2/pMBXS1341 lines, generated from transformations of winter Camelina WDH2 with genetic construct pMBXS1341, after glufosinate spray.
  • A Pictures of untransformed control (no glufosinate treatment, left image) and events of WDH2/pMBXS1341 treated with 6X or 4X commercial levels of glufosinate (middle and right image).
  • WDH2/pMBXS1341 lines were tolerant to the high levels of herbicide treatment.
  • FIG.37 shows phenotypes of WDH3/pMBXS1391 lines, generated from transformations of WDH3 with genetic construct pMBXS1391, after glufosinate and group 2 herbicide foliar applications.
  • A Pictures of an event of WDH3/pMBXS1391 with no glufosinate treatment (control, left image) and an event of WDH3/pMBXS1391 treated (from left to right) with 4X commercial levels of glufosinate, 1X commercial levels of a group 2 imidazolinone (IMI) herbicide imazamox/imazethapyr, and 1X commercial levels of group 2 sulfonyl urea (SU) herbicide thifensulfuron-methyl/tribenuron-methyl.
  • IMI imidazolinone
  • SU group 2 sulfonyl urea
  • IMI imidazolinone
  • WDH3 control is severely impaired/dead after herbicide application whereas WDH3/pMBXS1391 event has only minor blemishing.
  • IMI herbicide contained 15.05 g active ingredient per hectare of Imazamox and 15.05 g active ingredient per hectare of Imazethapyr.1X commercial rates of SU herbicide contained 7.5 g active ingredient per hectare of Tribenuron and 7.5 g active ingredient per hectare of Thifensulfuron.1X commercial levels of glufosinate spray are equivalent to 0.53 lbs active ingredient per acre, corresponding to 0.60 kg active ingredient per hectare.
  • transgenic Camelina plants comprise an insertion of a heterologous gene encoding phosphinothricin acetyltransferase within their genome.
  • the transgenic Camelina plants were obtained by transforming parental-line Camelina plants with the heterologous gene, conducting self-pollination of the transformed parental-line Camelina plants to obtain T1 seed of the transformed parental-line Camelina plants, obtaining T1 generation Camelina plants from the T1 seed, and conducting one or more rounds of self-pollination of the T1 generation Camelina plants or progeny thereof to obtain the transgenic Camelina plants.
  • the transgenic Camelina plants are homozygous for the insertion.
  • the transgenic Camelina plants exhibit no decrease in seed yield when treated with glufosinate in comparison to the parental-line Camelina plants that have not been treated with glufosinate.
  • the transgenic Camelina plants exhibit increased seed yield in comparison to the parental-line Camelina plants that have not been treated with glufosinate, and this is so even when the transgenic Camelina plants are treated with glufosinate.
  • the transgenic Camelina plants provide a basis for genetically engineering Camelina stacked for glufosinate and group 2 herbicide tolerance.
  • the transgenic Camelina plants also provide a basis for developing hybrid seed technology in Camelina.
  • plant includes whole plant, mature plants, seeds, shoots and seedlings, and parts, propagation material, plant organ tissue, protoplasts, callus and other cultures, for example cell cultures, derived from plants belonging to the plant subkingdom Embryophyta, and all other species of groups of plant cells giving functional or structural units, also belonging to the plant subkingdom Embryophyta.
  • mature plants refers to plants at any developmental stage beyond the seedling.
  • seedlings refers to young, immature plants at an early developmental stage.
  • crops and “plants” are used interchangeably.
  • a “genetically modified plant” refers to non-naturally occurring plants or crops engineered as described throughout herein.
  • a “control plant” means a plant that has not been modified as described in the present disclosure to impart an enhanced trait or altered phenotype.
  • a control plant is used to identify and select a modified plant that has an enhanced trait or altered phenotype.
  • a control plant can be a plant that has not been modified or has not been genome edited to express or to inhibit its endogenous gene product.
  • a suitable control plant can be a non-transgenic or non-edited plant of the parental line used to generate a transgenic plant, for example, a wild-type plant devoid of a recombinant DNA or a genome edit.
  • a suitable control plant can also be a transgenic plant that contains recombinant DNA that imparts other traits, for example, a transgenic plant having enhanced herbicide tolerance.
  • a suitable control plant can in some cases be a progeny of a hemizygous transgenic plant line that does not contain the recombinant DNA, known as a negative segregant, a null segregant, or a negative isogenic line.
  • seed oil content refers to amount of oil per mature seed weight and is typically expressed as a percentage.
  • seed yield refers to weight of seeds produced per plant and is typically expressed in grams per plant.
  • oil yield refers to weight of oil produced per plant and is typically expressed as grams per plant.
  • Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • “Native gene” refers to a gene as found in nature with its own regulatory sequences.
  • a “Cis-genic gene” is a chimeric gene where the DNA sequences making up the gene are from the same plant species or a sexually compatible plant species where the cis-genic gene is deployed in the same species from which the DNA sequences were obtained. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.
  • Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure. [0098] As used herein the term “coding sequence” refers to a DNA sequence which codes for a specific amino acid sequence.
  • regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • gene includes protein coding regions of the specific genes and the regulatory sequences both 5’ and 3’ which control the expression of the gene.
  • Codon degeneracy refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.
  • the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein.
  • the skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for increased expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity).
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percent sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
  • percent sequence identity means the value determined by comparing two aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percent sequence identity.
  • “Homeologs” are pluralities of genes (e.g.
  • Polyploidy is a heritable condition of an organism having more than two complete sets of chromosomes (Woodhouse et al., 2009, Nature Education, 2, 1). For example, a “tetraploid” has four sets of chromosomes. A “hexaploid” has six sets of chromosomes.
  • Allopolyploidy is a type of whole-genome duplication by hybridization followed by genome doubling (Glover et al., 2016).
  • Allopolyploidy typically occurs between two related species, and results in the merging of the genomes of two divergent species into one genome.
  • an “allotetraploid” is an alloploid that has four sets of chromosomes.
  • An “allohexaploid” is a hexaploid that has six sets of chromosomes.
  • Autopolyploidy is a type of whole-genome duplication based on doubling of a genome within one species.
  • “Diploidization” of a polyploid is a process that involves genomic reorganization, restructuring, and functional alternations in association with polyploidy, generally resulting in restoration of a secondary diploid-like behavior of a polyploid genome (del Pozo et al., 2015, Journal Experimental Botany, 66, 6991). Most polyploid plants have lost their polyploidy over time through diploidization (del Pozo et al., 2015).
  • PREFERRED EMBODIMENTS [0108] As noted above, a transgenic Camelina plant genetically modified to be tolerant to glufosinate without exhibiting a decrease in seed yield is disclosed.
  • the transgenic Camelina plant comprises an insertion of a heterologous gene encoding phosphinothricin acetyltransferase within the genome of the transgenic Camelina plant.
  • the heterologous gene encoding phosphinothricin acetyltransferase encodes one or more of (i) phosphinothricin acetyltransferase of Streptomyces hygroscopicus, (ii) a phosphinothricin acetyltransferase that is at least 80% identical to phosphinothricin acetyltransferase of Streptomyces hygroscopicus, (iii) phosphinothricin acetyltransferase of Streptomyces viridochromogenes, or (iv) a phosphinothricin acetyltransferase that
  • the phosphinothricin acetyltransferase that is at least 80% identical to phosphinothricin acetyltransferase of Streptomyces hygroscopicus can be, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to phosphinothricin acetyltransferase of Streptomyces hygroscopicus.
  • the phosphinothricin acetyltransferase that is at least 80% identical to phosphinothricin acetyltransferase of Streptomyces viridochromogenes can be, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to phosphinothricin acetyltransferase of Streptomyces viridochromogenes.
  • the transgenic Camelina plant was obtained by transforming a parental-line Camelina plant with the heterologous gene, conducting self-pollination of the transformed parental-line Camelina plant to obtain T1 seed of the transformed parental-line Camelina plant, obtaining a T1 generation Camelina plant from the T1 seed, and conducting one or more rounds of self-pollination of the T1 generation Camelina plant or progeny thereof to obtain the transgenic Camelina plant.
  • the transgenic Camelina plant is homozygous for the insertion.
  • the transgenic Camelina plant exhibits no decrease in seed yield when treated with glufosinate in comparison to the parental-line Camelina plant that has not been treated with glufosinate.
  • the transgenic Camelina plant has not undergone cross- pollination.
  • the parental-line Camelina plant was obtained from Camelina germplasm 10CS0043, directly or indirectly.
  • the parental-line Camelina plant is a doubled haploid Camelina plant.
  • the parental-line Camelina plant is Camelina line spring line DH12, or winter lines WDH2 or WDH3.
  • the parental-line Camelina plant exhibits an increase in seed yield relative to a progenitor plant from which the parental-line Camelina plant was derived, the parental-line Camelina plant comprising: (a) a first homeolog of the SUGAR- DEPENDENT1 (SDP1) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a wild-type allele; and (b) a second homeolog of the SDP1 gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein: (i) the wild- type allele encodes an active SDP1 triacylglycerol lipase and is identical to an allele of the first homeolog of the SDP1 gene from the progenitor plant; and (ii) the mutant allele does not encode an active SDP1 triacylglycerol lipase and includes one or more additions
  • the parental-line Camelina plant expresses about 20% to 80% of SDP1 triacylglycerol lipase activity in seeds relative to the progenitor plant. This is based on the transgenic Camelina plant not having a full complement of wild-type alleles of homeologs of SDP1, and particularly not having wild-type alleles of the second homeolog of SDP1. In some embodiments, the transgenic Camelina plant expresses about 30% to 70% of SDP1 triacylglycerol lipase activity in seeds relative to the progenitor.
  • the transgenic Camelina plant expresses about 30% to 40%, about 40% to 50%, about 50% to 60%, or about 60% to 70% of SDP1 triacylglycerol lipase activity in seeds relative to the progenitor. Also for example, in some embodiments the transgenic Camelina plant expresses about 30% to 36%, about 45% to 55%, or about 63% to 70% of SDP1 triacylglycerol lipase activity in seeds relative to the progenitor. [0118] In some embodiments, the increase in seed yield is at least 9%. [0119] As noted above, the first homeolog of the SDP1 gene is homozygous for the wild- type allele.
  • the transgenic Camelina plant is homozygous for the wild- type allele based on including two identical copies of a wild-type allele.
  • the identical wild- type alleles may be derived, for example, from a single wild-type allele of a progenitor plant.
  • the transgenic Camelina plant is homozygous for the wild-type allele based on including a first wild-type allele and a second wild-type allele that are not identical to each other.
  • the non-identical wild-type alleles may differ, for example, based on differences in the nucleotide sequences of the non-identical alleles that are sufficiently minor as to have no corresponding phenotype with respect to SDP1 triacylglycerol lipase activity.
  • the second homeolog of the SDP1 gene is homozygous for the mutant allele.
  • the transgenic Camelina plant is homozygous for the mutant allele based on including two copies of the mutant allele that are identical.
  • the identical mutant alleles may be based, for example, on breeding the transgenic Camelina plant to homozygosity with respect to a particular mutant allele.
  • the transgenic Camelina plant is homozygous for the mutant allele based on including a first mutant allele and a second mutant allele that are not identical to each other.
  • the non-identical mutant alleles may differ, for example, based on having different additions, deletions, and/or substitutions of one or more nucleotides relative to each other, with the additions, deletions, and/or substitutions of each being sufficiently severe to cause a loss of function of the SDP1 triacylglycerol lipase encoded by each.
  • the active SDP1 triacylglycerol lipase has a sequence that comprises SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35.
  • the one or more additions, deletions, or substitutions of one or more nucleotides comprise one or more of a frameshift mutation, an active site mutation, a nonconservative substitution mutation, or an open-reading-frame deletion mutation in the mutant allele relative to the allele of the second homeolog of the SDP1 gene from the progenitor plant.
  • the parental-line Camelina plant further comprises a third homeolog of the SDP1 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the SDP1 gene is homozygous for a mutant allele.
  • the third homeolog is homozygous for a wild-type allele. In some of these embodiments, the third homeolog is homozygous for a mutant allele. In some of these embodiments, the third homeolog is heterozygous for a wild- type allele and a mutant allele.
  • the parental-line Camelina plant further comprises a first homeolog of the SUGAR-DEPENDENT1-LIKE (SDP1-L) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein the mutant allele of the first homeolog of the SDP1-L gene does not encode an active SDP1-L triacylglycerol lipase and includes one or more additions, deletions, or substitutions of one or more nucleotides relative to an allele of the first homeolog of the SDP1-L gene from the progenitor plant.
  • SDP1-L gene is discussed in detail in Examples 6-11 below.
  • the parental-line Camelina plant further comprises a second homeolog of the SDP1-L gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the second homeolog of the SDP1-L gene is homozygous for a mutant allele.
  • the parental-line Camelina plant further comprises a third homeolog of the SDP1-L gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the SDP1-L gene is homozygous for a mutant allele.
  • the parental-line Camelina plant further comprises a first homeolog of the TRANSPARENT TESTA2 (TT2) gene, occurring in its natural position within the genome of the parental-line Camelina plant and being homozygous for a mutant allele, wherein the mutant allele of the first homeolog of the TT2 gene does not encode an active TT2 transcription factor and includes one or more additions, deletions, or substitutions of one or more nucleotides relative to an allele of the first homeolog of the TT2 gene from the progenitor plant.
  • the TT2 gene is discussed in detail in Examples 6-11 below.
  • the parental-line Camelina plant further comprises a second homeolog of the TT2 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the second homeolog of the TT2 gene is homozygous for a mutant allele.
  • the parental-line Camelina plant further comprises a third homeolog of the TT2 gene occurring in its natural position within the genome of the parental-line Camelina plant, wherein the third homeolog of the TT2 gene is homozygous for a mutant allele.
  • the parental-line Camelina plant is Camelina line E3902. Camelina line E3902 is discussed in detail in Examples 6-11 below.
  • the transgenic Camelina plant exhibits a decrease in mucilage in comparison to the parental-line Camelina plant. This is discussed with respect to Camelina line E3902 in Example 4 below. [0128] In some embodiments, the transgenic Camelina plant exhibits an increase in seed yield when treated with glufosinate in comparison to the parental-line Camelina plant that has not been treated with glufosinate. [0129] In some embodiments, the transgenic Camelina plant is tolerant to glufosinate applied at 0.53 pounds or more of active ingredient per acre (0.60 kg or more of active ingredient per hectare).
  • the transgenic Camelina plant is tolerant to glufosinate applied at 0.53 pounds of active ingredient per acre (0.60 kg of active ingredient per hectare) to 2.12 pounds of active ingredient per acre (2.40 kg of active ingredient per hectare).
  • the transgenic Camelina plant is further genetically modified to be tolerant to Group 2 herbicides, the transgenic Camelina plant further comprising a gene encoding acetohydroxy acid synthase mutated to provide tolerance to Group 2 herbicides within the genome of the transgenic Camelina plant.
  • Group 2 herbicides are discussed in detail in Examples 2 and 3 below.
  • the acetohydroxy acid synthase comprises a mutated Arabidopsis acetohydroxy acid synthase comprising one or more of the following mutations: (i) P197S, (ii) P197S and W574L, or (iii) W574L and S653N, with numbering of the mutations based on positions of amino acids in wild-type Arabidopsis acetohydroxy acid synthase.
  • the gene encoding acetohydroxy acid synthase comprises a wild-type promoter of Arabidopsis acetohydroxy acid synthase.
  • the transgenic Camelina plant is tolerant to one or more of the Group 2 herbicides Imazamox or chlorsulfuron.
  • the transgenic Camelina plant is further genetically modified to express barnase targeted to anthers of the transgenic Camelina plant, thereby leading to male sterility of the transgenic Camelina plant. Methods for hybrid seed production in Camelina based on using barnase/barstar is discussed in Example 5 below.
  • the transgenic Camelina plant is further genetically modified to express barstar targeted to anthers of the transgenic Camelina plant, thereby leading to a male fertility restorer line. This is also discussed in Example 5 below.
  • the transgenic male sterile Camelina plant is further genetically modified to express at least one more herbicide resistance gene than a male fertility restorer line, for simplified removal of male fertility restorer line. This is also discussed in Example 5 below.
  • GENETIC MODIFICATION OF PLANTS Methods of Plant Transformation [0134] Known transformations methods can be used to genetically modify a plant with respect to one or more gene sequences of the invention using transgenic, cis-genic, or genome editing methods.
  • Plant transformation vectors generally include one or more coding sequences of interest under the transcriptional control of 5’ and 3’ regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal, and a selectable or screenable marker gene.
  • Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA sequence and include vectors such as pBIN19.
  • Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001, as well as the binary vector pCIB 10 and hygromycin selection derivatives thereof (see, for example, U.S. Patent No 5,639,949).
  • Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences are utilized in addition to vectors such as the ones described above which contain T-DNA sequences.
  • the choice of vector for transformation techniques that do not rely on Agrobacterium depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non-Agrobacterium transformation include pCIB3064, pSOG 19, and pSOG35. (See, for example, U.S. Patent No 5,639,949).
  • DNA fragments containing the transgene and the necessary regulatory elements for expression of the transgene can be excised from a plasmid and delivered to the plant cell using microprojectile bombardment-mediated, or alternatively, nanotube-mediated methods.
  • Protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.
  • Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome are described in US 2010/0229256 A1 to Somleva & Ali and US 2012/0060413 to Somleva et al.
  • the transformed cells are grown into plants in accordance with conventional techniques (see, for example, McCormick et al., 1986, Plant Cell Rep.5: 81-84).
  • Stable Arabidopsis transformants can be obtained by several in planta methods including vacuum infiltration (Clough & Bent, 1998, The Plant J.16: 735-743), transformation of germinating seeds (Feldmann & Marks, 1987, Mol. Gen. Genet.208: 1-9), floral dip (Clough and Bent, 1998, Plant J.16: 735-743), and floral spray (Chung et al., 2000, Transgenic Res.9: 471- 476).
  • rapeseed and radish vacuum infiltration, Ian and Hong, 2001, Transgenic Res., 10: 363-371; Desfeux et al., 2000, Plant Physiol.123: 895-904), Medicago truncatula (vacuum infiltration, Trieu et al., 2000, Plant J.22: 531-541), camelina (floral dip, WO/2009/117555 to Nguyen et al.), and wheat (floral dip, Zale et al., 2009, Plant Cell Rep.28: 903-913).
  • the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the DNA construct for introducing the targeted insertion of the DNA sequence elements producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.
  • the cells that have been transformed may be grown into plants in accordance with conventional techniques (see, for example, McCormick et al. Plant Cell Reports 5:81- 84(1986)).
  • Transgenic plants can be produced using conventional techniques to express any genes of interest in plants or plant cells (Methods in Molecular Biology, 2005, vol.286, Transgenic Plants: Methods and Protocols, Pena L., ed., Humana Press, Inc. Totowa, NJ; Shyamkumar Barampuram and Zhanyuan J.
  • RNA or an RNA molecule to be introduced into the organism is part of a transformation vector.
  • a large number of such vector systems known in the art may be used, such as plasmids.
  • the components of the expression system can be modified, e.g., to increase expression of the introduced nucleic acids. For example, truncated sequences, nucleotide substitutions or other modifications may be employed.
  • transgene comprising a DNA molecule encoding a gene of interest is preferably stably transformed and integrated into the genome of the host cells. Transformed cells are preferably regenerated into whole fertile plants. Detailed description of transformation techniques are within the knowledge of those skilled in the art.
  • Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser & Fraley, 1989, Science 244: 1293-1299). In one embodiment, promoters are selected from those of eukaryotic or synthetic origin that are known to yield high levels of expression in plants and algae.
  • promoters are selected from those that are known to provide high levels of expression in monocots.
  • Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No 6,072,050, the core CaMV 35S promoter (Odell et al., 1985, Nature 313: 810-812), rice actin (McElroy et al., 1990, Plant Cell 2: 163-171), ubiquitin (Christensen et al., 1989, Plant Mol. Biol.12: 619-632; Christensen et al., 1992, Plant Mol.
  • tissue-preferred promoters can be used to target gene expression within a particular tissue.
  • Tissue- preferred promoters include those described by Van Ex et al., 2009, Plant Cell Rep.28: 1509- 1520; Yamamoto et al., 1997, Plant J.12: 255-265; Kawamata et al., 1997, Plant Cell Physiol.38: 792-803; Hansen et al., 1997, Mol. Gen.
  • Any of the described promoters can be used to control the expression of one or more of the genes of the invention, their homologs and/or orthologs as well as any other genes of interest in a defined spatiotemporal manner.
  • Expression Cassettes [0150] Nucleic acid sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter active in plants. The expression cassettes may also include any further sequences required or selected for the expression of the transgene.
  • transcription terminators include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
  • transcription terminators can then be transferred to the plant transformation vectors described infra.
  • a variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and the correct polyadenylation of the transcripts. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tm1 terminator, the nopaline synthase terminator and the pea rbcS E9 terminator.
  • a phenotype of the transgenic plant may be measured as a percentage of individual plants within a population.
  • the yield of a plant can be measured simply by weighing.
  • the yield of seed from a plant can also be determined by weighing.
  • the increase in seed weight from a plant can be due to a number of factors, an increase in the number or size of the seed pods, an increase in the number of seed or an increase in the number of seed per plant.
  • In the laboratory or greenhouse seed yield is usually reported as the weight of seed produced per plant and in a commercial crop production setting yield is usually expressed as weight per acre or weight per hectare.
  • a recombinant DNA construct including a plant-expressible gene or other DNA of interest is inserted into the genome of a plant by a suitable method.
  • Suitable methods include, for example, Agrobacterium tumefaciens-mediated DNA transfer, direct DNA transfer, liposome-mediated DNA transfer, electroporation, co-cultivation, diffusion, particle bombardment, microinjection, gene gun, calcium phosphate coprecipitation, viral vectors, and other techniques.
  • Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens.
  • transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium alternative methods can be used to insert DNA constructs into plant cells.
  • a transgenic plant can be produced by selection of transformed seeds or by selection of transformed plant cells and subsequent regeneration.
  • the transgenic plants are grown (e.g., on soil) and harvested.
  • above ground tissue is harvested separately from below ground tissue. Suitable above ground tissues include shoots, stems, leaves, flowers, grain, and seed. Exemplary below ground tissues include roots and root hairs.
  • whole plants are harvested and the above ground tissue is subsequently separated from the below ground tissue.
  • Genetic constructs may encode a selectable marker to enable selection of transformation events. There are many methods that have been described for the selection of transformed plants [for review see (Miki et al., Journal of Biotechnology, 2004, 107, 193- 232) and references incorporated within].
  • Selectable marker genes that have been used extensively in plants include the neomycin phosphotransferase gene nptII (U.S. Patent Nos. 5,034,322, U.S.5,530,196), hygromycin resistance gene (U.S. Patent No.5,668,298, Waldron et al., (1985), Plant Mol Biol, 5:103-108; Zhijian et al., (1995), Plant Sci, 108 :219-227), the bar gene encoding resistance to phosphinothricin (U.S. Patent No.5,276,268), the expression of aminoglycoside 3’-adenyltransferase (aadA) to confer spectinomycin resistance (U.S.
  • Patent No.5,073,675 the use of inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase (U.S. Patent No.4,535,060) and methods for producing glyphosate tolerant plants (U.S. Patent No.5,463,175; U.S. Patent No.7,045,684).
  • selectable markers include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al., (1983), EMBO J, 2 :987-992), methotrexate (Herrera Estrella et al., (1983), Nature, 303 :209-213 ; Meijer et al, (1991), Plant Mol Biol, 16:807-820); streptomycin (Jones et al., (1987), Mol Gen Genet, 210 :86-91) ; bleomycin (Hille et al., (1990), Plant Mol Biol, 7 :171-176) ; sulfonamide (Guerineau et al., (1990), Plant Mol Biol, 15:127-136); bromoxynil (Stalker et al., (1988), Science, 242:419-423); glyphosate (Shaw et al., (1986), Science, 233:478-481);
  • Patent No.5,767,378 describes the use of mannose or xylose for the positive selection of transgenic plants.
  • Methods for positive selection using sorbitol dehydrogenase to convert sorbitol to fructose for plant growth have also been described (WO 2010/102293).
  • Screenable marker genes include the beta-glucuronidase gene (Jefferson et al., 1987, EMBO J.6: 3901-3907; U.S. Patent No.5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., 1995, Trends Biochem. Sci.20: 448-455; Pan et al., 1996, Plant Physiol.112: 893-900).
  • Transformation events can also be selected through visualization of fluorescent proteins such as the fluorescent proteins from the nonbioluminescent Anthozoa species which include DsRed, a red fluorescent protein from the Discosoma genus of coral (Matz et al. (1999), Nat Biotechnol 17: 969-73). An improved version of the DsRed protein has been developed (Bevis and Glick (2002), Nat Biotech 20: 83-87) for reducing aggregation of the protein. [0159] Visual selection can also be performed with the yellow fluorescent proteins (YFP) including the variant with accelerated maturation of the signal (Nagai, T. et al.
  • YFP yellow fluorescent proteins
  • the plants modified for enhanced performance may be combined or stacked with input traits by crossing or plant breeding.
  • Useful input traits include herbicide resistance and insect tolerance, for example a plant that is tolerant to the herbicide glyphosate and that produces the Bacillus thuringiensis (BT) toxin.
  • Glyphosate is a herbicide that prevents the production of aromatic amino acids in plants by inhibiting the enzyme 5- enolpyruvylshikimate-3-phosphate synthase (EPSP synthase).
  • EPSP synthase in a crop of interest allows the application of glyphosate as a weed killer without killing the modified plant (Suh, et al., J. M Plant Mol. Biol.1993, 22, 195-205).
  • BT toxin is a protein that is lethal to many insects providing the plant that produces it protection against pests (Barton, et al. Plant Physiol.1987, 85, 1103-1109).
  • herbicide tolerance traits include but are not limited to tolerance to Dicamba by expression of the dicamba monoxygenase gene (Behrens et al, 2007, Science, 316, 1185), tolerance to 2,4-D and 2,4-D choline by expression of a bacterial aad-1 gene that encodes for an aryloxyalkanoate dioxygenase enzyme (Wright et al., Proceedings of the National Academy of Sciences, 2010, 107, 20240), glufosinate tolerance by expression of the bialophos resistance gene (bar) or the pat gene encoding the enzyme phosphinotricin acetyl transferase (Droge et al., Planta, 1992, 187, 142), as well as genes encoding a modified 4-hydroxyphenylpyruvate dioxygenase (HPPD) that provides tolerance to the herbicides mesotrione, isoxaflutole, and tembotrione (Siehl et al., Plant dic
  • Genome editing can also be used to accomplish genetic modification of plants according to the invention.
  • An advantage of using genome editing technologies is that the regulatory body in the United States views genome editing as an advanced plant breeding tool and may provide more favorable or no regulation for the technologies.
  • Recent advances in genome editing technologies provide an opportunity to precisely remove genes, edit control sequences, introduce frame shift mutations, etc., to significantly alter the expression levels of targeted genes and/or the activities of the proteins encoded thereby.
  • Plants engineered using this approach may be defined as non-regulated by USDA-APHIS providing the opportunity to continually improve the plants.
  • Genome editing can be accomplished by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) or CRISPR/Cpf1.
  • CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR/Cpf1 Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA).
  • Cas CRISPR-associated
  • sgRNA CRISPR-mediated nucleic acid cleavage
  • At least two classes (Class I and II) and six types (Types I-VI) of Cas proteins have been identified across a wide range of bacterial hosts.
  • One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers).
  • the non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer).
  • the Type II CRISPR/Cas is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences.
  • the mature crRNA: tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition.
  • Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.
  • Cas9 is thus the hallmark protein of the Type II CRISPR-Cas system, and a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
  • the Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases.
  • the HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA.
  • Heterologous expression of Cas9 together with an sgRNA can introduce site- specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms.
  • DSBs site- specific double strand breaks
  • the single guide RNA is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease.
  • sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA.
  • the sgRNA guide sequence located at its 5’ end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities.
  • the canonical length of the guide sequence is 20 bp.
  • sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3.
  • Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.
  • the Cas9 enzyme and sgRNA can be introduced to the cells to be edited using multiple methods. Genetic transformation of an expression construct encoding the sgRNA and the Cas9 enzyme (FIG.30) can be used to edit the cells. Subsequent removal of the transgenes encoding the sgRNA and the Cas9 enzyme can be achieved through segregation yielding plants with only the genome edit.
  • the sgRNA can be synthesized in vitro and introduced into cells, often in the form of Ribonucleoprotein complexes (RNPs) that contain Cas9 protein to promote cleavage of the target genomic DNA at the “guide target sequence”.
  • RNPs Ribonucleoprotein complexes
  • TALENs transcription activator- like effector nucleases
  • CRISPR/Cas9 clustered Regularly Interspaced Short Palindromic Repeats
  • ZFN zinc-finger nucleases
  • Camelina with glufosinate tolerance To produce a Camelina line that is tolerant to over-the-top-spray applications of glufosinate for weed control, construct pMBXS1341 (SEQ ID NO:1; FIG.1) was constructed containing the bar gene, which provides tolerance to glufosinate. Plasmid pMBXS1341 was transformed into spring Camelina lines DH12 and E3902 using previously described procedures. E3902 is an edited line previously described as line 17-3902 in Yield10’s international patent application PCT/US2020/043063, filed on July 22, 2020 and published as WO2021/016348, which is incorporated herein by reference.
  • DH12 is a doubled haploid line generated from a large seeded Camelina sativa germplasm 10CS0043 (abbreviated WT43, obtained from Kevin Falk at Agriculture and Agri-Food Canada).
  • Glufosinate 300 mg/L was applied to T1 plants to screen for the bar gene at the fully expanded leaf stage. Lines were grown for several generations and homozygous (T3 generation) lines were obtained. [0167] Testing for glufosinate tolerance in events was performed in a greenhouse by spraying T2 plants with two different commercial glufosinate solutions at rates equivalent to 2X or 4X the rates that are typically used on canola in commercial fields. Commercial sprays contain 150 g/L glufosinate with surfactants.
  • the 1X rate is the recommended rate of in-crop application for glufosinate tolerant canola.
  • the first herbicide treatment was performed at the 2-5 leaf stage.
  • the second treatment was performed prior to bolting. All engineered events expressing the bar gene showed herbicide tolerance in the field when sprayed with 1X and 2X glufosinate (FIG.4).
  • Control plots, planted with either E3902 or DH12 without the bar gene (designated C in FIG.4), were bare one week after the second spray with glufosinate.
  • the harvested seed yield (kg/ha) was calculated for each event tested.
  • FIG.5 and FIG.6 show the seed yields and standard deviations of selected best events from the trial.
  • Group 2 herbicides are a broad class of herbicides that inhibit the enzyme acetohydroxy acid synthase (AHAS), also called acetolactate synthase (ALS), and include imidazolinones (IMIs), sulfonylureas (SUs), triazolopyrimidines, and triazolinones.
  • AHAS acetohydroxy acid synthase
  • ALS acetolactate synthase
  • IMIs imidazolinones
  • SUs sulfonylureas
  • triazolopyrimidines triazolopyrimidines
  • Triazolinones Triazolinones.
  • Camelina is very sensitive to Group 2 herbicides. In some agricultural lands favorable for Camelina growth, application of Group 2 herbicides in the prior growth season(s) can leave soil residues that severely compromise Camelina growth. The length of time that members of the Group 2 family persist in the soil varies with the herbicide, but some have
  • AHAS gene from Glycine max containing P197A (proline to alanine) and W574L (tryptophan to leucine) mutations was used based on a construct described in US Patent 7,951,995.
  • the conventional numbering of these mutations is based on the positions of these amino acids in the Arabidopsis AHAS protein.
  • the soybean gene encoding AHAS with these amino acid substitutions was reported to provide tolerance to Group 2 herbicides (US Patent 7,951,995).
  • Construct pMBXO128 contains the AHAS gene from soybean codon optimized for Arabidopsis (designated GmAHAS-P197A/W574L in FIG.7 (A)) whereas pMBXO133 contains a non-codon optimized AHAS gene from soybean (designated gm-hra in FIG.7 (B) and US Patent 7,951,995).
  • Constructs pMBXO128 and pMBXO133 were transformed into both DH12 and E3902 backgrounds and T1 seeds were isolated. Glufosinate (300 mg/L) was applied to T1 plants to screen for the bar gene at the fully expanded leaf stage.
  • Example 3 Camelina engineered for stacked glufosinate and group 2 herbicide tolerance using the Arabidopsis AHAS mutant genes. [0174] Since no Group 2 tolerance herbicide tolerance was obtained in Camelina using the soybean gene encoding AHAS with P179A and W574L mutations in pMBXO128 and pMBXO133 constructs, a series of new constructs were made with the mutations in the Arabidopsis AHAS gene (TABLE 2).
  • pMBXS1391 (FIG.10, SEQ ID NO: 4), pMBXS1393 (FIG.12, SEQ ID NO: 5), pMBXS1397 (FIG.13, SEQ ID NO: 6), pMBXS1395 (FIG.14, SEQ ID NO: 7), pMBXS1399 (FIG.15, SEQ ID NO: 8), and pMBXO138 (FIG.16, SEQ ID NO: 9) were transformed into Camelina and T1 plantlets were isolated. Plants were screened by applying glufosinate (300 mg/L) to screen for the bar gene at the fully expanded leaf stage.
  • Group 2 herbicides were incorporated into soil 13 days before planting as described in FIG.18. Stand counts and crop injury data were taken 10 and 14 days after planting, respectively. Crop injury was compared visually for events and controls and numerical numbers were assigned. Plots were then sprayed with glufosinate 18 days after planting, as described in FIG.19, and crop injury data were taken 27 days after planting. TABLE 3. Stacked herbicide tolerant lines and controls chosen for field trials * All genetic constructs also contain an expression cassette for the bar gene imparting glufosinate herbicide tolerance as shown in the referenced figures in TABLE 3.
  • IMI soil residue results Significant injury to control line E3902 is apparent when IMI residues are incorporated into the soil whereas events engineered for both group 2 herbicide and glufosinate tolerance have no injury to very low levels of injury (FIG.18 (A)).
  • WA-HT1 a line that has a mutation for IMI tolerance (Hulbert et al., 2018) has low levels of injury (FIG.18 (A)).
  • control plots with E3902 or WA-HT1 contain very few plants, as neither line contains glufosinate tolerance, whereas events engineered for both group 2 herbicide and glufosinate tolerance have little injury (FIG.19) and good plant stands (FIG.20).
  • Seed yields are listed in TABLE 5. TABLE 4. Seed yield (kg/ha) of stacked herbicide tolerant lines grown on SU pretreated soil and treated with over-the-top spray of glufosinate Herbicide treatments are described in FIG.18 (B) and FIG.19 (B). *Means followed by same letter do not significantly differ (P ⁇ 0.05, Duncan’s New MRT) TABLE 5. Seed yield (kg/ha) of stacked herbicide tolerant lines grown on IMI pretreated soil and treated with over-the-top spray of glufosinate Herbicide treatments are described in FIG.18 (A) and FIG.19 (A). *Means followed by same letter do not significantly differ (P ⁇ 0.05, Duncan’s New MRT) Example 4.
  • Mucilage is a polysaccharide that swells and surrounds the seed upon hydration. It is also an antinutritional that, when in seed meal used for feeds, can reduce the absorption of nutrients (Gajardo et al., 2017, 57, 1-12). Mucilage has been shown to reduce digestibility of other seed meals in finfish (Drew et al., 2007) and broiler chickens (Alzueta et al., 2002). Reducing seed mucilage will thus have benefits in use of the seed meal remaining after oil extraction in feed.
  • the transcription factor TRANSPARENT TESTA2 coordinates the gene expression of enzymes for the proanthocyanidins (PAs) in the seed coat and fatty acid biosynthesis in the embryo (Chen et al., 2012). While TT2 activates the biosynthesis pathway of PAs in the seed coat, it represses the expression of the fatty acid biosynthesis pathway enzymes in the embryo (Chen et al., 2012). Arabidopsis TT2 knockout plants have been shown to have significant reductions in the expression of GL2 and MUM4, that can affect the production of mucilage (Wang et al., 2014).
  • GLABRA2 encodes a transcription factor that controls partitioning of imported sucrose between mucilage production and seed oil biosynthesis (Shi et al., 2012; Shen et al, 2006).
  • MUCILAGE MODIFIED 4 MUM4
  • MUM4 MUCILAGE MODIFIED 4
  • Edited line E3902 contains edits in the three alleles of tt2 in Camelina (Yield10’s international patent application PCT/US2020/043063, line 17-3902), in addition to edits in the sdp1 and sdp1-like genes.
  • the mucilage layer was visualized by staining with ruthenium red and measuring the size of the mucilage layer at the width and length of the seeds. This allowed comparison of the mucilage layer between edited line E3902 and wild-type line WT43. Dry seeds of E3902 and WT43 were imbibed in 0.03% ruthenium red solution for three minutes and rinsed with water once.
  • Hybrid seed production in Camelina [0181] Creation of hybrid plants and their subsequent breeding can be used to improve performance of plants (Labroo, M. R. et al, 2021) including for output traits such as seed yield. Heterosis can occur in hybrid plants where plants grown from hybrid seed can outperform the parental lines.
  • FIG.21 and FIG.22 describe (i) the production of male sterile lines using barnase, a strong ribonuclease that when targeted to the anthers leads to male sterility (Szeluga et al., 2023); (ii) the propagation of male sterile lines by pollination with a maintainer line (M); (iii) the production of a restorer line (R) containing an expression cassette for barstar, a ribonuclease inhibitor that can restore male fertility (Szeluga et al., 2023); and (iv) the planting of male sterile and restorer lines in the field to produce F 1 hybrid Camelina seed.
  • FIG.21 uses mechanical or manual removal of Restorer lines.
  • FIG.22 has stacked herbicide tolerance (glufosinate and Group 2 tolerance) and can thus use the second herbicide tolerance that is unique to the Male Sterile line to remove Restorer lines from the field before seed formation.
  • Camelina can be transformed with a genetic construct such as pMBXS1440 (FIG.23, SEQ ID No: 10), pMBXS1446 (FIG.24; SEQ ID NO: 11) or pMBXS1450 (FIG.25; SEQ ID NO: 12).
  • pMBXS1440 FIG.23, SEQ ID No: 10
  • pMBXS1446 FIG.24; SEQ ID NO: 11
  • pMBXS1450 FIG.25; SEQ ID NO: 12
  • Each of these constructs has an expression cassette for barnase (Hartley et al., 1988), a strong ribonuclease, whose expression can be targeted to the anther with an anther specific promoter. This leads to male sterility in the plant.
  • the constructs also have an expression cassette for bar conferring glufosinate tolerance.
  • Each construct also includes a barstar gene cassette, that produces the Barstar protein, a ribonuclease inhibitor that can restore male fertility. While barstar expression cassettes are typically associated with the restorer line, the barstar expression cassette in constructs pMBXS1440 (FIG.23, SEQ ID No: 10), pMBXS1446 (FIG.24; SEQ ID NO: 11) and pMBXS1450 (FIG.25; SEQ ID NO: 12) is driven by a weak pNOS promoter, for low level expression of the Barstar protein. This low level expression of the Barstar protein is designed to increase transformation efficiency as previously described by Bayer Crop Science (2016, USDA Petition No.16_23501p).
  • barstar gene (Hartley et al., 1988) in the vector backbone of pMBXS1450 (FIG. 25; SEQ ID NO: 12) was added upstream of an efficient E. coli ribosome binding site (RBS; Gerngross et al., 1994) and the kanamycin resistance coding sequence, so that barstar and kanamycin are expressed as an operon, Alternatively, barstar and kanamycin can be expressed separately using their own regulatory sequences with the barstar gene inserted upstream of the kanamycin gene.
  • RBS E. coli ribosome binding site
  • pMBXS1440 (FIG.23, SEQ ID No: 10), pMBXS1446 (FIG.24; SEQ ID NO: 11) and pMBXS1450 (FIG.25; SEQ ID NO: 12) differ in expression cassettes for a mutated AHAS gene that leads to group 2 herbicide tolerance either in the length of the promoter or the mutations in the AHAS gene that confer herbicide tolerance.
  • pMBXS1440 (FIG.23, SEQ ID No: 10), pMBXS1446 (FIG.24; SEQ ID NO: 11) or pMBXS1450 (FIG.25; SEQ ID NO: 12) can be transformed into Camelina using floral dip procedures previously described by Lu and Kang (2008).
  • the line chosen for floral dip to produce the male sterile line is ideally an elite inbred line or doubled haploid line that is expected to provide heterosis when crossed with the restorer line.
  • seeds of Camelina sativa germplasm are sown directly into 4 inch (10 cm) pots filled with soil in the greenhouse.
  • Agrobacterium strain GV3101 (pMP90) is transformed with the genetic construct for male sterile line production such as pMBXS1440 (FIG.23, SEQ ID No: 10), pMBXS1446 (FIG.24; SEQ ID NO: 11) or pMBXS1450 (FIG.25; SEQ ID NO: 12) using electroporation.
  • a single colony of GV3101 (pMP90) containing the genetic construct of interest is obtained from a freshly streaked plate and is inoculated into 5 mL LB medium. After overnight growth at 28 °C, 2 mL of culture is transferred to a 500-mL flask containing 300 mL of LB and incubated overnight at 28 °C. Cells are pelleted by centrifugation (6,000 rpm, 20 min), and diluted to an OD600 of ⁇ 0.8 with infiltration medium containing 5% sucrose and 0.05% (v/v) Silwet-L77 (Lehle Seeds, Round Rock, TX, USA). Camelina plants are transformed by floral dip using the transformation construct as follows.
  • T1 seeds are harvested and germinated in soil.
  • Plantlets are screened for tolerance to glufosinate and/or group 2 herbicides, the expected tolerances for transformed plants based on the expression cassettes of plasmids pMBXS1440 (FIG.23, SEQ ID No: 10), pMBXS1446 (FIG.24; SEQ ID NO: 11) or pMBXS1450 (FIG.25; SEQ ID NO: 12).
  • Transgenic events are identified.
  • T1 lines are grown in the greenhouse and pollinated with male fertile plants such as the Maintainer Line. Agronomic and yield evaluation of multiple plants is performed in the T2 generation on single copy lines.
  • T3 seed is collected and seed yield and oil content are determined.
  • the oil content of T3 seeds is measured using published procedures for preparation of fatty acid methyl esters (Malik et al.2015, Plant Biotechnology Journal, 13, 675-688). Based on these procedures, an elite Male Sterile line is isolated that has a single copy insert and agronomic properties that are equivalent or better than the original line prior to transformation. [0189] For propagation and maintenance of the male sterile line, male sterile plants are pollinated with the same genotype as used for the transformation (maintainer line, M, FIG. 22). The seeds harvested from this cross are a mixture of heterozygous transgenic seeds and seeds with the genotype of the maintainer line, with an expected ratio of 1:1 (FIG.22 (B)). [0190] (B) Generation of Restorer Line.
  • a Restorer Line is produced from an elite genotype that is expected to provide heterosis when crossed with the Male Sterile line.
  • the selected elite Camelina genotype can be transformed with genetic constructs pMBXS1480 (FIG.26, SEQ ID NO: 143) or pMBXS1481 (FIG.27, SEQ ID NO: 14). These constructs primarily differ in the expression cassette for herbicide tolerance.
  • Genetic construct pMBXS1480 contains an expression cassette for bar, conferring tolerance to glufosinate, whereas construct pMBXS1481 contains an expression cassette for a mutated AHAS sequence, conferring tolerance to Group 2 herbicides.
  • FIG.22 describes use of a restorer line that has been transformed with an expression cassette for bar.
  • constructs pMBXS1480 (FIG.26, SEQ ID NO: 3) or pMBXS1481 (FIG.27, SEQ ID NO: 14) are transformed into the elite Camelina genotype chosen for the restorer line using the Camelina floral dip procedures described above and T1 seeds are isolated.
  • T1 plantlets are selected by spraying glufosinate.
  • T1 plantlets are selected by applying Group 2 herbicides such as sulfonylureas or imidazolinones to the plants. T1 plantlets with herbicide tolerance are propagated in the greenhouse and T2 seed are isolated. Agronomic and yield evaluation of multiple plants is performed in the T2 generation on single copy lines. T3 seed is collected and seed yield and oil content are determined. The oil content of T3 seeds is measured using published procedures for preparation of fatty acid methyl esters (Malik et al. 2015). [0192] (C) Hybrid seed production. In this example, use of the restorer line with glufosinate tolerance, lines generated with pMBXS1480 (FIG.26), will be described.
  • Group 2 herbicides such as sulfonylureas or imidazolinones
  • Seeds harvested from selected male sterile plants and restorer lines are grown. This can either be done by planting alternating rows of male sterile lines with restorer lines (FIG.22 (D)) or broadcasting mixtures of male sterile line and restorer line seed. Before flowering, the field is sprayed with a glufosinate containing herbicide to keep the male sterile line and restorer line but remove the contaminating plants from the maintainer line that were produced in FIG.22 (B). After flowering, rows with the restorer line can be removed by spraying with a group 2 herbicide as detailed in FIG.22.
  • Camelina line E3902 is an edited line previously described as line 17-3902 in Yield10’s international patent application PCT/US2020/043063, filed on July 22, 2020 and published as WO2021/016348.
  • TAG degradation is an essential process for seed germination.
  • Genes involved in the production of oil in plants include, among others, the following: (i) SUGAR-DEPENDENT1 (also termed “SDP1” or “sdp1”) and SUGAR- DEPENDENT1-LIKE (also termed “SDP1-L,” “sdp1-L,” “SDP1-Like” or “sdp1-like”) genes, which encode oil body-associated triacylglycerol lipases (Eastmond, 2006, Plant Cell, 18, 665); (ii) TRANSPARENT TESTA2 (also termed “TT2” or “tt2”) genes, which encode a transcription factor that coordinates gene expression for fatty acid biosynthesis in the embryo and proanthocyanidins in the seed coat (Chen et al., Plant Physiology, 2012, 160, 1023); and (iii) genes encoding biotin/lipoyl attachment domain-containing (also termed “BADC” or “badc”) proteins, which are negative regulators of the
  • Oil catabolism is initiated by triacylglycerol lipases that hydrolyze fatty acids off the glycerol backbone for subsequent conversion into sugars or amino acids via ⁇ -oxidation, glyoxylate cycle, and gluconeogenesis.
  • triacylglycerol lipases SDP1 and SDP1-L, have been identified in Arabidopsis thaliana. Both enzymes together contribute over 95% of triacylglycerol lipase activity during seed germination (Eastmond, 2006, Plant Cell, 18, 665).
  • Knockout mutants of SDP1 in Arabidopsis thaliana were delayed in germination due to reduced rates of oil degradation, but had no phenotype once photosynthesis contributed to carbon supply, and SDP1-like null mutants had no growth and developmental phenotype (Kelly et al., 2011, Plant Physiology, 157, 866). Both genes are also highly expressed during seed maturation and desiccation in Arabidopsis thaliana, suggesting their involvement in oil loss during desiccation. Desiccated seeds of the Arabidopsis SDP1 null mutant sdp1-5 were larger and had 11.5% higher seed weight per seed as compared to wild- type seeds.
  • TT2 a transcription factor that coordinates the gene expression of enzymes for the proanthocyanidins (PAs) in the seed coat and fatty acid biosynthesis in the embryo
  • PAs proanthocyanidins
  • FUSCA3 transcription factor
  • Null mutants of TT2 lack the dark brown color of the condensed tannins (oxidized PAs) in the maternal seed coat and are therefore easily identified in T 2 seeds (Debeaujon et al., 2003, The Plant Cell Online, 15, 2514). Analysis of seed composition showed that TT2 knockout lines contain up to 79% more fatty acids (based on seed dry weight) compared to wild-type while their protein content was reduced by more than 50%. Most of the increased fatty acids were found to be long-chain (C20) and very-long chain fatty acids (C22; C24) (Chen et al., 2012; Wang et al., 2014).
  • PCT/US2020/043063 discloses that three copies of SDP1, corresponding to SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 as disclosed herein, were identified on chromosome 8, chromosome 13, and chromosome 20, respectively. Subsequent improved sequencing results indicate that SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 are actually on chromosome 20, chromosome 13, and chromosome 8, respectively. This is reflected in Examples 7-11 below.
  • Example 7 Identification of the Camelina orthologs of the Arabidopsis SUGAR- DEPENDENT1 (sdp1) and SUGAR-DEPENDENT1-like (sdp1-like) genes.
  • Triacylglycerol (TAG) content in oil seeds is highest during the late seed maturation phase, but in many species declines during the following desiccation phase. This loss can account for about 10% of the maximum oil content in Brassica napus seeds grown in the greenhouse or in the field (Chia et al., 2005, Journal of Experimental Botany, 56, 1285; Kelly et al., 2013, Plant Biotechnology Journal, 11, 355). Oil catabolism is initiated by triacylglycerol lipases that hydrolyze fatty acids off the glycerol backbone for subsequent conversion into sugars or amino acids via ⁇ -oxidation, glyoxylate cycle and gluconeogenesis.
  • SDP1 SUGAR-DEPENDENT1
  • SDP1-like Two oil body-associated triacylglycerol lipases SUGAR-DEPENDENT1 (SDP1) and SUGAR-DEPENDENT1-LIKE (SDP1-like) have been identified in Arabidopsis thaliana. Both enzymes together contribute over 95% of triacylglycerol lipase activity during seed germination (Eastmond, 2006, Plant Cell, 18, 665). [0202] The sdp1 and sdp1-like genes were selected for editing in Camelina sativa to reduce the turnover of TAGs that occurs in mature seeds, both to prevent yield loss and to prevent the undesirable accumulation of free fatty acids in oil.
  • GenBank was searched for genes annotated as sdp1 or sdp1-like in the Camelina sativa DH55 genome and by using the Genbank BLAST search tool using the Arabidopsis SDP1 and SDP1-like proteins as queries. Eight sequences were identified and are listed in TABLE 7. Two of these sequences (SEQ ID NO: 16, SEQ ID NO: 17) were annotated in Genbank as triacylglycerol SDP1 lipases. The remaining six sequences were annotated as triacylglycerol lipase SDP1-like, SDP1L, or SDP1L-like.
  • FIG.29A-D shows a Clustal O multiple sequence alignment of the Arabidopsis SDP1 and SDP1-like protein and the Camelina orthologs found through this analysis.
  • the three copies of SDP1 were identified on chromosome 8 (XM_010425338.2, SEQ ID NO: 15), chromosome 13 (XM_010453992.2, SEQ ID NO: 16), and chromosome 20 (XM_010492596.2, SEQ ID NO: 17).
  • the Arabidopsis SDP1 protein closely aligns with the identified Camelina SDP1 proteins on Chromosomes 8, 13 and 20 (FIG.29A-D).
  • the copy on chromosome 8 had been previously annotated as an SDP1-like lipase in Genbank (TABLE 7).
  • the three copies of the sdp1-like genes were found in the Camelina genome on chromosome 4 (XM_010506334.2, SEQ ID NO: 21), chromosome 6 (XM_010518019.2, SEQ ID NO: 22), and chromosome 9 (XM_010429278.2, SEQ ID NO: 23).
  • a protein isoform for the gene on chromosome 6 was also predicted (SEQ ID NO: 38) in GenBank which was larger by 36 amino acid residues due to an extra internal sequence.
  • GenBank GenBank which was larger by 36 amino acid residues due to an extra internal sequence.
  • the Arabidopsis SDP1-like protein closely aligns with the identified Camelina SDP1-like proteins on Chromosomes 4, 6 and 9 (FIG.29A-D). TABLE 7.
  • the annotation of the gene as SDP1 or SDP1- like was based on sequence similarity analysis, as well as syntenic analysis.
  • Example 8 Genome editing of the Camelina sativa SUGAR-DEPENDENT1 (sdp1) gene encoding a triacylglycerol lipase.
  • the large seeded C. sativa germplasm 10CS0043 (abbreviated WT43) that was obtained from a breeding program at Agriculture and Agri-Food Canada was used for genome editing of the sdp1 gene target.
  • FIG.30 shows the genetic elements required for editing and how they interact with genomic DNA to achieve an edit.
  • Genetic construct pMBXS1107 (FIG.31 (A), SEQ ID NO: 19), a binary vector containing expression cassettes to produce an sgRNA to target the sdp1 genes, a plant-codon optimized Cas9 (pcoCas9, Li et al., 2013.
  • DsRed gene which encodes a red fluorescent protein from the Discosoma genus of coral (Matz et al., 1999, Nat Biotechnol 17, 969), was constructed. DsRed expression can be used to distinguish transformed T1 seeds from untransformed seeds using a fluorescence microscope or by shining light of the correct wavelength on the seeds and viewing through the appropriate filter.
  • Construct pMBXS1107 (FIG.31 (A)) was designed with the Guide sequence SDP1- #71 (SEQ ID NO: 18; TABLE 8) fused to DNA encoding the RNA scaffold (FIG.30) to allow formation of the functional sgRNA for editing all three copies of the sdp1 gene.
  • a single colony of GV3101 (pMP90) containing the construct of interest was obtained from a freshly streaked plate and was inoculated into 5 mL LB medium. After overnight growth at 28 o C, 2 mL of culture was transferred to a 500-mL flask containing 300 mL of LB and incubated overnight at 28 o C. Cells were pelleted by centrifugation (4,000 rpm, 20 min), and diluted to an OD600 of ⁇ 0.8-1.0 with infiltration medium containing 5% sucrose and 0.05% (v/v) Silwet-L77 (Lehle Seeds, Round Rock, TX, USA).
  • Plants of Camelina sativa germplasm WT43 were transformed by “floral dip” using the pMBXS1107 transformation construct as follows. Pots containing plants at the flowering stage were placed inside a 460 mm height vacuum desiccator (Bel-Art, Pequannock, NJ, USA). Inflorescences were immersed into the Agrobacterium inoculum contained in a 500-ml beaker. A vacuum (85 kPa) was applied and held for 5 min. Plants were removed from the desiccator and were covered with plastic bags in the dark for 24 h at room temperature. Plants were removed from the bags and returned to normal growth conditions within the greenhouse for seed formation (T1 generation of seed).
  • T1 seeds were screened by monitoring the expression of DsRed, a marker on the T- DNA in plasmid vector pMBXS1107 (FIG.31 (A)) allowing the identification of transgenic seeds.
  • DsRed expression in the seed was visualized by fluorescent microscopy using a Nikon AZ100 microscope with a TRITC-HQ(RHOD)2 filter module (HQ545/30X, Q570LP, HQ610/75M) as previously described (Malik et al., 2015, Plant Biotechnology Journal, 13, 675).
  • T1 generation DsRed+ seeds were selected and planted in soil. Plantlets were grown in a greenhouse under supplemental lighting.
  • Tissue was harvested from plants with 3-4 leaves and amplicon sequencing was used to identify edited lines.
  • Amplicon sequencing allows a survey of the different types of edits in a plant (i.e. deletions, insertions) as well as a determination of the number of alleles of the target gene that are edited. A fee for service provider was used to perform amplicon sequencing work.
  • the analysis of amplicon sequencing data from wild-type WT43 plants showed that each sdp1 allele was represented in almost equal numbers (i.e. approximately 33% of sequences correspond to each allele, TABLE 9). The slight deviation from the expected 33% for each allele may be due to a slight bias during PCR for the alleles present on different chromosomes. TABLE 9.
  • Amplicon sequencing reads for sdp1 alleles in WT43 control line [0210] Amplicon sequencing data for the T1 lines transformed with pMBXS1107 showed edits mostly in the form of 1 to 6 base pair deletions or single base pair insertions in the sdp1 gene. The T1 generation line with the highest percentage of edited alleles contained 13.86% editing (line NS56, TABLE 10). TABLE 10. Summary of Amplicon sequencing reads for sdp1 gene edits in select representative T1 lines transformed with pMBXS1107. 2 For a completely edited chromosomal allele, a value of approximately 33% is expected.
  • T2 lines After confirmation of edits in T1 lines, select lines were advanced by growing the plants to produce T2 generation seed. The segregation of the transformed T-DNA sequences (includes expression cassettes for the DsRed marker gene, Cas9 enzyme, and sgRNA) from the edited line was monitored with loss of the visible DsRed marker in T2 seeds and amplicon sequencing verification that the edit was retained in the T2 DsRed- lines. At this point in line development, edits were not yet homozygous and often required at least one additional cycle of breeding to achieve a homozygous edit. [0212] T2 lines were allowed to produce T3 seeds that were planted in the greenhouse to generate T3 lines. Tissue from T3 lines was harvested and edits were characterized by amplicon sequencing.
  • T4 seeds were harvested from T3 homozygous edited plants and a total of eleven lines with homozygous editing were compared to wild-type control plants for seed yield and oil content. Results from the best plants are summarized in TABLE 12. The highest yielding plants were those that contained edits in only the SDP1 gene located on chromosome 13 (NS14 lines, TABLE 12) leaving the copies on chromosomes 8 and 20 intact. Seed yields in these plants increased by up to 39% over the wild-type unedited control plants (TABLE 12).
  • TABLE 12 Summary of T4 seed production from best T3 lines edited in SDP1 gene 1 Wild-type data is from the average of six wild-type control plants. 2 Oil per plant for each line (calculated from seed yield and seed oil content). 3 Symbol “X” denotes complete editing of the chromosomal allele of the gene and “_” denotes the wild-type sequence of the chromosomal allele of the gene. [0214] The results in TABLE 12 suggest that it is difficult to edit all three homeologs of sdp1 in Camelina.
  • Construct pMBXS1126 (FIG.31 (B), SEQ ID NO: 20) was designed with the Guide sequence SDP1- like #4 (SEQ ID NO: 24, TABLE 13) fused to DNA encoding the RNA scaffold (FIG.30) to allow formation of the functional sgRNA for editing all three copies of the sdp1-like gene. TABLE 13. Guide sequences for editing the SDP1-like gene. [0216] Camelina WT43 was transformed with pMBXS1126, using the Camelina transformation procedures described above, and 48 T1 lines were obtained. Analyses of amplicon sequencing data showed that edits obtained were mostly in the form of insertions of 1 base pair or deletions of 1-30 base pairs.
  • T1 lines with a high percentage of edits 38 to 79% total editing in all sdp1-like alleles
  • Four T1 lines with a high percentage of edits 38 to 79% total editing in all sdp1-like alleles
  • transgene-free progenies of OA05 showed a high percentage of editing.
  • Other transgene-free lines (16-4073, 16-4083, and 16-4092) showed complete editing of sdp1-like alleles on chromosomes 4 and 6 and 0% editing on chromosome 9.
  • T3 seeds were planted in the greenhouse and five T3 transgene-free plants derived from T2 plant 16-4076 showed 100% editing in all three alleles by amplicon sequencing. Two of these T3 generation 100% edited lines, lines 17-0607 and 17-0609 (TABLE 14), were propagated further for yield assessment. T3 lines 17-0607 and 17-0609 differ in the nature of their edits with 17-0607 having homozygous and 17-069 having heterozygous edits in chromosomes 6 and 9 in terms of sequence (TABLE 14). These lines were allowed to set seed which was sown in the greenhouse.
  • T4 progeny plants were grown in the greenhouse and amplicon sequencing was performed.
  • the nature of edits in the six T4 progeny plants of 17-0607 was similar to the parental T3 plant 17-0607 showing stable inheritance of all edits (TABLE 14).
  • the T4 progeny of T3 line 17-0609 showed 5 plants with one type of editing (an insertion of nucleotide ‘A’ in the sdp1-like alleles on chromosomes 6 and 9 and an insertion of ‘T’ in the allele on chromosome 4) and one plant with a different editing pattern (an insertion of ‘T’ in all three alleles of sdp1-like gene).
  • T3 plant 17-0609 was heterozygous for editing in the alleles on chromosome 6 and 9 and therefore showed segregation in the nature of edits in the T4 plant population (TABLE 14).
  • the editing in the form of a 1 base pair insertion in all of the edited lines in TABLE 14 will produce a truncated polypeptide leading to a non-functional SDP1-like protein.
  • TABLE 14 Summary of edits in best lines edited in the sdp1-like gene 1 Amplicon sequencing reads showed two kinds of edits on chromosome which segregated in the next generation. 2 Bold underlined letters indicate the edit in the sequence.
  • T4 edited lines progeny of lines 17-0607 and 17-0609, TABLE 14
  • the wild-type plants were taller than the edited plants by the end of flowering, and remained taller during seed filling and maturation.
  • the plants of the two edited lines were 3-4 cm shorter than the wild- type plants as determined by height measurements of mature plants.
  • T4 seed weights from T3 plants of the two lines, 17-0609 and 17-0607 were 4% and 7.9% higher than that of the wild-type plants.
  • the increase in 1000 seed weights of 7.9% in plants of line 17-0607 as compared to the wild-type plants was statistically significant.
  • TABLE 15 Summary of T4 seed production from best T3 lines edited in sdp1-like gene 1 Wild-type data is from the average of six wild-type control plants. 2 Performed with three replicated seed samples from each plant. *Statistically significant compared to wild-type plants. 3 Symbol “X” denotes complete editing of the chromosomal allele of the gene and “_” denotes the wild-type sequence of the chromosomal allele of the gene.
  • the tt2 gene encodes TRANSPARENT TESTA2, a transcription factor that coordinates the gene expression of enzymes for the proanthocyanidins in the seed coat and fatty acid biosynthesis in the embryo (Chen et al., Plant Physiol, 2012, 160, 1023; Wang et al., Plant J., 2014, 77, 757).
  • the sdp1 and sdp1-like genes were previously described in Examples 6-8. While TT2 activates the biosynthetic pathway for proanthocyanidins in the seed coat, it represses the expression of the fatty acid biosynthetic pathway enzymes in the embryo by inhibiting the activity of the transcription factor FUSCA3.
  • tt2 Arabidopsis thaliana null mutants
  • expression of FUSCA3 is increased and leads to an increase in fatty acid biosynthesis in the seed embryo (Chen et al., Plant Physiol, 2012, 160, 1023).
  • Arabidopsis mutants of tt2 lack the dark brown color of the condensed tannins (oxidized PAs) in the maternal seed coat (Wang et al., Plant J., 2014, 77, 757).
  • the objective of the present research was to determine if stacking edits of sdp1, sdp1-like, and tt2 would provide a benefit for seed oil content and/or seed yield.
  • tt2 gene Three copies of the tt2 gene were identified in Camelina, one on chromosome 10 (SEQ ID NO: 25), one on chromosome 11 (SEQ ID NO: 26), and one on chromosome 12 (SEQ ID NO: 27) (TABLE 16).
  • Genetic construct pMBXS1140 (FIG.33, SEQ ID NO: 30) was designed with three separate expression cassettes for the Guide sequences shown in TABLE 16 to target editing of all three copies of the sdp1, sdp1-like, and tt2 genes.
  • each of these Guides are fused to DNA encoding the RNA scaffold (FIG.30) to allow formation of the functional sgRNA for target specific editing.
  • Construct pMBXS1140 also contains an expression cassette for Cas9 and an expression cassette for DsRed. Construct pMBXS1140 was transformed into Camelina using the procedures described above and 44 T1 lines were obtained. Amplicon sequencing of select T1 lines showed editing for all the three gene targets (TABLE 17). TABLE 16. Guide sequences for multiplex editing of the sdp1, sdp1-like, and tt2 genes in Camelina. [0223] As expected, amplicon sequencing data showed different editing efficiency for the three genes in the edited lines obtained from the pMBXS1140 transformation.
  • Event OG31 T1 line 17-0309 The highest editing was observed in event OG31 T1 line 17-0309 with 69% total editing of the sdp1 gene, >99% editing in the sdp1-like gene, and 92% editing in the tt2 gene targets (TABLE 17) .
  • Event OG15 T1 line 17-0293 showed editing of 49% in the sdp1 gene, 84.7% in the sdp1-like gene, and 46% editing in the tt2 gene target. Since all nine alleles of the three genes were edited in these two T1 lines, they were advanced to produce T2 seed.
  • TABLE 17 Summary of percent editing from amplicon sequencing data for eleven T1 lines transformed with pMBXS1140.
  • 1 % editing indicates the sum of total editing of all three alleles of a gene.
  • T2 plants were generated and amplicon sequencing was performed on select T2 plants.
  • T2 transgene-free plants of the OG31 line, identified by loss of DsRed expression, were isolated that showed high editing in the sdp1, sdp1-like, and tt2 targets with some plants possessing 100% editing of tt2 or sdp1-like (TABLE 18).
  • T3 seeds were harvested from the T2 plants and analyzed for seed yield and oil content.
  • T2 line 17-1013 was chosen for advancement to generate stable homozygous edited lines as illustrated in FIG.35. The advancement of seeds from select lines of the two sets was prioritized based on the nature of edits.
  • T3 lines 17-2596 and 17-2617, progeny of line T1 line 17-0309 were completely homozygous for edits in two genes (sdp1-like and tt2 in 17-2596, sdp1 and tt2 in 17-2617) and heterozygous for editing for one allele of the third gene (sdp1 in 17-2596 and sdp1-like in 17-2617) and were categorized as higher priority lines.
  • These T3 edited plants showed normal phenotype similar to the wild-type plants during the growth cycle. All lines with 100% editing in tt2 displayed a non-pigmented seed coat phenotype, such as shown for earlier generation lines in FIG.34.
  • T4 progeny plants of line 17-2596 and 17-2617 were grown out in a randomized complete block design and subsequent amplicon sequencing of these lines showed that they segregated in different patterns as expected from Mendelian segregation.
  • the stably edited lines 17-3902, 17-3909 and 17-3919 were selected for further study (FIG.35).
  • Line 17-3919 is the only plant that showed 100% editing in sdp1, sdp1-like and tt2 genes.
  • the lines with the highest increase in milligrams of oil produced per individual seed also have a lower number of total harvested seeds per plant.
  • This observed yield drag upon increasing oil content suggests that there may not be enough carbon or reducing power available in the plant to both significantly increase oil content and produce a normal amount of seeds.
  • the plants producing a low seed number also tend to flower longer than wild-type controls. This suggests that the plant can sense that it has not produced the typical number of seeds and thus extends its reproductive phase in an attempt to produce more seed.
  • additional gene targets may need to be added to the lines edited in sdp1, sdp1-like, and tt2. TABLE 19.
  • T5 seed yield and oil content for T5 seed of select edited lines 1 Symbol “X” denotes complete editing of the chromosomal allele of the gene and “_” denotes the wild- type sequence of the chromosomal allele of the gene. [0227] To determine the carbon partitioning between the seed oil and seed protein of the edited lines, one gram T5 seed samples obtained from T4 lines 17-3902 and 17-3909 (FIG. 35) were submitted for protein analysis to determine if protein content is altered with increased oil.
  • the levels of free fatty acids in oil extracted from seeds from line 17-3902 can be measured using the American Oil Chemists’ Society standardized method AOCS Ac-541 and compared to levels in oil extracted from wild-type seeds.
  • the presence of the tt2 mutation in line 17-3902 may also lower fiber content in seeds, which may improve the digestibility of Camelina meal used as animal feed. Fiber content in seeds can be measured using standard methods for generation of acid detergent fiber (ADF).
  • Holtzapple describes standardized methods for preparing and measuring acid detergent fiber from plant material (M.T. Holtzapple, in Encyclopedia of Foods Sciences and Nutrition, Editors: Luiz Trugo and Paul M. Finglas, Second Edition, 2003) which can be used in this invention to measure acid detergent fiber.
  • WDH2 was found to be a cold tolerant line of winter Camelina that was able to survive harsh Canadian winter conditions.
  • WDH2 and WDH3 were transformed with genetic construct pMBXS1341 (SEQ ID NO: 1) using the procedures described above for spring Camelina with the exception of a vernalization step for winter Camelina.
  • SEQ ID NO: 1 genetic construct pMBXS1341
  • T2 plants were screened with herbicide and vector backbone-free homozygous plants were advanced to produce T3 seeds.
  • 1X commercial sprays contained 150 g/L glufosinate with surfactants to deliver the recommended 1X commercial rate used for in-crop canola applications (0.53 lbs active ingredient per acre, corresponding to 0.60 kg active ingredient per hectare).
  • WDH2/pMBXS1341 screening 48 single copy T1 events were generated and tested using the procedures described above. These lines were propagated in the green house and 20 single copy, vector backbone-free T2 events were chosen for further testing.
  • FIG.36 shows examples of glufosinate tolerance in T2 events of WDH2/pMBXS1341 with tolerance observed at up to 6X commercial levels of spray of glufosinate containing herbicide.
  • WDH3/pMBXS1341 screening 10 single copy T1 events were generated and tested using the procedures described above. These lines were propagated in the green house and 8 single copy, vector backbone-free T2 events were chosen for further advancement. T3 homozygous seeds were obtained and T3 plants were planted in the greenhouse and sprayed with 4X commercial levels of spray of glufosinate containing herbicide. The performance of these events compared to control line WDH3 is shown in TABLE 22. T3 events have also been planted in the field tents for seed production. TABLE 22.
  • T1 plants were screened with 300mg/l glufosinate and single copy lines were identified.
  • T2 single copy plants were sprayed with 1X commercial rates of glufosinate, IMI herbicide (Imazamox and Imazethapyr), and SU herbicides (Tribenuron and Thifensulfuron).
  • IMI herbicides contained 15.05 g of active ingredient per hectare of Imazamox and 15.05 g of active ingredient per hectare of Imazethapyr.
  • SU herbicide contained 7.5 g of active ingredient per hectare of Tribenuron and 7.5 g of active ingredient per hectare of Thifensulfuron.
  • FIG.37 (A) shows phenotypes of a representative event of WDH3/pMBXS1391 separately treated with 4X commercial levels of glufosinate, 1X commercial levels of IMI (imazamox/imazethapyr), or SU (thifensulfuron/tribenuron) herbicides showing tolerance to all three herbicides.
  • FIG.37 (B) shows dead plants from control WDH3 treated with glufosinate.
  • FIG.37 (C) shows survival of WDH3/pMBXS1391 with treatment of 1X commercial levels IMI herbicide (Imazamox and Imazethapyr) whereas control line WDH3 is severely impaired.
  • FIG.37 (D) shows survival of WDH3/pMBXS1391 with treatment of 1X commercial levels of SU herbicides (Tribenuron and Thifensulfuron) whereas control line WDH3 is severely impaired/dead.
  • Homozygous backbone-free T2 herbicide tolerant plants were advanced to produce T3 seeds that were planted in field trials for further evaluation. T3 seeds were also planted in the cages for pure seed production.
  • winter Camelina lines that are tolerant to glufosinate, IMIs, and SUs can be made with any of the constructs listed in TABLE 3.

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Abstract

Une plante de Camelina transgénique génétiquement modifiée pour être tolérante au glufosinate sans présenter une diminution du rendement en graines est divulguée. L'installation de la plante de Camelina transgénique comprend une insertion d'un gène hétérologue codant pour la phosphinothricine acétyltransférase dans le génome de l'installation de la plante de Camelina transgénique. La plante de Camelina transgénique a été obtenue par transformation d'une plante de Camelina de lignée parentale avec le gène hétérologue, réalisation d'une autopollinisation de la plante de Camelina de lignée parentale transformée pour obtenir T1 graine de la plante de Camelina de lignée parentale transformée, obtention d'une plante de cameline de génération T1 à partir de la graine T1 et réalisation d'un ou plusieurs cycles d'autopollinisation de la plante de Camelina de génération T1 ou de sa descendance pour obtenir la plante de Camelina transgénique. L'installation de la plante de Camelina transgénique est homozygote pour l'insertion. L'installation de la plante de Camelina transgénique ne présente pas de diminution du rendement en graines par rapport à l'installation de la plante de Camelina de lignée parentale.
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