WO2010147636A1 - Camelina sativa résistant aux herbicides - Google Patents

Camelina sativa résistant aux herbicides Download PDF

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WO2010147636A1
WO2010147636A1 PCT/US2010/001704 US2010001704W WO2010147636A1 WO 2010147636 A1 WO2010147636 A1 WO 2010147636A1 US 2010001704 W US2010001704 W US 2010001704W WO 2010147636 A1 WO2010147636 A1 WO 2010147636A1
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als
plant
seq
gene
promoter
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PCT/US2010/001704
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English (en)
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Seppo Kaijalainen
Kimmo Koivu
Viktor Kuvshinov
Eric Murphy
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Huttenbauer, Samuel, Jr.
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Priority to CA2758247A priority Critical patent/CA2758247A1/fr
Priority to EP10789853.8A priority patent/EP2458973A4/fr
Priority to AU2010260518A priority patent/AU2010260518A1/en
Publication of WO2010147636A1 publication Critical patent/WO2010147636A1/fr
Priority to US13/200,951 priority patent/US20130117887A1/en

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    • 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
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • This invention relates to herbicide resistant crop plants. More specifically this invention relates to herbicide resistant Camelina sativa plants.
  • Camelina sativa (L. Crantz) belongs to the family Brassicaceae in the tribe Sisymbrieae and both spring- and winter forms are in production. It is a low-input crop adapted to low fertility soils. Results from long-term experiments in Central Europe have shown that the seed yields of Camelina sativa are comparable to the yields of oil seed rape.
  • Camelina sativa is a minor crop species, very little has been done in terms of its breeding aside from testing different accessions for agronomic traits and oil profiles.
  • Camelina sativa seeds due to the high oil content of Camelina sativa seeds (varying between 30 - 40%), there has been a renewed interest in Camelina sativa oil.
  • Camelina sativa seeds have high content of polyunsaturated fatty acids, about 50 - 60% with an excellent balance of useful fatty acids including 30 - 40% of alpha-linolenic acid, which is omega- 3 oil. Omega- 3 oils from plants metabolically resemble marine omega- 3oils and are rarely found in other seed crops.
  • Camelina sativa seeds contain high amount of tocopherols (appr. 600 ppm) with a unique oxidative stability.
  • Camelina sativa is well suited to marginal soils, this plant species offers an alternative crop that can be grown and harvested in large quantities.
  • improvements in Camelina sativa such as herbicide resistance, increased protein quality, increased oil content, and enhanced agronomic characteristics are lacking.
  • herbicides used in grain production can carry over, resulting in loss of Camelina yields.
  • Herbicides commonly used in the Pacific Northwest America are listed in Table 1 below. Areas where these herbicides are used cannot be used for Camelina cultivation before herbicide residues are degraded. Factors that affect degradation include climate factors such as moisture, and temperature as well as soil pH. In Pacific Northwest America the period that the soil contains herbicide residues may last several years.
  • This invention is aimed to resolve the existing problems: creating an herbicide resistant Camelina sativa line would allow access for Camelina cultivation in areas, where growing Camelina will offer a cropping choice in areas in which choices for rotation crops are limited. Moreover, this invention is aimed to provide herbicide resistant Camelina sativa plants and cultivars.
  • Fig. 1 illustrates the procedure for DNA cloning.
  • Fig. 2 illustrates cloning of plant transformation vector for ALS constructs used in this disclosure
  • Fig. 3 illustrates cloning of Arabidopsis thaliana (A.t) ALSl -gene and regions up- and down-stream from the gene.
  • Fig. 4 illustrates cloning of pC0300-P-At-ALS containing ALS promoter region of Arabidopsis thaliana.
  • Fig. 5 illustrates cloning of pC0300-PT-At- ALS construct containing promoter region (P) and transcription termination region (T) of ALS gene.
  • Fig. 6 illustrates cloning of pC0300-T-At-ALS without promoter region.
  • Fig. 7 illustrates cloning of pC0300-P35 S-At-ALS plant transformation vector containing 35S promoter of Cauliflower Mosaic Virus and Transcription termination region of the ALS gene of Arabidopsis thaliana.
  • Fig. 8 illustrates cloning of pVKl-At-ALS containing coding region of ALS gene amplified in PCR on genomic DNA of Arabidopsis thaliana.
  • Fig. 9 illustrates cloning of Papaver rhoeas ALS 3'UTR downstream of A.t. ALSl-gene in pVKl cloning vector.
  • Fig. 10 illustrates mutagenesis of Arabidopsis thaliana ALSl gene.
  • Fig. 11 illustrates cloning of mutated ALSl gene into plant transformation vectors.
  • Fig. 12 illustrates the mutation (removal) of SEC61 putative TATA-boxes
  • Fig. 13 In vitro leaf segment test of two transgenic C. sativa cv. Celine lines VK6-Al-No24 (shown as 24 on Petri dishes) and VK6-Al-No26 (shown as 26 on Petri dishes).
  • leaf segments of herbicide resistant natural mutant o ⁇ Camelina microcarpa (Cm.) and leaf segments of susceptible C. sativa (Cs.) were used.
  • Leaf segments of transgenic plants are resistant to the herbicides, while non- transgenic Camelina sativa is susceptible.
  • the media in Petri dishes contains herbicides in various concentrations from 0 mg/1 in control plate to 0.001, 0.01, 0.1, 1.0 mg/1 of IMI, MSU and CSU.
  • Fig. 15. Shows a Southern Analysis blot often transgenic ALS-carrying plants (columns 1-10). Column 11 presents plasmid DNA carrying the gene sequence, cut with restriction enzymes and mixed with non-transgenic plant DNA as positive control (BC + M). Column 12 presents a negative control being DNA of non-transgenic Camelina sativa plant DNA mixed with 1 kb Marker (Fermentas) (BC + lkb M). The line does not show any signal.
  • Figure 16A shows seedlings of transgenic lines bearing triple (APW) mutated ALS gene: ALS 11.3 (VK10A5.3) and ALS22.3 (VK13A15.12) of the fourth generation (T3) of Camelina sativa v. Blane Creek.
  • the seedlings were grown one week on various concentrations of herbicides MSU, CSU and IMI. The concentrations are shown in mg/1. Control plate has no herbicide in the grown media.
  • Figure 16B shows seedlings of transgenic lines bearing double (PW) mutated ALS gene: ALS7.3
  • FIG. 16C shows seedlings of transgenic lines bearing A-mutated ALS gene: A3 (VK9A3.4.1) of the second generation (Tl) of Camelina sativa v. Calena, as well as A9.1 (VK14A9.1) and A9.2 (VK14A9.4.2) of the second generation (Tl) Camelina sativa v. Blane Creek.
  • the seedlings were grown one week on the various concentrations of herbicides MSU, CSU and IMI. The concentrations are shown in mg/1. Control plate has no herbicide in the grown media.
  • Figure 16 D shows seedlings of transgenic lines bearing P-mutated ALS gene: A4 (VK9A4.4.2) of the second generation (Tl) of Camelina sativa v. Calena and AlO (VK14A10.16) of the second generation (Tl) Camelina sativa c. Blane Creek.
  • the seedlings were grown one week on various concentrations of herbicides MSU, CSU and IMI. The concentrations are shown in mg/1.
  • Figure 16 E shows seedlings of transgenic lines bearing W-mutated ALS gene: A2 (VK10A2.2) of the second generation (Tl) of Camelina sativa v. Blane Creek and ALS23.3 (VK10A2.3) of the fourth generation (T3) of Camelina sativa v. Blane Creek.
  • the seedlings were grown one week on various concentrations of herbicides MSU, CSU and IMI. The concentrations are shown in mg/1. Control plate has no herbicide in the grown media.
  • Figure 16 F shows seedlings of wild type control seeds of Camelina sativa c. Blane Creek. The seedlings were grown one week on the various concentrations of herbicides MSU, CSU and IMI. The concentrations are shown in mg/1. Control plate has no herbicide in the grown media. Description of the invention
  • the present invention provides methods for producing novel Camelina plants and cultivars resistant to herbicides.
  • the present invention provides novel Camelina plants, tissues and seeds that contain modified acetolactate synthase (ALS) genes and proteins that are resistant to inhibition by herbicides that normally inhibit ALS enzyme.
  • ALS acetolactate synthase
  • ALS-targeting herbicides inhibit acetolactate synthase (ALS), which is required for production of essential branched-chain amino acids such as valine, leucine, and isoleucine.
  • ALS-inhibiting herbicides are sulfonylureas (SU), imidazolinones (IMI), triazolopyrimidines (TP), pyrimidinylthiobenzoates (PTB), and sulfonylamino-carbonyl-triazolinones (SCT). Mutations in some crop plants, for example tobacco, corn and soybean have been found conferring ALS herbicide resistance in those plants. As Camelina sativa has not been a major crop plant, such information is not available for it.
  • ALS -inhibiting herbicides control a wide spectrum of grass and broad leaf weeds at very low application rates. As is shown in Table 1 , the commonly used herbicides in Pacific Northwest in the United States of America to control weeds are members of ALS herbicides. Accordingly, this invention provides Camelina plants resistant to ALS-inhibiting herbicides, thereby providing cultivars for large-scale production in this area.
  • ALS mutants in the nature (e.g. ALS mutant has been documented in Arabidopsis thaliana); however, in conditions where herbicides are not present the mutant enzymes do not function as effectively as normal ALS enzymes. This fact causes cost for fitness for mutated plants in the field.
  • This disclosure provides transgenic Camelina sativa lines that are transformed with artificial ALS enzyme variations giving resistance to IMI and SU herbicides.
  • the transgenic plants according to this invention do not suffer fitness cost, because they have their normal ALS enzymes functional in conditions where herbicide is not present.
  • the additional transgenic ALS mutant works during herbicide contact and allows survival of the plant in those conditions.
  • This invention discloses use of Arabidopsis thaliana (A.t) ALS natural gene cassette comprising promoter, gene coding region and terminator region in creating the transgenic Camelina plants. Because Arabidopsis thaliana has only a single ALS gene per genome, it is functional in all organs and development stages. Although the ALS promoter is strong enough to confer sufficient enzymatic activity with one gene copy only, we have also chosen to use the 35S as an alternative promoter strategy because it strongly and constitutively (ubiquitously) expresses ALS in various organs.
  • TATA-less pALS or -TATA promoters
  • -TATA TATA-less pALS
  • Herbicide - ALS binding is dependent on specific 3D structures of the herbicide and of ALS protein. Generally speaking, protein structure can be changed by mutating its amino acids. Resistance to ALS- inhibiting herbicides is formed by mutated 3D structure of ALS protein and reduced binding of specific herbicide chemical to certain area of the protein.
  • Figure 1 illustrates the scheme of cloning plant transformation vectors containing ALS gene according to this disclosure.
  • Multiple cloning sites (containing several restriction sites) for further cloning of acetolactate synthase gene (ALS-MCS) was cloned by amplifying fragment formed by phosphorylated primers ALS-MCS-F (SEQ ID NO:1) and ALS-MCS-R (SEQ ID NO:2) and cloning it into Xmnl-Pmel digested dephosphorylated pC0300 to make pC0300-ALS-MCS vector carrying the multiple cloning site as is shown in Fig. 2.
  • A.t. ALSl gene putatively containing promoter of ALSl gene, was amplified in PCR using primers P-At-ALS-Fl (SEQ ID NO: 3) and P-At-ALS-Rl (SEQ ID NO: 4).
  • Another 2069 bp fragment, putatively containing A.t. ALSl gene CDS was amplified in PCR using primers At-ALS-Fl (SEQ ID NO: 5) and At-ALS-Rl (SEQ ID NO: 6).
  • ALSl 3'UTR was amplified in PCR using primers T-At-ALS-Fl (SEQ ID NO:7) and T-At-ALS-Rl (SEQ ID NO:8) .
  • Genomic DNA of A.t. CoI-O was used as a template in PCR using Fusion Polymerase (Finnzymes) in reaction conditions according to manufacturer's recommendations.
  • Fig. 3 illustrates cloning of Arabidopsis thaliana (A.t) ALSl -gene and regions up- and down-stream from the gene.
  • PCR-product which was obtained by using primers P-At-ALS-Fl (SEQ ID NO: 3) and P-At-ALS-Rl (SEQ ID NO:4) was precipitated and digested with Xbal and Ncol restriction enzymes and cloned into plant transformation vector pC0300- ALS-MCS, which was opened by using the same restriction enzymes and the vector was dephosphorylated.
  • the new clone was named as pC0300-P-At-ALS.
  • Figure 4 illustrates cloning of pC0300-P-At-ALS containing ALS promoter region of Arabidopsis thaliana (SEQ ID NO: 9)
  • the PCR-product was obtained by using primers T-At-ALS-Fl (SEQ ID NO:7) and T-At-ALS-Rl (SEQ ID NO: 8) , and putatively was precipitated and digested with Agel and Mfel restriction enzymes, and cloned into plant transformation vector pC0300-P-At-ALS. MCS, which was opened by using Aarl and Mfel restriction enzymes and dephosphorylated.
  • the new clone was named pC0300-PT-At-ALS.
  • Figure 5 illustrates cloning of pC0300-PT-At-ALS construct containing promoter region (R)(SEQ ID NO:9) and transcription termination region (T) ( SEQ ID NO: 10) of ALS gene.
  • the PCR-product which was obtained by using primers T-At-ALS-Fl (SEQ ID NO: 7) and T-At- ALS-Rl 9 ( SEQ ID NO:8) was precipitated and digested with Agel and Mfel restriction enzymes, and cloned into plant transformation vector pC0300- ALS-MCS, which was opened by using Aarl and Mfel restriction enzymes and dephosphorylated.
  • the new clone was named pC0300-T-At-ALS.
  • Figure 6 illustrates cloning of pC0300-T-At-ALS.
  • P35S was cut from pC1301 by using EcoRI and Ncol restriction enzymes and cloned into pC0300-T-
  • FIG. 7 illustrates cloning of pC0300-P35S T-At-ALS plant transformation vector containing 35S promoter of Cauliflower Mosaic Virus (SEQ ID NO: 11) and Transcription termination region of the ALS gene of Arabidopsis thaliana (SEQ ID NO: 10).
  • Figure 8 illustrates cloning of pVKl -At-ALS containing coding region of ALS gene amplified in PCR on genomic DNA of Arabidopsis thaliana.
  • A.t. ALSl gene was amplified from At. genomic DNA by using primers At-ALS-Fl (SEQ ID NO: 5) and At-ALs-Rl (SEQ ID NO: 6).
  • the 2069 bp product was cut by using Ncol and Agel restriction enzymes, gel-purified and cloned into pVKl vector, which was cut with Ncol and Agel restriction enzymes dephosphorylated and gel-purified.
  • the construct was named pVK 1-At- ALS.
  • mRNA of Arabidopsis thaliana (the source plant for ALS gene) and Camelina sativa ALS genes are very close to each other by their sequence, which brings the problem of how to detect foreign mutated gene in transgenic plant.
  • transgenic ALSl transcripts e.g. using Northern hybridization or PCR-based methods
  • 103 bp piece of ALS 3'UTR of Papaver rhoeas (SEQ ID NO: 14), (which had clear sequence difference with A. t. ALSl 3'UTR) was cloned into Agel- site close after A.t. ALSl gene's STOP codon.
  • the Papaver rhoeas ALS 3'UTR-fragment was cloned by amplifying two overlapping oligonucleotides, Pr3'-F (SEQ ID NO: 12) and Pr3'-R (SEQ ID NO: 13).
  • the product had one nucleotide error comparing to the origin sequence, which was not repaired.
  • the oligonucleotides carried sites for Eco31I, outside cutting restriction enzyme. After cutting with Eco31I and cloning into pVKl-At-ALS Agel-site, the 5'-site was removed and 3'-site restored for Agel restriction enzyme.
  • the construct was named pVKl-At-ALS-Pr3'.
  • Figure 9 illustrates cloning of Papaver rhoeas ALS 3'UTR downstream of A.t. ALSl -gene in pVKl cloning vector.
  • Mutagenesis was conducted by using PCR and primers carrying the mutating nucleotides.
  • A122T mutation was conducted by amplifying two pieces of A.t. ALSl gene.
  • Primers M13F (SEQ ID NO: 15) and ALS-A 122T-R (SEQ ID NO: 16) were used to amplify the 5' of the mutagenized fragment, and ALS-A122T-F (SEQ ID NO: 17) and ALS-MIuI-R (SEQ ID NO: 18) the 3 '-part of the fragment.
  • the alanine to threonine mutation was in the middle of ALS-Al 22T-F (SEQ ID NO: 17) and ALS-Al 22T- R (SEQ ID NO: 16) primers, which were complementary to each other.
  • PCR was conducted by using Ncol-linearized pVKl -At-ALS-Pr3' as a template by using high fidelity Phusion polymerase (Finnzymes) according to manufacturer's recommendations. After gel-purification, the fragments were used as templates in secondary PCR by using primers M13-F (SEQ ID NO: 15) and ALS-MIuI-R (SEQ ID NO: 18), and the complementary ends of the fragments.
  • the product carried Ncol and MIuI restriction sites, which were used to clone the mutagenized fragment into pVKl-At-ALS-Pr3' to make pVKl-At-ALS-A122T as shown in Figure 10.
  • P197S mutation was created by amplifying a piece of A.t. ALSl gene.
  • Primers M13F (SEQ ID NO: 15) and ALS-P 197S-R (SEQ ID NO: 19) were used to amplify the mutagenized fragment.
  • the proline to serine mutation was in the middle of P197S-R primer.
  • PCR was conducted by using Ncol-linearized pVKl-At-ALS-Pr3' as template by using high fidelity Phusion polymerase (Finnzymes) according to the manufacturer's recommendations.
  • the product carried Ncol and MIuI restriction sites, which were used to clone the mutagenized fragment into pVKl-At-ALS-Pr3' to make pVKl -At-ALS-P 197S.
  • W574L mutation was created by amplifying two pieces of A.t. ALSl gene.
  • Primers ALS-Nhel-F (SEQ ID NO: 20) and ALS-W574L-R (SEQ ID NO: 21) were used to amplify the 5' of the mutagenized fragment and ALS-W574L-F (SEQ ID NO:22) and M13-R (SEQ ID NO:23) the 3 '-part of the fragment.
  • the tryptophan to leucine mutation was in the middle of ALS-W574L-F (SEQ ID NO: 22) and W574L-R (SEQ ID NO: 21) primers, which were complementary to each other.
  • the fragments were used as templates after gel-purification in secondary PCR using primers ALS-Nhel-F (SEQ ID NO: 20), Ml 3-R (SEQ ID NO: 23), and the complementary ends of the fragments.
  • the product carried Nhel and Agel restriction sites, which were used to clone to clone the mutagenized fragment into pVKl-At-ALS-Pr3' to make pVKl-At-ALS-W574L.
  • A122T, P197S -double mutation was created by swapping the region between Eco ⁇ ll and MIuI from pVKl -ALS-P 197S to pVKl-At-A122T to make pVKl-AtALS-A122T-P197S.
  • A122T, 574L -double mutation was created by swapping the region between Xbal and Nhel -sites from pVKl-At-A122T to VKl -At-ALS-W574L to make pVKl -At-ALS-Al 22T- W574L.
  • Pl 97S, W5474L -double mutation was created by swapping the region between Xbal and Nhel -sites from pVKl-At-ALS-P197S to pVKl- At-ALS-W574L to make pVKl-P197S-W574L.
  • the mutation carrying all three mutations was created by swapping the region between Nhel and Xbal -sites from pVKl -At-ALS-Al 22T-P197S to pVKl-At-ALS-W574L to make pVKl-At-ALS-A122T- P197S-W574L.
  • Figure 11 illustrates cloning of mutated ALSl gene into plant transformation vectors. All versions of mutated ALSl gene were cut out from pVKl vector by using restriction enzymes Ncol and Agel, and cloned into pC0300-PT-At-ALS plant vector, which was digested by using Ncol and Aarl restriction enzymes and dephosphorylated. As an example it is shown construction of pC0300-PT-At-ALS- A122T-P197S-W574L.
  • A.t. ALSl 5'UTR contains SEC61 gene (SEQ ID NO:25) close upstream of ALSl CDS (SEQ ID NO:24).
  • SEC61 gene SEQ ID NO:25
  • SEQ ID NO:24 close upstream of ALSl CDS
  • WebGene HCTATA was used to predict the TATA-boxes of SEC61 gene.
  • Figure 12 illustrates the mutation (removal) of SEC61 putative TATA-boxes. Resulting mutated P-At-ALS sequence is according to SEQ ID NO:33.
  • P-At-ALS-SacI-F (SEQ ID NO: 26) and mTATA-R (SEQ ID NO: 27) were used to amplify the 5'- fragment of the TATA-region, mTATA-F (SEQ ID NO:28) and mTATA-R (SEQ ID NO:29) the middle-part of the region and mTATA-F (SEQ ID NO:30) and At-ALS-PvuI-R (SEQ ID NO:31) the 3 '-part of the region.
  • the seeds of Camelina sativa plant grown in greenhouse were sterilized by immersing in 70% ethanol for 1 min and then treating for 5 minutes in 2.5% active Cl (Na-hypoclorite) with an addition of Tween-20 (1 drop per 100 ml). After sterilization the seeds were washed three times in sterile water and placed on solid Murashige and Skoog (MS) agar medium (Murashige and Skoog, Physiol. Plant. 15:472-493, 1962) without sugars for germination. Sterilized seeds were germinated and grown 10 days on solid MS- medium without hormones. Green leaves served as a source of explants for transformation procedure.
  • MS Murashige and Skoog
  • the explants were washed with water containing Ticarcillin (Duchefa) 200 mg /1.
  • the surfaces of the explants were dried on filter paper and the explants were placed on MS medium supplemented with hormones 0.7-1.5 mg/1 6-benzylaminopurine (BAP), 0.3-1.0 mg/1 alpha- naphthaleneacetic acid (NAA) and 100 mg/1 Ticarcillin.
  • BAP 6-benzylaminopurine
  • NAA alpha- naphthaleneacetic acid
  • IMI Imazamox
  • MSU Metsulfuron-methyl
  • CSU Chlorsulfuron
  • Figure 13 shows one of such in vitro leaf segment test of two transgenic C. sativa cv. Celine lines VK6-Al-No24 and VK6-Al-No26 transformed with Al construct (35S promoter - ALS W574 mutant - 35S terminator).
  • Al construct 35S promoter - ALS W574 mutant - 35S terminator.
  • leaf segments of herbicide resistant natural ALS mutant of Camelina microcarpa that has broad resistance to class-2 herbicides and leaf segments of susceptible Camelina sativa. From the figure it can be clearly seen that leaf segments of transgenic plants are resistant to the herbicides, while non-transgenic Camelina sativa is susceptible.
  • FIG. 14 shows IMI and MSU 10 day-tests applied to the shoots recovered after transformation of Blane Creek. Green and well-developed shoots are most evidently transgenic with good expression of the mutant ALS gene. Such shoots were transferred to fresh MS agar with the same herbicides to prove the resistance of the shoots.
  • the herbicide resistance test can also be performed by using surface sterilized seeds that are germinated on the herbicide containing media similar way than leaf explants are grown. Growth of the seedlings is then followed and effects of variable herbicide concentrations are monitored, as it is shown in Figure 16 and Table 3.
  • Total genomic DNA was isolated from leaf tissue of transgenic and non-transgenic Camelina sativa plants by using DNeasy Plant Mini Kit according to the supplier's instructions (Qiagen). The presence of the ALS gene in the herbicide resistant plants was determined by PCR analysis by using 24 nucleotides long primers specific to the promoter sequences of ALS and hpt genes. PCR reaction mix contained approximately 1 ng/ ⁇ l of template DNA and DyNAzyme polymerase (Finnzymes) was used for amplification. PCR program consisted of: 94 0 C for 2 min; 30 cycles of 94° C. for 30 sec, 48°C for 30 sec and 72°C for 2 min.
  • Plant total RNA was isolated from approximately 20 mg leaf samples by using E.Z.N.A Plant RNA kit (Omega Bio-Tek). 250 ng of each sample were denatured in Glyoxal/DMSO RNA loading buffer (Ambion) containing SYBR nucleic acid stain (Molecular Probes).
  • RNA sample 1 ⁇ g was reverse transcribed with RevertAid RNaseH- M-MLV reverse transcriptase 200 u (Fermentas) in 25 ⁇ l reactions consisting in addition to the enzymes, own IX buffer, ImM dNTPs, 2 ⁇ M random nonamer primers (Sigma- Aldrich), 1.5 ⁇ l D(+) trehalose (saturated at room temperature), 800 mM D(+) sorbitol, 1O u SUPERase-in RNase inhibitor (Ambion). Samples were incubated 25° C, 5 min; 37° C, 5 min; 42° C, 5 min; 55° C, 5 min; 93° C, 3 min.
  • the primers for RT-PCR were designed to match the 103 nucleotide sequence (SEQ ID NO: 14) from transcription terminator region of ALS gene of Papaver rhoeas (which was introduced close downstream of stop codon the mutated Arabidopsis ALS gene specifically for this purpose). Most of the RNA samples produced the right size amplification product confirming the right mRNA expression of foreign ALS gene.
  • Total genomic DNA was isolated from leaf tissue o ⁇ Camelina sativa plants by CTAB extraction and
  • RNA probes were synthesized using T7 RNA polymerase on the PCR product carrying promoter of Arabidopsis ALS gene and labeled with digoxigenin-11-UTP. The membrane was hybridized and developed according to the supplier's instructions (Boehringer, Mannheim, The DIG user's guide for filter hybridization): prehybridized at 50° C.
  • RNA probe was 150 ng/ml. After hybridization the membrane was washed in SSC buffers, blocked and detected by using "Anti-Digoxigenin-AP alkaline phosphatase (Boehringer, Mannheim). Chemiluminescent detection was conducted with CSPD-substrate and the membrane was exposed to X-ray film.
  • the Xbal cut 5.8 kb fragment contains whole ALS gene including promoter and part of terminator regions. Therefore the right size T-DNA insert should look as one 5.8 kb band in Fig. 15. If one recognition site of the Xbal restriction enzymes absence in the case of defective insert, the size of the detected band should differ 5.8 kb.
  • Seedlings of the developed transgenic lines of Camelina sativa, cultivars Blane Creek and Calena second (Tl) and fourth (T3) generations were exposed to various concentration of three herbicides: imidazolinone (IMI), chlorsulfuron (CSU) and metsulfuron (MSU). Resistance of the transgenic lines was compared to control non-transgenic seedlings of C. sativa cv. Blane Creek.
  • IMI imidazolinone
  • CSU chlorsulfuron
  • MSU metsulfuron
  • Seeds were sterilized in 2% Na-hypocloride and placed on wet filter paper in sterile conditions to allow germination. On the next day well-germinated seedlings were transferred on Petri dishes containing MS (Murasige-Scoog) 0.7 % agar (without sucrose or hormones). Each dish contained one from the three herbicides in particular concentration (0.0001, 0.001, 0.01, 0.1, 1.0, 10, 100 mg/1). After one week of cultivation in vitro (+25 0 C/+ 18 0 C; light/dark) the seedlings were photographed (as shown on the figures 16A- 16F). The highest survival concentration was found for each transgenic line and for each herbicide and compared to the non-transgenic control. The results are combined in the Table 3.
  • VKl 2 transformation series 17 th March 2009, Blane Creek, constructs: A2 (pCambia0300 PaIs-
  • ALS(W574)-Tals A5 (pCambia0300 PaIs-ALS (A122,P197,W574)-Tals) and A8 (pCambia0300
  • VKl 3 transformation series 26 th March 2009, Blane Creek, constructs: Al 1 (pCambia0300 Pals(-
  • VK12A2 A2 (W574) NO 26 40 12 46 30
  • VK12A2 A2 (W574) YES 29 33 14 48 42

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Abstract

Cette invention porte sur une nouvelle plante résistante aux herbicides, sur un procédé de transformation de Camelina sativa pour une résistance aux herbicides et sur un procédé pour une régénération in vitro améliorée de plantes transformées.
PCT/US2010/001704 2009-06-15 2010-06-15 Camelina sativa résistant aux herbicides WO2010147636A1 (fr)

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CA2758247A CA2758247A1 (fr) 2009-06-15 2010-06-15 Camelina sativa resistant aux herbicides
EP10789853.8A EP2458973A4 (fr) 2009-06-15 2010-06-15 Camelina sativa résistant aux herbicides
AU2010260518A AU2010260518A1 (en) 2009-06-15 2010-06-15 Herbicide resistant Camelina sativa
US13/200,951 US20130117887A1 (en) 2010-06-15 2011-10-05 Herbicide resistant Camelina Sativa

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US26871609P 2009-06-15 2009-06-15
US61/268,716 2009-06-15

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015024957A1 (fr) * 2013-08-21 2015-02-26 Bayer Cropscience Nv Plantes mutantes tolérantes aux herbicides inhibiteurs d'als
WO2020140146A1 (fr) * 2019-01-02 2020-07-09 Linnaeus Plant Sciences Inc. Plantes de camelina sativa résistantes aux herbicides et polypeptides variants d'acétohydroxyacide synthase de cameline

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007024782A2 (fr) * 2005-08-24 2007-03-01 Pioneer Hi-Bred International, Inc. Compositions assurant une tolerance a de multiples herbicides et methodes d'utilisation
WO2007149069A2 (fr) * 2006-06-15 2007-12-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Résistance aux herbicides inhibant l'acétolactate synthase
US20090083882A1 (en) * 2005-11-07 2009-03-26 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Method for increasing the total oil content in oil plants

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI110009B (fi) * 2000-11-13 2002-11-15 Unicrop Ltd Camelina sativan transformaatiojärjestelmä
US20090151023A1 (en) * 2000-11-13 2009-06-11 Viktor Kuvshinov Transformation system for Camelina sativa

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007024782A2 (fr) * 2005-08-24 2007-03-01 Pioneer Hi-Bred International, Inc. Compositions assurant une tolerance a de multiples herbicides et methodes d'utilisation
US20090083882A1 (en) * 2005-11-07 2009-03-26 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Method for increasing the total oil content in oil plants
WO2007149069A2 (fr) * 2006-06-15 2007-12-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Résistance aux herbicides inhibant l'acétolactate synthase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HANSON ET AL.: "Resistance of Camelina microcarpa to acetolactate synthase inhibiting herbicides.", EUROPEAN WEED RESEARCH SOCIETY WEED RESEARCH., vol. 44, 2004, pages 187 - 194, XP008149004 *
See also references of EP2458973A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015024957A1 (fr) * 2013-08-21 2015-02-26 Bayer Cropscience Nv Plantes mutantes tolérantes aux herbicides inhibiteurs d'als
WO2020140146A1 (fr) * 2019-01-02 2020-07-09 Linnaeus Plant Sciences Inc. Plantes de camelina sativa résistantes aux herbicides et polypeptides variants d'acétohydroxyacide synthase de cameline

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AU2010260518A1 (en) 2011-11-03
EP2458973A4 (fr) 2013-05-01
EP2458973A1 (fr) 2012-06-06

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