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WO1998044139A1 - Procede ameliore de transformation des vegetaux sar - Google Patents

Procede ameliore de transformation des vegetaux sar Download PDF

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Publication number
WO1998044139A1
WO1998044139A1 PCT/US1998/006109 US9806109W WO9844139A1 WO 1998044139 A1 WO1998044139 A1 WO 1998044139A1 US 9806109 W US9806109 W US 9806109W WO 9844139 A1 WO9844139 A1 WO 9844139A1
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WIPO (PCT)
Prior art keywords
cells
plant
genes
gene
group
Prior art date
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PCT/US1998/006109
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English (en)
Inventor
Michael E. Horn
Gerald E. Hall, Jr.
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Mycogen Plant Science, Inc.
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Application filed by Mycogen Plant Science, Inc. filed Critical Mycogen Plant Science, Inc.
Priority to JP54185498A priority Critical patent/JP2001518782A/ja
Priority to EP98914334A priority patent/EP0970230A1/fr
Priority to AU68716/98A priority patent/AU6871698A/en
Priority to CA002283463A priority patent/CA2283463A1/fr
Priority to BR9807899-2A priority patent/BR9807899A/pt
Publication of WO1998044139A1 publication Critical patent/WO1998044139A1/fr

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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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • 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
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/822—Reducing position variability, e.g. by the use of scaffold attachment region/matrix attachment region (SAR/MAR); Use of SAR/MAR to regulate gene expression

Definitions

  • the present invention relates to the use of scaffold attachment regions (SARs), also known as matrix attachment regions (MARs), in transformation of cells and tissues.
  • SARs scaffold attachment regions
  • MARs matrix attachment regions
  • SARs/MARs are AT-rich genomicDNA sequences that occur in eukaryotic genomes (see Boulikas [1993] J Cell Biochem. 52:14). SARs are non-coding DNA sequences that flank structural genes and stabilize the transcriptionprocess. It is also known that SARs bind to certain components of the proteinaceous scaffold material that encompasses nuclear DNA. SARs have been found to improve the expression of heterologous genes in transformed plants (Allen et al. [1993] Plant Cell 5:603).
  • SARs in plant expression cassettes advantageously decreases the time required to recover a stably transformed plant.
  • the SAR-positive clones invariably appear in culture faster as compared to SAR- negative clones which, in turn, allows for quicker regeneration. Shorter times in culture leads to easier regeneration, less abnormalities in the plants, higher fertility, better seed set, etc. Quicker transformation/regeneration processes are highly desirable from a commercial standpoint due to the fact that shorter time periods allow for dramatically increased throughput of events which result in quicker identification of commercial candidates.
  • Figure 1 shows Southern blots of maize callus samples transformed with SAR- positive plant expression cassettes compared with controls. Treated callus was plated on bialaphos selection and bialaphos-resistantcolonies appeared after 7 to 12 weeks. Callus was extracted for DNA and separated on an agarose gel as per Materials and Methods. Lane 1 : DNA sizing standards; Lanes 2-12: DNA from selected colonies arising on bialaphos selection; Lanes 13 : DNA from PAT+ control callus known to contain the PAT gene; Lane 14: DNA from PAT- control callus known not to contain the PAT gene; Lane 15: plasmid DNA corresponding to the equivalent of 1 copy; Lane 16: plasmid DNA corresponding to the equivalent of 3 copies.
  • Figure 2 shows Southern analysis of DNA from regenerated T 0 plants from callus (Fig. 1) derived from transfection with pAGM 607 plasmid DNA.
  • Lane 1 DNA sizing standards
  • Lane 2 plasmid DNA corresponding to the equivalent of 1 copy
  • Lanes 3-1 1 not relevant
  • Lane 12 DNA from PAT+ control leaf tissue known to contain the PAT gene
  • Lane 13 DNA from PAT- control leaf tissue known not to contain the PAT gene
  • Lanes 15-27 DNA from leaves of T 0 plants regenerated from Southern positive callus.
  • Figure 3 shows Southern analysis of DNA from leaf tissue from plants segregating for the PAT gene in the ST2-1 derived T, generation.
  • Lane 1 DNA sizing standards
  • Lane 2 blank
  • Lanes 3-1 1 DNA from leaf tissue of plants judged to be sensitive to the herbicide
  • Lane 22 DNA from PAT- control leaf tissue known not to contain the PAT gene
  • Lane 23 Lane 12: DNA from PAT+ control leaf tissue known to contain the PAT gene
  • Lane 24 plasmid DNA corresponding to the equivalent of 1 copy
  • Lane 25 plasmid DNA corresponding to the equivalent of 3 copies
  • Lanes 26-28 not relevant.
  • Figure 4 shows a nucleotide sequence of a SAR polynucleotide which can be used according to the present invention.
  • Figure 5 shows a restriction map of plasmid pAGM243.
  • Figure 6 shows a restriction map of plasmid pAGM285A.
  • Figure 7 shows a restriction map of plasmid pAGM607.
  • Figure 8 shows a restriction map of plasmid pAGM608.
  • the present invention concerns methods and materials for increasing frequency of recovery of stable transformation events in plant transformation processes and for increasing the number of low copy number transformants, as well as reducing or eliminating the occurrence of gene silencing throughout subsequent generations descended from the original transformant.
  • SARs are used in a plant expression cassette to provide a plant transformation process that produces a greater percentage of stable transformants and a greater percentage of low copy number transformants than are obtained with SAR-negative plant expression cassettes.
  • a method of the subject invention comprises transforming a cell or tissue with a SAR polynucleotide sequence operably linked to a polynucleotide of interest that encodes a protein, polypeptide, or peptide.
  • SARs useful with the subject invention include, but are not limited to, SARs originating from plants and animals.
  • SAR-encoding polynucleotides useful with the subject invention include, for example, SAR isolated from tobacco (Hall et al, [1991] Proc. Natl. Acad. Sci. USA 88:9320).
  • a SAR polynucleotide useful in practicing the present invention comprises the nucleotide sequence shown in Figure 4, or a functional fragment or mutant thereof.
  • the SAR polynucleotide sequence is provided in the form of an
  • a SAR polynucleotide sequence is operably linked at either and or both ends of the polynucleotideof interest. Transformation can be accomplished using known methods, including, for example, particle bombardment or biolistics transformation with DNA coated microparticles, Agrobacterium-mediated transformation, electroporation, microinjection, magnetophoresis,silicon carbide whiskers,PEG mediated transformation, and protoplast transformation.
  • plant cells are transformed with the polynucleotides of the invention by electroporation according to standard techniques known in the art. See, for example, Pescitelli, S.
  • Transformed plant cells can be selected and then cultured under suitable conditions according to routine practice to generate transformed plantlets and plants.
  • nucleotide sequences of the subject invention can be truncated and/or mutated such that certain of the resulting fragments and/or mutants of the original full-length sequence can retain the desired characteristics of the full-length sequence.
  • restriction enzymes are well known by ordinarily skilled artisans which are suitable for generating fragments from larger nucleic acid molecules.
  • BaB 1 exonuclease can be conveniently used for time-controlled limited digestion of DNA. See, for example, Maniatis et ⁇ /. (1982)
  • mutant sequences of the full length sequence, or fragments thereof can be easily produced with site directed mutagenesis. See, for example, Larionov, O.A. and Nikiforov, V.G. (1982) "Directed Mutagenesis” Genetika 18(3):349-59; Shortle, D., DiMaio, D., and Nathans, D. (1981 ) "Directed Mutagenesis” wro. Rev. Genet. 15:265- 94; both incorporated herein by reference. The skilled artisan can routinely produce deletion-, insertion-, or substitution-typemutations and test whether the resulting mutants contain the desired characteristics of the full length wild-type sequence, or fragments thereof.
  • a polynucleotide comprising a polynucleotide sequence that has substantially the same sequence as a SAR polynucleotide is operably linked to a polynucleotidethat encodes a protein, polypeptide, or peptide.
  • Any desired polynucleotide sequence can be employed to transform cells or tissue according to the present invention.
  • genes used for example, as (i) selective markers (antibiotic and/or herbicide resistance genes), (ii) reporter genes (e.g.,GUS), (iii) insecticide resistance genes (R.t. delta endotoxins) and (iv) any other genes that improve the value or use of a plant.
  • Such genes can include, but are not limited to, glucuronidase,phosphinothricin,N-acetyltransferase, green fluorescent protein (GFP), luciferase, Pat/bar, and glyphosate resistance genes (NPTII, HPT, biomoxila resistance gene, AHAS, ALS, cyanomide hydrolase, adenine deaminase, 2,4-D monooxygeanse).
  • the encoded protein, polypeptide or peptide can be one that is naturally present in the transformed cell or it can be heterologous to the transformed cell.
  • a regulatory sequence such as, for example, a promoter sequence that can regulate transcription of the polynucleotide sequence
  • the SAR polynucleotide sequence is a polynucleotide component of an expression cassette on a suitable vector.
  • Vectors useful with the SAR polynucleotides of the invention are known in the art and can be prepared and/or selected according to standard techniques.
  • the methods and materials of the present invention can be used to transform cells or tissue from any organism, and preferably from a eucaryotic organism.
  • the cells are plant cells. Any plant cell competent to be transformed can be employed in the present invention. It is preferred to employ plant cells that are readily regenerable into whole plants. Suitable plant cells include embryogenic suspension cells, non-embryogenic suspension cells (except in corn where these cells are not regenerable), plant explants, germline cells ( pollen, ovules, meristem domes, megaspore cells, embryos cells, egg cells and embryosacs), microspore cells and callus tissue cells, both compact callus and friable callus.
  • Preferred cells include embryogenic callus, suspension cells (embryogenic suspension) and callused immature zygotic embryos.
  • Particularly preferred plant cells are early embryogenic suspension and young callus (still attached to the zygotic embryo) cells of from about 3-14 and preferably from about 5-10, days old. Wounding the plant cells and/or treating the plant cells prior to being subjected to electroporation is unnecessary and is in fact expressly avoided.
  • embryogenic suspension tissues it is preferred to gently break up the tissue into small clumps or into fine aggregates as small as possible without damaging the embryogenic suspension cells. This can be done by sieving the tissue through a screen, such as, for example, pushing tissue through a 1,000 micron (u) sieve with a spatula or pestle. The finer the aggregates or suspension of undamaged cells, the more efficient the present process.
  • Plant tissue useful with the invention includes, but is not limited to, callus, meristematic, leaf, shoot, root, and embryonic tissue.
  • the present invention is applicable to any plant species including angiosperms (dicots, monocots) and gymnosperms.
  • Suitable crops include corn, wheat (especially Type C wheat callus), sorghum, rice, pearl millet, sugar cane, orchardgrass and other Gramineae plants; soybean, peanuts, alfalfa and other members of the Luguminoseae family; cotton, kenaf, and other members of the Malvaceae family; poppy and other members of the Papavaraceae family; cannabis and other members of the Cannabinaceae family; tea and other members of the Theaceae family; rape (canola), vegetables and oilseed crops and other members of the Cruciferae family; sunflower, safflower and other members of the Compositae family; coffee and other members of the Rubiaceae family; cacao, theobroma and other members of the Byttneriaceae family; fruits and vegetables, trees, orchard crops, and turf grass.
  • Preferred crops include cotton, tomato, sugarbeet, potato, peanut, alfalfa, rice, wheat and especially corn (maize
  • the polynucleotide sequences employed in the present invention comprise any sequences which have a 5' promoter region, a structural gene region and a 3 ' nontranslated region (polyadenylation site) which can be expressed in plants.
  • the polynucleotide sequences can be modified in any manner (extra codons, deletion of codons, changed codons, etc.) as long as gene expression is not prohibited.
  • the polynucleotides inserted into the plants according to the present invention can include any desired gene whether eukaryotic or procaryotic in nature. Usually, more than one gene will be inserted into plant cells which are transformed for agronomic purposes.
  • One gene will typically be a selective marker gene (antibiotic resistance gene or an herbicide resistance gene) in order to easily detect transformants from non-transformed cells. Additional genes can also be added to the plant cell genome to impart an additional property, to suppress an existing property (via "antisense” mechanism) or to amplify a known property of the plant cells and the whole plants regenerated therefrom.
  • the genes can be expressed in specific tissues by the use of tissue specific promoters. The genes can be constructed according to techniques well known to one skilled in the art.
  • Gene constructs may exist as single gene expression cassettes comprised of a promoter, a structural gene coding sequence and a sequence to permit the addition of poly-adenine (poly-A) residues.
  • the promoter is necessary to initiate transcription of the DNA coding for the structural gene into RNA.
  • the promoter may be derived from a variety of sources, as long as it is functional in the cells to be transformed, and may be modified to enhance expression by the addition or deletion of sequences.
  • the DNA may contain intron sequences, either outside or within the coding region for the protein. The removal of these introns and the addition of the poly-A sequence results in the production of a mature messenger RNA (mRNA) which can be translated into the corresponding protein.
  • Gene expression cassettes may be linked in groups of two or more.
  • Polycistronic expression cassettes in which a single mRNA may code for more than one protein, may also be used.
  • expression cassettes may be used to produce an "antisense" RNA from the transcription of a strand of DNA which is opposite to the strand of DNA coding for a protein.
  • promoters active in plants include maize ubiquitin promoter
  • the untranslated leader sequence, including the first intron, of the maize ubiquitin gene may be incorporated, particularly for use in monocot cells.
  • the 35S promoter of Cauliflower Mosaic Virus (Murray et al., [1991] Plant Molecular Biology 16: 1035-1050) or the T-DNA Mas2 promoter of the mannopine synthase gene (Leung et al. , [ 1991 ] Molecular
  • the 35S promoter may contain a deletion with the addition of an upstream enhancer sequence and an intron in the untranslated leader region (Last et al, [1991] Theoretical & Applied Genetics 81 : 581-588) and the Mas2 promoter may also contain a deletion to enhance expression (Leung et al. , supra).
  • Examples of structural genes include reporter genes such as that coding for GUS, or -glucuronidase, (Jefferson et al, [1987] EMBO Journal 6:3901-3907), a selectable marker gene such as that coding for PAT, or phosphinothricinN-acetyltransferase,which confers resistance to the active ingredient of the commercial herbicide Basta (Droge et al, [1992] Planta 187:142-151), or genes which result in expression of a value-added phenotypic trait. Examples of the latter type gene includes those derived from Bacillus thuringiensis (B.t.) which confer resistance to insects such as lepidoptera (Adang et al ,
  • B.t. genes may be reconfigured to enhance their expression in plant cells (Adang et al, U.S. Patent No. 5,380,831 issued January 10, 1995). While herbicide resistance genes serve the purpose of selecting transformants, they also serve the valuable agronomic purpose of allowing herbicide use in the field in otherwise sensitive crops and/or preventing damage to otherwise sensitive crops planted to fields wherein those herbicides were used earlier in the field for weed control (Herbicide carryover).
  • genes of value for use in plants include genes isolated from Bacillus thuringiensis that code for delta-endotoxins as well as truncated and/or synthetic derivatives thereof; fungal resistance genes; oil biosynthesis genes; anti-sense genes and genes responsible for nutritional and/or fiber quality.
  • the DNA and cells are reacted according to the present invention in a suitable buffered medium that is preferably iso-osmotic. See for example, Wong and Neumann's F-medium, Biochemistry and Biophysics Research Communications, Vol. 107, pp.
  • a preferred buffer medium is EPR Buffer (555 mM glucose, 4 mMCaCL, 10 mM Hepes buffer, pH 7.2).
  • EPR Buffer 555 mM glucose, 4 mMCaCL, 10 mM Hepes buffer, pH 7.2.
  • the temperature at which the electroporation process takes place is not critical although it is preferred to cool the DNA/plant cell mixture immediately prior to and after the application of the electric field. Any culturing or regenerating steps are conducted under conditions (including temperature) well known to those of ordinary skill in the art. Heat shock treatments, i.e., 37° C for 10 minutes, of the DNA and plant cell mixture can also be employed.
  • the electroporation step of the present invention is accomplished by applying an electric field to the DNA/cell mixture according to well-known techniques. Any electric field can be employed. Electric pulses can be from 25-5,000 volts (V) or more depending on the current employed. Preferred methods include rectangular pulse generating systems and capacitor discharge systems. The capacitor discharge system creates pulses of exponentially decaying voltages.
  • DNA and the plant cells are incubated together at room temperature for at least about 10 minutes and preferably for 20-90 minutes.
  • the DNA/cell mixture is then transferred in aliquots to electroporation cuvettes and optionally cooled on ice prior to applying an electric field to it.
  • the electric field strength can vary depending on a variety of factors, such as, for example, the particular plant species being transformed, the particular type (including age) of cells being employed in the transformation process, the type of electric field being employed including the length of exposure time of the plant cells to the electric field, the concentration and type of DNA, etc.
  • One of ordinary skill in the art can easily determine the optimum process conditions by employing routine titration experiments.
  • electroporation conditions include a 250-1500 ⁇ F capacitor, 25-500 or more volts and a pulse time of from 50-500 msec. Discharge should be from 25-250 volts. Especially preferred conditions are 850 ⁇ F, 150 V and a pulse time of 200 msec.
  • the electroporation cuvettes can be optionally placed on ice for about 10 minutes. The cuvettes are then allowed to stand at room temperature for at least about 5 minutes and a small aliquot of cell culture medium is added thereto. Samples are then pipetted from the cuvettes and placed in 2 ml of culture medium in a well of a six-well plate. The treated cells are maintained in culture and regenerated employing standard culturing and plants regenerating techniques.
  • SAR polynucleotides contemplated within the scope of the present invention encompass known SARs, including functional fragments and allelic variants of a SAR, as well as any SAR that may be identified in the future so long as the SAR retains substantially the same biological activity as SARs exemplified herein.
  • SARs can be prepared from natural sources or synthesized using standard techniques known in the art, such as an automated DNA synthesizer.
  • the SAR polynucleotides of the subject invention also encompass variant sequences containing mutations in the natural sequences. These mutations can include, for example, nucleotide substitutions, insertions, and deletions as long as the variant SAR sequence retains substantially the same biological activity as the natural SAR sequences of the present invention.
  • the subject invention also concerns cells and tissue transformed using the methods of the invention. Plants, plantlets, and plant seeds transformed to express heterologous genes according to the methods of the described herein are also contemplated within the scope of the invention.
  • Plant Extraction Buffer for callus or ten volumes for leaf material and micro fuging the crude extract for 5 min. Then the supernatant was transferred and microfuged again for 5 min. Extracts were then diluted 10-fold with grinding buffer prior to adding to the microtiter plate. After washing the plates, lOO ⁇ l of l ⁇ g/ml protein A purified goat anti- PAT IgG in Ab Buffer was added and the plates incubated for one hour at room temperature with gentle shaking. The plates were washed and 1 OO ⁇ l of 1 :30,000 dilution of anti-goat antibody conjugated to alkaline phosphatase (Pierce) was added and the plates incubated for one hour at room temperature with gentle shaking.
  • Pierce alkaline phosphatase
  • Southern Blotting and Pre-hybridization were performed essentially as described in Murray, M., et al, [1992] Plant Molecular Biology Reporter, vol. 10(2). Briefly, genomic DNA (5-1 Oug) which has been digested with the appropriate restriction enzyme(s) and resuspended in IX loading buffer is loaded into an agarose/TAE gel (0.85%). The DNA is separated by electrophoresis (75 W/4h) and the gel is then stained (0.1 ug/ml EtBr in 10 mM NaPO) for 30 min. and photographed. The gel is then denatured for 20 min. (150 mM NaPO). The separated DNA is then transferred onto nylon membrane via capillary action overnight.
  • the nylon membrane is then baked for 2 h at 80°C, blocked for 2 h (2% SDS, 0.5% BSA. 1 mM EDTA, 1 mM Orthophenanthroline) and allowed to pre-hybridize for 2 h ( 100 mM Na phosphate buffer (pH 7.8), 20 mM Napyrophosphate,5mM EDTA, 1 mM l,10 orthophenanthroline,0.1% SDS, 10% dextran sulfate 500 ug/ml heparin sulfate, 50 ug/ml yeast RNA, 50 ug/ml herring sperm DNA).
  • DNA template to be used as a probe is labeled with P dCTP using a Prime-It RmT Random Priming Labeling Kit (Stratagene). Labeling efficiency of the probe was measured and approximately 1 X 10 6 CPM/ML is added to the prehybridized membrane.
  • the membrane is hybridized with the probe at 65°C for 12-16 h. The membranes are then washed 3X to remove unbound probe (5mM Na phosphate (pH 7.8), 1.25 mM Na pyrophosphate, 0.25 mM EDTA, 0.1% SDS) and exposed to Kodak scientific imaging film.
  • Unbound probe 5mM Na phosphate (pH 7.8), 1.25 mM Na pyrophosphate, 0.25 mM EDTA, 0.1% SDS
  • Immature zygotic embryos were isolated from 12-day old cobs of the genotype Hill. They were plated on callus initiation medium (CIM), scutellum side up, which contained N 6 salts, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and lO ⁇ M AgNO 3 , incubated in the dark for 8 days at 27-29 °C and then examined for Type II callus formation. 250 callused embryos were chosen and placed into sterile 1.5ml micro- cuvettes, at five embryos per cuvette. 20 ⁇ l of AGM285A plasmid DNA (l ⁇ g/ ⁇ l) was introduced, followed by 200 ⁇ l of EPM buffer (80 mM KC1, 5 mM CaCl 2 , 10 mM hepes,
  • One cuvette was inoculated with pAGM243 (comprising the gene Another cuvette contained no plasmid DNA.
  • the contents of the cuvettes were agitated and then allowed to stand for 1 hr at room temperature. Electroporation was carried out using the gene ZAPPER 450/2500 (IBI) at 850 ⁇ F, 150V for one pulse. The cuvettes were then allowed to stand for 20 min at room temperature before removing the embryos and replating on CIM, scutellum side up, and returned to the dark at 27-29 °C.
  • IBI ZAPPER 450/2500
  • the cuvettes were then allowed to stand for 20 min at room temperature before removing the embryos and replating on CIM, scutellum side up, and returned to the dark at 27-29 °C.
  • the embryos treated with pAGM243 were placed into GUS stain and 9 days later were observed for GUS+ events. Those embryos showed 242 GUS+ events or 48.4 GUS+
  • CMM callus maintenance medium
  • Example 5 Field trials with the maize-SAR+ material showed that 13 out of 19 events gave segregation ratios consistent with heterozygous insertions (3:1 ratio in self crosses; 1 :1 ratio in outcross to wild type).
  • transgenic plants exhibit a relatively high frequency of gene silencing of the transgene as a given transgenic line is advanced from one generation to the next.
  • Gene silencing is a major problem in the commercialization of transgenic crops. It occurs at a rate of less than 0.1% up to 3% and sometimes more. SARs have now been shown to prevent this phenomenon when used at the 3' and 5' ends of the inserted transgene.
  • Transgenic maize lines containing Ubiquitin promoter-phosphinothricin acetyl transferase-now terminator (Ubi-pat-nos) with the Rb7 SAR regions attached at both ends prevent gene silencing in transgenic corn produced by the methods described in this patent or other known transformation methods over several generations in the field.
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO : 1 :
  • TCAAGTGTTA CTAAAATGCG TCAATCTCTT TGTTCTTCCA TATTCATATG TCAAAATCTA 300
  • AATATTCATC TAACAAAAAA AAAACCAGAA AATGCTGAAA ACCCGGCAAA ACCGAACCAA 660

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Abstract

La présente invention concerne l'utilisation des régions SAR (Scaffold Attachment Regions), également connues comme régions MAR (Matrix Attachment Regions), dans des cassettes d'expression pour améliorer l'efficacité transformationnelle de telles cassettes d'expression au cours du processus transformationnel.
PCT/US1998/006109 1997-03-28 1998-03-27 Procede ameliore de transformation des vegetaux sar WO1998044139A1 (fr)

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Application Number Priority Date Filing Date Title
JP54185498A JP2001518782A (ja) 1997-03-28 1998-03-27 改良ｓａｒ植物形質転換法
EP98914334A EP0970230A1 (fr) 1997-03-28 1998-03-27 Procede ameliore de transformation des vegetaux sar
AU68716/98A AU6871698A (en) 1997-03-28 1998-03-27 Improved sar plant transformation process
CA002283463A CA2283463A1 (fr) 1997-03-28 1998-03-27 Procede ameliore de transformation des vegetaux sar
BR9807899-2A BR9807899A (pt) 1997-03-28 1998-03-27 Processo de transformação de plantas por sar aperfeiçoado

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US4285297P 1997-03-28 1997-03-28
US5442297P 1997-07-31 1997-07-31
US60/054,422 1997-07-31
US60/042,852 1997-07-31

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WO2000006757A1 (fr) * 1998-07-31 2000-02-10 Mycogen Plant Science, Inc. Procede ameliore de transformation de plantes a l'aide de regions d'attache d'echafaudage
EP1003849A4 (fr) * 1997-06-03 2004-06-16 Univ North Carolina State Procede pour attenuer la variabilite de l'expression des transgenes dans les cellules vegetales
WO2004076662A1 (fr) * 2003-02-26 2004-09-10 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences Sequences de regions associees de support et leur utilisation
US10947555B2 (en) 2004-04-30 2021-03-16 Dow Agrosciences Llc Herbicide resistance genes
US11371055B2 (en) 2005-10-28 2022-06-28 Corteva Agriscience Llc Herbicide resistance genes
US11685928B2 (en) 2020-09-30 2023-06-27 Nobell Foods, Inc. Recombinant fusion proteins for producing milk proteins in plants
US11840717B2 (en) 2020-09-30 2023-12-12 Nobell Foods, Inc. Host cells comprising a recombinant casein protein and a recombinant kinase protein

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1003849A4 (fr) * 1997-06-03 2004-06-16 Univ North Carolina State Procede pour attenuer la variabilite de l'expression des transgenes dans les cellules vegetales
WO2000006757A1 (fr) * 1998-07-31 2000-02-10 Mycogen Plant Science, Inc. Procede ameliore de transformation de plantes a l'aide de regions d'attache d'echafaudage
WO2004076662A1 (fr) * 2003-02-26 2004-09-10 Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences Sequences de regions associees de support et leur utilisation
US10947555B2 (en) 2004-04-30 2021-03-16 Dow Agrosciences Llc Herbicide resistance genes
US11149283B2 (en) 2004-04-30 2021-10-19 Dow Agrosciences Llc Herbicide resistance genes
US11299745B1 (en) 2004-04-30 2022-04-12 Dow Agrosciences Llc Herbicide resistance genes
US11371055B2 (en) 2005-10-28 2022-06-28 Corteva Agriscience Llc Herbicide resistance genes
US11685928B2 (en) 2020-09-30 2023-06-27 Nobell Foods, Inc. Recombinant fusion proteins for producing milk proteins in plants
US11840717B2 (en) 2020-09-30 2023-12-12 Nobell Foods, Inc. Host cells comprising a recombinant casein protein and a recombinant kinase protein
US11952606B2 (en) 2020-09-30 2024-04-09 Nobell Foods, Inc. Food compositions comprising recombinant milk proteins
US12077798B2 (en) 2020-09-30 2024-09-03 Nobell Foods, Inc. Food compositions comprising recombinant milk proteins
US12139737B2 (en) 2020-09-30 2024-11-12 Nobell Foods, Inc. Host cells comprising a recombinant casein protein and a recombinant kinase protein
US12241109B2 (en) 2020-09-30 2025-03-04 Nobell Foods, Inc. Host cells comprising a recombinant casein protein and a recombinant kinase protein

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CA2283463A1 (fr) 1998-10-08
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EP0970230A1 (fr) 2000-01-12
AR012202A1 (es) 2000-09-27
JP2001518782A (ja) 2001-10-16

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