Classifications

• • 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
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
  • C12N9/1088—Glutathione transferase (2.5.1.18)
• • 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

Definitions

• GSTIIIc glutathione-S-transferase IIIc
• plants in whose genome the new GSTIIIc DNA was incorporated have an increased resistance to herbicides, preferably heteroaryloxyacetamide herbicides, in comparison with the corresponding “starting plants”
• the GSTIIIc DNA as Xbal / BamHI fragment was purified from the vector pET3a-GSTIIIc and cloned into the plasmid Bluescript-SKII (Xbal / BamHI-linearized; Stratagene).
• the GSTIIIc DNA was then isolated from the plasmid Bluescript-SKII-GSTIIIc obtained in this way via an Sstl / Smal restriction cleavage and ligated into the vector pRTlOl (Sstl / Smal-linerized; Töpfer et al. 1987).
• the Escherichia coli strain pET3a-GSTIIIc was obtained from the German Collection of Microorganisms (DSM), Mascheroder Weg lb, D-38124 Braunschweig, Federal Republic of Germany in accordance with the provisions of the Treaty of Budapest on the international recognition of the deposit of microorganisms for the Purposes of patent procedures deposited (Filing date: January 17, 1995).
• the strain received the deposit number DSM 9677.
• Buffer B 1.0 M potassium phosphate pH 7.3
• Gradient 0-100% B in 500 ml
• I I ⁇ m reaction mixture (bulk reaction mix) consisting of:
• the mRNA is removed by alkaline hydrolysis.
• the synthesized cDNA is precipitated and used as a template for the polymerase chain reaction.
• the shoot cultures are kept in a culture room at 24-26 ° C under 12 h light (1000-3000 lux).
• leaf protoplasts approx. 2 g of leaves (approx. 3-5 cm long) are cut into small pieces (0.5 cm x 1 cm) with a fresh razor blade.
• the leaf material is in 20 ml enzyme solution consisting of K3
• the GSTIIIc DNA was then purified as an EcoRI / Smal fragment from the vector pRTl01 -GSTIIIc and linearized into the expression vector pSS (EcoRI / Smal, cloned (Voss A et al, Molec. Breeding 1: 15-26 (1995)) .
• the Zeil suspension is at a density of 5 x 10 4 / ml in K3 medium (0.3 m sucrose) with 1 mg / 1 NAA (naphthyl-1-acetic acid), 0.2 mg / 1 kinetin and 500 mg / 1 of the cephalosporin antibiotic cefotaxime.
• K3 medium 0.3 m sucrose
• NAA naphthyl-1-acetic acid
• kinetin 500 mg / 1 of the cephalosporin antibiotic cefotaxime.
• the cell suspension is diluted every week with fresh K3 medium and the osmotic value of the medium is gradually increased by 0.05 M sucrose (approx. 60 mOsm / kg) per Week reduced.
• a modified "bead type culture” technique (Shillito et al. 1983) is used for the culture and selection of kanamycin-resistant colonies described below.
• K3 medium 0.3 M sucrose + hormones; 1.2% (Seaplaque) LMT agarose (low melting agarose, marine Colloids) are mixed in 5 cm Petri dishes, for this purpose agarose is dry autoclaved and briefly boiled in the microwave after adding K3 medium.
• T L -DNA gene 5 controls the tissue- specific expression of chimaeric genes carried by a noval type of Agrobacterium linary vector. Mol. Gen. Genet. (1986) 204: 338-396
• the plants are grown at a temperature of 23 ° C and a relative humidity of 70 - 80%.
• the treatment with the herbicidal compound mentioned above, formulated as 70 WP (wettable powder), takes place 24 hours after laying out the seeds at concentrations which correspond to an application rate of 2,000-4,000 g of active ingredient / ha.
• This international depository accepts the microorganism designated under I, which it received on 19 95 - 01 - 17 (date of first deposit) 1 .

Applications Claiming Priority (3)

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• 1995
  • 1995-01-23 DE DE19501840A patent/DE19501840A1/de not_active Withdrawn
• 1996
  • 1996-01-10 CN CN96191572A patent/CN1169160A/zh active Pending
  • 1996-01-10 US US08/875,034 patent/US5968796A/en not_active Expired - Fee Related
  • 1996-01-10 WO PCT/EP1996/000068 patent/WO1996023072A1/de not_active Application Discontinuation
  • 1996-01-10 EP EP96900929A patent/EP0805865A1/de not_active Withdrawn
  • 1996-01-10 CA CA002210901A patent/CA2210901A1/en not_active Abandoned
  • 1996-01-10 BR BR9606780A patent/BR9606780A/pt not_active Application Discontinuation
  • 1996-01-10 JP JP8522574A patent/JPH10512451A/ja active Pending
  • 1996-01-10 AU AU44849/96A patent/AU4484996A/en not_active Abandoned
  • 1996-01-10 RU RU97114451/13A patent/RU2169196C2/ru not_active IP Right Cessation
  • 1996-10-01 UA UA97084350A patent/UA51642C2/ru unknown

Non-Patent Citations (1)

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