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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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  • 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
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • 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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
• • 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
  • C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8281—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 biotic stress resistance, pathogen resistance, disease resistance for bacterial 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
  • C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—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 biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

• the present invention relates to a method for increasing the resistance of crop plants to bacterial and fungal pathogens, characterized in that a plant is produced using molecular genetic methods in which the activity of the enzyme flavanone-3-hydroxylase is reduced.
• the method is further characterized in that the enzyme flavanone-3-hydroxylase by molecular biological methods (eg anti-sense construct, co-suppression, the expression of specific antibodies or the expression of specific inhibitors) in whole or in part, continuously or temporarily, in the entire plant or in parts of the plant is inhibited in its activity.
• molecular biological methods eg anti-sense construct, co-suppression, the expression of specific antibodies or the expression of specific inhibitors
• the present invention furthermore relates to plants with increased resistance to bacterial and fungal pathogens, characterized in that the activity of the enzyme flavanone-3-hydroxylase is reduced by molecular genetic methods.
• the productivity of crops can be reduced in many ways by stress factors. These include: viral diseases, bacterial and fungal pathogens, damaging insects, nematodes, snails, bite, heat, cool, cold, lack of water, too high water content in the soil, salinity, too high radiation intensity, too high ozone content, competition for light , Water and nutrients due to accompanying flora, improper or inapplicable herbicide applications (especially in fruit crops), treatments with herbicides, insecticides, fungicides, bioregulators or foliar fertilizers with insufficient selectivity, foliar applications of plant protection products or fertilizers during intensive sun exposure.
• the object of the invention was to find a simple and inexpensive method for permanently improving the resistance 15 to bacterial and fungal pathogens, in particular in crop plants.
• Cimectacarb 25 tere designation: Cimectacarb are used as bioregulators to inhibit plant growth. Their bioregulatory effect arises from the fact that they block the biosynthesis of gibberellins that promote length growth. They inhibit due to their structural relationship
• Luteoliflavan does not normally occur in apple tissue and eriodyctiol occurs as an intermediate of the flavonoid substance echse only in small amounts.
• the expected flavonoids catechin and cyanidin were not detectable in the treated tissue or occurred only in significantly reduced amounts (p Ro melt et al, lecture 8 th International Workshop on Fire Blight, Kusadasi, Turkey, 12th-15th October over 1998).
• prohexadione-Ca, trinexapac-ethyl and other acylcyclohexadiones inhibit 2-oxoglutaric acid-dependent hydroxylases, which are important in the metabolism of phenolic substances. These are primarily chalcone synthetase (CHS) and flavanone-3-hydroxylase (F3H) (W. Heller and G. Forkmann, Biosynthesis, in: The Flavonoids, Harborne, JB (ed.), Chapman and Hall, New York, 1988). However, it cannot be excluded that acylcyclohexadiones also inhibit other, previously unknown, 2-oxoglutaric acid-dependent hydroxylases.
• CHS chalcone synthetase
• F3H flavanone-3-hydroxylase
• the flavonoids eriodictyol, proanthocyanidins which are substituted on the C atom 3 with hydrogen, e.g. Luteforol, luteoliflavan, apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers are increasingly formed from the above-mentioned and structurally related substances.
• flavanon-3-hydroxylase F3H
• Increased concentrations of the phenols hydroxycinnamic acid (p-coumaric acid, ferulic acid, sinapic acid), salicylic acid or umbelliferone, including the homogeneous and heterogeneous oligomers and polymers formed from them, are reduced after reducing the enzyme activity of the enzyme flavanone-3-hydroxylase (F3H ) in plants detected.
• the concentration of the chalcones, such as phloretin, and the stilbene, such as resveratrol also increases.
• the concentration of the glycosides of the flavonoids, the phenolic compounds, the chalcones and the stilbene is also increased by reducing the enzyme activity of the enzyme flavanon-3-hydroxy-lase.
• the process according to the invention for increasing the resistance to attack by bacterial and fungal pathogens by reducing the flavonone-3-hydroxylase enzyme activity can be successfully carried out on the following crop plants: wheat, barley, rye, oats, rice, corn, millet, sugar cane, Banana, tomato, tobacco, bell pepper, potato, rapeseed, sugar beet, soya, cotton, fruit trees from the rosacea family, such as apple and pear, plum, plum, peach, nectarine and cherry, and grapevines.
• the method according to the invention is particularly suitable for increasing the resistance to Venturia inaequalis in apple and pear and to Botrytis cinerea in grapevines.
• Plants whose flavanone 3-hydroxylase was reduced with the help of molecular genetic methods also showed an increased resistance to attack by Erwinia amylovora and other phytopathogenic bacteria.
• the most important phytopathogenic bacteria can be found in the publication "European Handbook of Plant Diseases", Eds. Smith, I.M., Dunez, J., Lelliott, R.A. Phillips, D.H. and Archer, S.A. Blackwell Scientific Publications, 1988.
• the method according to the invention is particularly suitable for increasing the resistance to the following phytopathogenic fungi:
• Erysiphe graminis (powdery mildew) on cereals Erysiphe cichoracearum and Sphaerotheca fuliginea on pumpkin plants Podosphaera leucotricha on apples, Uncinula necator on vines, Puccinia species on cereals,
• Rhizoctonia species on cotton, rice and lawn Ustilago species on cereals and sugar cane, Venturia species (scab) on apples and pears Helminthosporium species on cereals, Septoria species on wheat
• Botrytis cinerea (gray mold) on strawberries, vegetables, ornamental plants and vines, Cercospora arachidicola on peanuts, Pseudocercosporella herpotrichoides on wheat and barley, Pyricularia oryzae on rice,
• Ripe tomato fruits from Lycopersicon esculentum Mill.cv. Moneymakers were washed, dried and, using a sterile blade, the pericarp of seeds, middle columnella and wooden parts freed.
• the pericarp (approx. 50 g) was frozen in liquid nitrogen.
• the material was then crushed in a mixer.
• the comminuted material was mixed with 100 ml homogenizing medium in a pre-cooled mortar.
• the suspension was then transferred to centrifuge cups by pressing through sterile gauze cloths. Then 1/10 vol 10% SDS was added and mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge cup closed and mixed well.
• the supernatant was transferred to a new reaction vessel. This was followed by three further phenol / chloroform extractions and one chloroform extraction. In the following, 1 vol 3 M NaAC and 2.5 vol ethanol were added. The nucleic acids were precipitated overnight at -20 ° C. The next morning, the nucleic acids were pelleted for 15 minutes at 10,000 rpm in a refrigerated centrifuge (4 ° C). The supernatant was discarded and the pellet resuspended in 5 ml of cold 3 M NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. The completely dried pellet was taken up in about 0.5 ml of sterile DEPC water and the RNA concentration was determined photometrically.
• RNA 20 ⁇ g of total RNA was first mixed with 3.3 ⁇ l of 3M sodium acetate solution, 2 ⁇ l of IM magnesium sulfate solution and made up to 100 ⁇ l of final volume with DEPC water. In addition, a microliter
• 5'-TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3.
• the sequence of the oligonucleotide derived using the peptide sequence DHQAW (amino acid 276281 in the sequence FL3H PETHY from Petunia hybridaj was as follows: 5'-CTT CAC ACA (C / G / T) GC (C / T) TG (A / G) G (A / G) TC-3.5. 5
• the PCR reaction was carried out using the Perkin-Elmer tTth polymerase according to the manufacturer's instructions. 1/8 of the cDNA was used as template (corresponds to 0.3 ⁇ g RNA). The PCR program was: 10
• the fragment was cloned into Promega's vector pGEM-T according to the manufacturer's instructions.
• the correctness of the fragment was checked by sequencing.
• the PCR fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends were blunt-ended using the T4 polymerase. This fragment
• Fragment A (529 bp) contains the 35S promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus).
• Fragment B the fragment of the F3H gene in antisense orientation.
• Fragment C (192 bp) contains the termination signal of the octopine syn
• the 5'RACE method (System for Rapid amplification of cDNA ends) was used to clone a larger fragment of the F3H.
• the cDNA first strand synthesis was carried out according to the manufacturer's instructions using the GSP-1 (gene-specific primer) 5'-TTCAC-CACTGCCTGGTGGTCC-3 '. Following RNase digestion, the cDNA was purified using the GlassMAX spin system from Life Tecgnologies TM according to the manufacturer's instructions.
• a cytosine homopolymer was added to the 3 'end of the purified single-stranded F3H cDNA using the terminal deoxynucleotydil transferase according to the manufacturer's instructions.
• the 5 'extended F3H cDNA was amplified using a second gene-specific primer (GSP-2) which binds in the region 3' before the GSP-1 recognition sequence and thus enables a "nested" PCR Manufacturer supplied "5'RACE abrided anchor primer” used, which is complementary to the homopolymeric dC tail of the cDNA.
• GSP-2 second gene-specific primer
• the cDNA fragment amplified in this way and designated as FSH extended was cloned into the vector pGEM-T from Promega according to the manufacturer's instructions.
• the identity of the cDNA was confirmed by sequencing.
• the F3He ⁇ en de c - cDNA fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends were transferred using the T4 polymerase and blunt ends.
• This fragment was cloned into a Smal (blunt) cut vector pBinAR (Höfgen and Willmitzer, 1990) (see Figure 3).
• This vector mediates resistance to the antibiotic kanamycin in plants.
• the DNA constructs obtained contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.
• Fragment A (529 bp) contains the 35S promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus).
• Fragment B the fragment of the F3H gene in the antisense orientation.
• Fragment C (192 bp) contains the termination signal of the octopine synthase gene.
• Tomato seeds (Lycopersicon esculentum Mill. Cv. Moneymaker) were incubated for 10 minutes in 4% sodium hypochlorite solution, then washed 3-4 times with sterile distilled water and on MS medium with 3% sucrose, pH 6.1 Germination designed. After a germination period of 7-10 d, the cotyledons could be used for the transformation.
• Cmm was cultured on yeast dextrose Ca agar (YDC) at 28 ° C for 2 days.
• the bacteria were washed off with sterile water and their cell density was determined.
• the cell density was adjusted to 10 6 cells / ml with sterile water.
• the injections were carried out with injection needles (No. 20), which were filled with the bacterial suspension. They were done in the
• Leaf axil of the top fully developed leaf of young plants, which had a total of 3-4 leaves.
• the infection was evaluated by assessing the developing phenotype.

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