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WO1993019181A1 - Matiere biologique - Google Patents

Matiere biologique Download PDF

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Publication number
WO1993019181A1
WO1993019181A1 PCT/EP1993/000702 EP9300702W WO9319181A1 WO 1993019181 A1 WO1993019181 A1 WO 1993019181A1 EP 9300702 W EP9300702 W EP 9300702W WO 9319181 A1 WO9319181 A1 WO 9319181A1
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WO
WIPO (PCT)
Prior art keywords
dna
plant
sequence
nematode
seq
Prior art date
Application number
PCT/EP1993/000702
Other languages
English (en)
Inventor
Nils Sandal
Kjeld Marcker
Willem Stiekema
Wouter Lange
René KLEIN-LANKHORST
Original Assignee
Sandoz Ltd.
Sandoz-Patent-Gmbh
Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H.
Steen, Per
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Sandoz Ltd., Sandoz-Patent-Gmbh, Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H., Steen, Per filed Critical Sandoz Ltd.
Publication of WO1993019181A1 publication Critical patent/WO1993019181A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8285—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 nematode resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a DNA region comprising the Beet Cyst Nematode Resistance Locus (BCNR Locus) which when present in a plant such as a Beta sp. is capable of conferring, to the plant, anti-phytopathogenic activity in the form of resistance to nematodes which are known to invade and damage the roots of the sugar beet plants.
  • the DNA region of the invention comprises one or more DNA sequences being single or being present in several repetitive sequences and being, in the wild Beta sp., closely linked to the BCNR Locus.
  • the invention also relates to a gene product or gene products encoded by the BCNR Locus having nematode resistance activity.
  • the invention relates to a genetic construct useful for the construction of a genetically transformed plant having an increased resistance to a phytopathogenic nematode as compared to untransformed plants.
  • the genetic construct comprises and is capable of expressing the BCNR Locus of the invention encoding the anti-phytopathogenic activity in the form of resistance to a phytopathogenic nematode, preferably in combination with several DNA regions encoding the same or analogous gene product or gene products also having anti-phytopathogenic activity in the form of resistance to a nematode.
  • the present invention relates to a genetically transformed plant, especially a genetically transformed sugar beet plant, in which a gene product having the anti-phytopathogenic activity in the form of resistance to a nematode is produced in an increased amount as compared to the untransformed plant so as to result in an increased resistance to phytopathogenic nematodes, especially nematodes of the genus Heterodera sp.
  • Most plants are susceptible to infection by pathogens such as nematodes and develop various undesirable disease symptoms upon infection which cause retarded growth, reduced yield and consequently economical loss to farmers.
  • the plants respond to infection with several defense mechanisms including phytoalexins, deposition of lignin-like material, accumulation of cell wall hydroxyproline-rich glycoproteins, pathogenesis related proteins (PR-proteins) and increase in the activity of several lytic enzymes.
  • PR-proteins pathogenesis related proteins
  • Some of these responses can be induced not only directly by infection but also in some cases by exposure to exogenous chemicals such as ethylene.
  • the full capacity of the defense mechanism of the plant is, however, normally delayed in relation to the onset of infection, and thus, the plant may be severely injured before its defense mechanism reaches its maximum capacity.
  • the defense mechanism of the plant may not in itself be sufficiently strong to effectively combat the infectious organism. This is in particular true for cultivated plants which have often been cultivated with the aim of increasing the yield, decreasing the climate susceptibility, decreasing the nutrient demand etc. Therefore, a normal and necessary procedure is to treat infected plants or plants susceptible to infection with a chemical, e.g. a nematocide either as a prophylactic treatment or shortly after infection. Another procedure is crop rotation which cannot, however, fully overcome the problem.
  • a chemical e.g. a nematocide
  • Beta sp. has been identified from wild Beta sp. possessing resistance against the nematode infection. This resistance is not present in the cultivated sugar beet which, however, possesses other very valuable features. Therefore, on object of the present invention is to provide plants, such as Beta sp., which have the features of the cultivated plant but which also possess anti-phytopathogenic activity in the form of resistance to a nematode.
  • a new method for localizing and characterizing the DNA region encoding the beet cyst nematode resistance is provided and thus, by the present invention it is possible to transfer this DNA region to plants after localization of the DNA region using the method according to the present invention. Thereby, a method for producing transgenic plants possessing the beet cyst nematode resistance is provided.
  • One aspect of the present invention is a DNA region comprising the Beet Cyst Nematode Resistance Locus, the DNA region additionally comprising at least one DNA sequence S which shows a homology of at least 60% with a DNA sequence S(A) from a plant (A) which exhibits nematode resistance, the sequence having been found by genomic subtraction subtracting, from the genome of the resistant plant, the genome of a non-resistant plant (B) of the same species, optionally followed by hybridization of the DNA resulting from the subtraction to DNA from the non-resistant plant (B) and to DNA from a corresponding plant (C) known to be nematode resistant, respectively, and the selection of clones containing DNA sequences from plant (A) which hybridize to DNA from plant (C) and not with DNA from plant (B).
  • a "Nematode resistant" plant is one which exhibits anti-phyto-pathogenic activity towards nematodes which are otherwise known to invade and damage roots. Such resistant plants may exhibit increased yield and/or growth, in comparison with nematode-sensitive such plants. In comparison with nematode sensitive plants, the nematode resistant plants may alternatively exhibit a more rapid onset of the plant defense mechanisms mentioned above, or they may be characterized by a reduced magnitude of such defense mechanisms, ie the need to produce such defenses is diminished as a consequence of the plant being resistant. Many methods of assessing nematode resistance are apparent to the skilled man, and one such method is described in Example 3.
  • the DNA region of the invention may be found using a plant (A) which is near isogenic to plant (B) or using a plant (C) which is a plant of the same species as plant (A) or is a species which exhibits nematode resistance to a higher extent than does plant (A).
  • the DNA region of the invention may be a DNA region comprising the Beet Cyst Nematode Resistance Locus, for example as assessible by the nematode resistance test defined herein, the DNA region additionally comprising at least one DNA sequence S which shows a homology of at least 60% with a DNA sequence selected from SEQ ID NO's.:1-14 as listed below.
  • N means undetermined nucleotide.
  • a sequence comprising about 1100 bp and having the following initial sequence:
  • a sequence comprising about 850 bp and having the following initial sequence:
  • a sequence comprising about 1000 bp and having the following initial sequence:
  • a sequence comprising about 1400 bp and having the following initial sequence:
  • CAGCCC-3' and the following terminal sequence :
  • a sequence comprising about 1400 bp and having the following initial sequence:
  • the homology between the DNA sequences is preferably at least 70%, more preferably at least 75%, still more preferably at least 80% and still more preferably at least 85%, most preferably at least 90% when examined using the hybridization technique under the conditions of 2.0 ⁇ SSC, 0.1% SDS at 65°C and under the washing conditions of 0.5 ⁇ SSC, 0.1% SDS at 50°C.
  • DNA sequences of the invention having a nucleotide sequence as shown in SEQ ID NO's.: 1-14 shown above are closely linked to the BCNR Locus and thus are very valuable markers for isolation of the BCNR Locus.
  • the DNA region comprising the BCNR Locus and one or several of the markers is preferably of a length of at the most 4 million, or at the most 2 million base pairs or at the
  • SUBSTITUTE SHEET most 1 million base pairs, preferably of a length of at the most 500,000 base pairs, more preferably of a length of at the most 200,000 base pairs, still more preferably of a length of at the most 100,000 base pairs, still even more preferably of a length of at the most 50,000 base pairs or more preferably a length of at the most 20,000 base pairs, even more preferable of a length of at the most 10,000 base pairs and most preferably of a length of at the most 5000 base pairs.
  • the DNA region preferably comprises a minimum of 1,000 bp, more preferably 2,000 bp, and still more preferably about 3,000 bp.
  • the genetic distance between the locus of the invention and a DNA sequence S or a DNA sequence having the nucleotide sequence as shown in any of SEQ ID NO's:1-14, as expressed in centiMorgan, is preferably at the most 1.0, more preferably at the most 0.2, more preferably at the most 0.15 and most preferably at the most 0.1.
  • BCNR Locus as assessible by the nematode resistance test defined herein which is in itself is a part of the region and which has preferably been found using the above described method is another aspect of the invention.
  • BCNR Locus contained in the DNA region of the invention encodes a gene product or gene products such as one or more polypeptides or one or more RNA transcript(s) having the anti-phytopathogenic activity in the form of resistance to a nematode such as has been identified in the various species of Beta such as B. patellaris or B. procumbens.
  • the nature of the anti-phytopathogenic activity is characteristic in that it reduces the damage of the plant tissue occurring when the tissue is infected with the phytopathogenic organisms.
  • the term "the anti-phytopathogenic activity in the form of resistance to a nematode” denotes the characteristic activity in a plant ascribable to the DNA region comprising the BCNR Locus or the DNA region, i.e. the capability of the gene product or gene products of the BCNR Locus to reduce or prevent the formation of cysts on the roots of a Beta sp.
  • the anti-phytopathogenic activity is preferably in the form of resistance to a nematode.
  • the BCNR Locus is defined as a DNA sequence comprising one or several genes, the gene product or gene products thereof being capable of conferring to the plant resistance as the gene product or gene products has/have anti-phytophatogenic activity.
  • the anti-phytopathogenic activity of the BCNR Locus may, for example, be assessed by using the biological screening assay 1 or screening assay 2 described in Example 3. In both biological screening assays, the effect of the BCNR Locus on the formation of nematode cysts on the roots, i.e. the capability of the BCNR Locus to retard or prevent this formation of cyst, is directly observed. When a positive result is obtained in any of these methods, i.e.
  • BCNR Locus activity may also be evaluated by microscopic examination of the formation of syncytia in the root tissue.
  • the anti-phytopathogenic activity of the BCNR Locus is a qualitative as well as a quantitative measure reflecting the ability of the gene product or gene products to prevent or retard the formation of cysts thereby reducing or inhibiting the spreading of emerging larvae.
  • the degree of resistance of a host plant may be of various strength ranging from a) non-resistant plants in which the phytopathogenic organism is capable of developing without being affected by the plant to b) partial resistance in which the resistance of the plant results in a reduced survival of the phytopathogenic organism with respect to the adult organism or with respect to the progeny as compared to the survival of the same organism in a non-resistant plant to c) complete resistance in which no phytopathogenic organism is capable of surviving or capable of producing offsprings. Normally, the resistance observed is varying degrees of partial resistance as described under b).
  • a) describes non-resistant Beta sp. in which the cysts on the roots are developed without being affected by the Beta sp.
  • b) describes resistant plants in which the formation of cysts on the roots is reduced as compared to the non-resistant Beta sp.
  • c) describes resistant Beta sp. in which no formation of cysts on the roots takes place.
  • the BCNR Locus comprises one or more genes encoding one or more gene products capable of conferring, to the host, resistance to varying degrees against phytopathogenic organisms such as nematodes.
  • the term "gene” is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3 '-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5'-upstream or 3 '-downstream region.
  • the 5'-upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter.
  • the 3'-downstream region comprises sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3' untranslated region.
  • the DNA region according to the invention may be produced using a DNA sequence S(A).
  • another important aspect of the present invention is a method for the production of the DNA region comprising a) selecting, from a genomic DNA library from nematode-resistant plants, DNA which hybridizes to a DNA sequence S(A) to a degree of 60% using the hybridization conditions of 2.0 ⁇ SSC, 0.1% SDS at 65°C and the washing conditions of 0.5 ⁇ SSC, 0.1% SDS at 50°C and, b) hybridizing the selected DNA to a cDNA region from a library of cDNA
  • cDNA regions being positioned in a distance of less than 2 million base pairs, preferably less than 1 million base pairs from the cDNA capable of hybridizing to the DNA selected from the hybridization with the DNA sequence S(A), and from which established cDNA region comprising the BCNR Locus the gene/genes in the Locus is/are expressed when inserted into the genome of a host plant which in itself is susceptible to infection with a nematode in such a way that resistance against the phytopatogenic nematode is conferred to the host plant.
  • the hybridization between the DNA from the genomic library and the DNA sequence S(A) is to a degree of preferably at least 70%, more preferably at least 75%, still more preferably at least 80% and still more preferably at least 85%, most preferably at least 90% using the hybridization technique under the hybridization conditions of 2.0 ⁇ SSC, 0.1% SDS at 65°C and the washing conditions of 0.5 ⁇ SSC, 0.1% SDS at 50°C.
  • the DNA region of the present invention which may be produced by the above described method is preferably derived from a member of the family Chenopodiaceae, Solanaceae, Apiaceae, Brassicaceae, Cucurbitaceae or Fabaceae.
  • the region may be derived from a com, alfalfa, oat, wheat, rye, rice, barley, sorghum, tobacco, cotton, sugar beet, fodder beet, red garden beet, leef beet, sunflower, carrot, bean, chenille, tomato, potato, soybean, oil seed rape, raddish, white mustard, cabbage, pepper, lettuce and pea.
  • sequences and subsequences as used herein with respect to sequences and subsequences according to the invention should of course be understood as not comprising these phenomena in their natural environment, but rather, e.g., in isolated, purified, in vitro or recombinant form.
  • Another aspect of the invention is the gene product or gene products encoded by the DNA region comprising the BCNR Locus.
  • the gene product or gene products confer(s) to the host plant resistance against a phytopathogenic nematode.
  • the gene product or gene products of the invention may be one or more polypeptides or may be one or more RNA transcripts.
  • the DNA region of the invention comprising the BCNR Locus or any of the DNA sequences SEQ ID NO's.:1-14, of the invention and especially a single stranded DNA or RNA sequence which is substantially complementary to either strand of such a DNA sequence may be used to isolate corresponding sequences from other plants, whereupon they, if desirable, may be modified as described herein.
  • the BCNR Locus of the invention may be fused to one or more second nucleotide sequences encoding a second polypeptide or part thereof under conditions which ensure that at least part of the DNA sequence of the invention is expressed in conjunction with the other nucleotide sequence(s), e.g. in the form of a fusion protein.
  • the BCNR Locus or part thereof possessing the nematode resistance activity as assessed by one of the screening assays as described in Example 3 may advantageously be fused to a 3'-terminal sequence encoding a signal peptide which gives rise to transport of the fusion protein expressed therefrom to specific organelles of the organism expressing the gene product or gene products.
  • One specific organ which may be of particular interest is the root.
  • a gene product or gene products encoded by the BCNR Locus preferably in a non-naturally occurring or recombinant form.
  • the gene product or gene products of the invention has/have the advantage that it/they may be easily produced in large quantities by use of well known conventional recombinant productions techniques, e.g. as described in Sambrook et al., 1989, and that it/they may be obtained in a form which is free from impurities normally associated with the naturally occurring gene product or gene products.
  • the gene product or gene products of the invention may be used as a constituent in an anti-phytopathogenic composition, e.g. as described below.
  • the BCNR Locus possesses very valuable features with respect to anti-phytopathogenic activity in the form of resistance to nematodes.
  • the use of a DNA region comprising the BCNR Locus encoding a polypeptide having the anti-phytopathogenic activity as defined above is expected to be very interesting in the construction of genetically modified plants having an increased resistance to phytopathogenic organisms as compared to untransformed plants.
  • the DNA region according to the can be inserted into the genome of a host plant which in itself is susceptible to infection by a phytopathogenic nematode in such a way that a gene/genes in the BCNR Locus is/are expressed, thereby conferring, to the host plant, resistance to infection by a phytopathogenic nematode.
  • Another aspect of the present invention relates to a genetic construct consisting of the DNA region comprising the BCNR Locus which genetic construct can then be used to genetically transfer a plant such as a cultivated plant in such a way that is becomes resistant to phytopathogenic nematodes.
  • the present invention relates to a genetic construct comprising a promoter functionally connected to a DNA region as defined according to the present invention or produced by the above described method of the invention, and a transcription terminator functionally connected to the DNA sequence.
  • the genetic construct may be used in the construction of a genetically modified plant in order to produce a plant showing an increased anti-phytopathogenic activity and thus an increased resistance towards phytopathogenic nematodes. It is contemplated that an increased effect will be observed in general when the gene product or gene products of the BCNR Locus is/are produced in larger amounts than what can be produced by only one Locus and therefore it may by advantageous to combine several BCNR Loci so as to obtain an increased expression of the gene product or gene products.
  • the present invention relates to a genetic construct comprising one or more copies of a DNA region according to the present invention or produced by the above described method of the invention, each of the DNA regions being functionally connected to a promoter and a transcription terminator capable of expressing the DNA region into functional gene products capable of conferring to the host plant resistance to a phytopathogenic nematode.
  • genetic constructs as defined above may be designed and prepared.
  • elements of the genetic constructs which may be varied are the number of copies of each of the DNA sequences of the genetic construct, the specific nucleotide sequence of each of the DNA sequences, the type of promoter and terminator connected to each DNA sequence, and the type of any other associated sequences, e.g. a C-terminal or N-terminal sequence (described below).
  • genetic constructs of the present invention may vary within wide limits. Normally, the combination of each of the above mentioned variable elements of the genetic construct to be chosen will depend, e.g. on the desired strength of the anti-phytopathogenic activity to be obtained which may be determined as a function of gene dosage and specific nucleotide sequence of each of the DNA sequences, and the type and strength of the promoter and terminator used for each DNA sequence.
  • a genetic construct of the invention which is to be expressed in a given organism such as a plant
  • the genetic construct of the invention is too large, it may be difficult to obtain a stable introduction thereof into the genome of the plant which may lead to excision of a part of or the entire genetic construct from the genome of the plant.
  • the genetic construct should be adapted so that the expression products therefrom are generally acceptable to the host organism.
  • the genetic construct of the invention as described above may be present on one or several DNA regions.
  • the use of more than one vector is discussed below. When the use of only one plant transformation vector is desirable, it is advantageous that the genetic construct is present on one DNA region.
  • the DNA sequence of the invention When a polypeptide encoded by the DNA sequence of the invention is to be expressed inan organism, e.g. in a plant, it is desirable that the DNA sequence further comprises anucleotide sequence encoding a signal sequence.
  • the signal sequence may be the natural signal sequence, or a signal sequence derived from DNA encoding another protein.
  • the signal sequence is to be functionally connected to the DNA sequence so that a polypeptide expressed from the resulting nucleotide sequence serves to direct a polypeptide encoded by the DNA sequence out of the endoplasmic reticulum of the cell in which it is produced.
  • the polypeptide may be directed to specific locations of the organism in which it is produced, e.g. to lysosomes or vacuoles, or the passenger polypeptide may be excreted into the intracellular space.
  • the signal sequence may be either N-terminally or C-terminally positioned.
  • N-terminal sequence to be used will e.g. depend on the particular organism and the part thereof, e.g. the specific cell or tissue, in which the polypeptide encoded by the DNA sequence of the invention is to be produced and to which part of the same cell or another location in the organism the polypeptide is to be transported.
  • a typical leader peptide has a core of hydrophobic amino acids and thus, a suitable leader sequence to be used in connection with the DNA sequence of the invention is a nucleotide sequence comprising a stretch of codons encoding hydrophobic amino acids.
  • each of the DNA sequences of the genetic construct of the invention or of a gene comprising such DNA sequences are accomplished by means of a regulatory sequence functionally connected to the DNA sequence or gene so as to obtain expression of said sequence or gene under the control of the inserted regulatory sequence.
  • the regulatory sequence is a promoter which may be constitutive or regulatable.
  • promoter is intended to mean a short DNA sequence to which RNA polymerase and/or other transcription initiation factors bind prior to transcription of the DNA to which the promoter is functionally connected, allowing transcription to take place.
  • the promoter is usually situated upstream (5') of the coding sequence.
  • promoter includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site.
  • regulatory sequences are, e.g. sequences which are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological conditions.
  • a “constitutive promoter” is a promoter which is subjected to substantially no regulation such as induction or repression, but which allows for a steady and substantially unchanged transcription of the DNA sequence to which it is functionally bound in all active cells of the organism provided that other requirements for the transcription to take place are fulfilled.
  • a “regulatable promoter” is a promoter the function of which is regulated by one or more factors. These factors may either be such which by their presence ensure expression of the relevant DNA sequence or may, alternatively, be such which suppress the expression of the DNA sequence so that their absence causes the DNA sequence to be expressed. Thus, the promoter and optionally its associated regulatory sequence may be activated by the presence or absence of one or more factors to affect transcription of each of the DNA sequences of the genetic construct of the invention.
  • regulatory sequences are upstream and downstream sequences involved in control of termination of transcription (transcription terminators) and removal of introns, as well as sequences responsible for polyadenylation, and initiation of translation.
  • transcription terminators termination of transcription
  • introns sequences responsible for polyadenylation, and initiation of translation.
  • the regulatory sequence is to function in a plant, it is preferably of plant origin.
  • tissue specific regulation may be regulated by certain intrinsic factors which ensure that genes encoding gene products specific to a given tissue are expressed.
  • tissue specific promoters are leaf specific promoters such as the chlorophyll a/b promoter and the AHAS promoter, and further root specific, stem specific, seed specific and petal specific promoters. Also factors such as pathogenic attack or certain biological factors have been shown to regulate promoters.
  • the regulatory sequence may be a BCNR Locus promoter, i.e. a promoter which is naturally found in connection with the gene or genes of the BCNR Locus and involved in the transcription thereof.
  • the regulatable promoter may be regulatable by a phytopatogenic nematode.
  • heat-response promoters and promoters involved in the developmental regulation of plants may be found to be of interest.
  • a suitable constitutive promoter is selected from the group consisting of plant promoters, fungal promoters, bacterial promoters, or plant virus promoters.
  • Suitable examples of plant virus promoters are promoters which may be derived from a cauliflower mosaic virus (CaMV). Such promoters are normally strong constitutive promoters. Examples of these are the CaMN 19S promoter and a CaMV 35S promoter (Odell et al., 1985).
  • promoters may be derived from the Ti-plasmid such as the octopine synthase promoter, the nopaline synthase promoter (Herrera-Estrella et al., 1983), the mannopine synthase promoter, and promoters from other open reading frames in the T-D ⁇ A such as ORF7- Further examples of suitable promoters are MAS/35S (Janssen and Gardner, 1989), MAS dual Tr 1,2 (Nelten et al., 1984) and a T-2 D ⁇ A gene 5 promoter (Konz and Schell, 1986).
  • the gene product is a polypeptide it may be advantageous that at least one of the D ⁇ A sequences of the genetic construct of the invention further comprises a 3'-terminal sequence encoding a signal peptide capable of directing the polypeptide encoded by the D ⁇ A sequence to a part of an organism in which it is to be expressed, e.g. the vacuole.
  • the 3'-terminal sequence may be the 3'-terminal extension normally associated with the D ⁇ A sequence, if any, or may be derived from the host in which the genetic construct is to be expressed or may be of another origin.
  • the natural promoter may be modified for the purpose, e.g. by modifications of the promoter nucleotide sequence so as to obtain a promoter functioning in another manner than the natural promoter, preferably being stronger.
  • each of the coding D ⁇ A sequences of the genetic construct of the invention is functionally connected to a transcription terminator.
  • the transcription terminator serves to terminate the transcription of the D ⁇ A into R ⁇ A and is preferably selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences and plant virus terminator sequences.
  • Suitable transcription terminators are a ⁇ OS and OCS transcription terminator sequence of the opine synthase genes of Agrobacterium (Herrera-Estrella et al., 1983), a 35S transcription terminator sequence of the cauliflower mosaic virus (Paszkowski et al., 1984), a PADG4 transcription terminator to the D ⁇ A gene 4 (Wing et al., 1989), and a PADG7 transcription terminator to the T-DNA gene 7.
  • One or more of the DNA sequences of the genetic construct of the invention may advantageously be functionally connected to an enhancer sequence which results in an increased transcription and expression of the DNA sequence(s).
  • Suitable enhancer sequences and means for obtaining an increased transcription and expression are known in the art.
  • the specific promoters and the specific terminators, respectively, to be connected with each of the DNA sequences of the genetic construct may be the same or different. It may be an advantage to use different promoters and terminators, respectively, because then the risk of recombinational events, which may lead to excision of parts of or the entire genetic construct, are avoided.
  • the present invention relates to a vector which is capable of replicating in a host organism and which carries a DNA region of the invention comprising a BCNR Locus, or a genetic construct of the invention.
  • the vector may either be one which is capable of autonomous replication, such as a plasmid, YAC, or one which is replicated with the host chromosome, such as a bacteriophage or integrated into a plant genome via the border sequences of Ti vectors.
  • the vector is an expression vector capable of expressing the DNA sequences in the organism chosen for the production.
  • the expression vector is a vector which carries the regulatory sequences necessary for expression such as the promoter, an initiation signal and a termination signal, etc. These regulatory sequences may be the ones carried by the genetic construct of the invention.
  • the present invention relates to a host organism which carries and which is capable of replicating or expressing an inserted DNA region as defined above.
  • inserted indicates that the DNA region has been inserted into the organism or an ancestor thereof by means of genetic manipulation, in other words, the organism may be one which did not naturally or inherently contain such a DNA region in its genome, or it may be one which naturally or inherently contains such a DNA region, but in a lower number so that the organism with the inserted DNA region has a higher number of such regions than its naturally occurring counterparts.
  • the DNA region carried by the organism may be part of the genome of the organism, or may be carried on a vector as defined above which is harboured in the organism.
  • the DNA region may be present in the genome or expression vector as defined above in frame with one or more second DNA regions encoding a second gene product or part thereof so as to encode a fusion gene product, e.g. as defined above.
  • the organism may be a higher organism such as a plant, or a lower organism such as a microorganism.
  • a lower organism such as a bacterium, e.g. a gram-negative bacterium such as a bacterium of the genus Escherichia, e.g. E. coli, or of the genus Pseudomonas, e.g. P. putida and P. fluorescens, or a gram-positive bacterium such as of the genus Bacillus, e.g. B. subtilis, or a yeast such as of the genus Saccharomyces or a fungus, e.g. of the genus Aspergillus, is useful for producing a recombinant polypeptide as defined above.
  • the recombinant production may be performed by use of conventional techniques, e.g. as described by Sambrook et al., 1989.
  • a microorganism producing the gene products conferring, to the host, anti-phytopathogenic activity in the form of nematode resistance may be used in combating soil phytopathogens, i.e. phytopathogens present in the soil and responsible for retarded growth or death of the plant.
  • phytopathogenic organisms are nematodes, in particular Heterodera schacthii
  • the organism may be a cell line, e.g. a plant cell line. Most preferably, the organism is a plant, i.e. a genetically modified plant such as will be discussed in further detail below.
  • the genetic construct is preferably to be used in modifying a plant.
  • the present invention also relates to a genetically transformed plant comprising in its genome a genetic construct as defined above.
  • the genetically transformed plant has an increased anti-phytopathogenic activity compared to a plant which does not harbour a genetic construct of the invention, e.g. an untransformed or natural plant or a plant which has been genetically transformed, but not with a genetic construct of the invention.
  • a constitutive expression of the gene products encoded by the genetic construct is desirable, but in certain cases it may be interesting to have the expression of the gene products encoded by the genetic construct regulated by various factors, for example by factors such as temperature, pathogens, and biological factors.
  • the plant to be transformed by the genetic construct of the invention may be a monocotyledonous as well as a dicotyledonous plant, since the genetic construct is expected to be active in such classes of plants.
  • monocotyledonous plants which may be transformed are corn, oat, wheat, rye, rice, barley and sorghum.
  • Non-limiting examples of dicotyledonous plants which may be genetically transformed are alfalfa, tobacco, cotton, sugar beet, fodder beet, red garden beet, leef beet, sunflower, carrot, canola, tomato, potato, soybean, oil seed rape, raddisg, white mustard, cabbage, pepper, lettuce, bean and pea.
  • the genetically transformed plant according to the invention has an increased resistance to pathogens such as phytopathogenic nematode as compared to plants which have not been genetically transformed according to the invention or as compared to plants which do not harbour the genetic construct as defined above.
  • the most important pathogen to be controlled according to the invention are represented by phytopathogenic nematodes.
  • the present invention relates to seeds, seedlings or plant parts obtained by growing the genetically transformed plant as described above. It will be understood that any plant part or cell derivable from the genetically transformed plant of the invention is to be considered within the scope of the present invention.
  • the genetic information is introduced into the plant by use of a vector system or by direct introduction, e.g.
  • the present invention relates to a transformation system comprising at least one vector which carries a genetic construct as defined above and which is capable of introducing the genetic construct into the genome of a plant such as a plant of the family Chenopodiaceae, in particular of the genus Beta, especially Beta vulgaris.
  • plant transformation systems are based on the use of plasmids or plasmid derivatives of the bacteria Agrobacterium.
  • the two best known Agrobacteria are Agrobacterium tumefaciens and Agrobacterium rhizogenes (plasmids thereof are in the following termed pTi and pRi, respectively).
  • the use of such plant transformation systems is based on the ability of the bacteria Agrobacterium to transfer a specific piece of DNA (T-DNA) to a plant cell in a wounded area.
  • T-DNA is located between specific border DNA sequences on the pTi or pRi which further carries virulence genes necessary for the transfer of the T-DNA to the plant.
  • the Agrobacterium transformation system mediates the transfer of any DNA sequence located between the "borders" and thus, it is possible to exchange the wild type Agrobacterium T-DNA with any desirable piece of DNA to be introduced into a plant.
  • the plant transformation system of the invention is based on disarmed Agrobacteria harbouring derivatives of the pTi or pRi from which the wild type T-DNA has been removed.
  • the vector system with which the plant is transformed comprises one or two plasmids.
  • the one-plasmid system also termed a co-integrate vector system
  • the T-DNA of pTi or pRi has been removed and replaced by the DNA to be transferred into the plant cell by use of homologous recombination.
  • the two-plasmid system also termed a binary vector system
  • both the T-DNA and the borders have been removed from the pTi or pRi.
  • pBI121 An example of a suitable plant transformation vector is pBI121 and derivatives thereof, e.g. as described by Jefferson 1987.
  • the vector to be used is provided with suitable markers, eucaryotic as well as procaryotic, e.g. genes encoding antibiotic resistance or herbicide resistance or glucoronidase (GUS), e.g. hygromycin or other known markers, e.g. the markers disclosed by Lindsey, 1990 and Reynaerts et al., 1988.
  • the marker is to be present so as to be able to determine whether the DNA insert has been inserted in the desired position in the plasmid and to be able to select plant cells transformed with the vector.
  • the genetic construct to be inserted in the plant is first constructed in a microorganism in which the plasmid can replicate and which is easy to manipulate.
  • a useful microorganism is E. coli, but other microorganisms having the above properties may be used.
  • a plasmid of a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
  • the plasmid harboring the genetic construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harboring the genetic construct of the invention, the DNA of which is subsequently transferred into the plant cell to be modified.
  • a suitable Agrobacterium strain e.g. A. tumefaciens
  • This transformation may be performed in a number of ways, e.g. as described in (An et al., 1988).
  • Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in (Butcher et al., 1980).
  • a plant to be infected is wounded, e.g. by cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasive or brushing the plant with a steel brush (e.g. as described in Example 15).
  • the wound is then inoculated with the Agrobacterium, e.g. in a suspension.
  • the infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
  • the inoculated plant or plant part is then subjected to selection and regeneration and grown on a suitable culture medium and allowed to develop into mature plants. This is accomplished by use of methods known in the art.
  • tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
  • Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
  • a genetically transformed plant may be performed as a double transformation event (introducing the genetic construct in two transformation cycles) or may be associated with use of conventional breeding techniques.
  • two genetically modified plants according to the present invention may be cross-bred in order to obtain a plant which contains the genetic construct of each of its parent plants.
  • the DNA region of the present invention and/or any of the DNA sequences SEQ ID NO's:1-14 may be used for diagnostic purposes, f.x. detection of the presence of a DNA region according to the invention.
  • the DNA sequences may also be used for detection of the BCNR Locus in a plant or detection of the presence of a DNA sequence according to the present invention which are closely linked to the BCNR Locus. This will be further explained in the following.
  • the messenger RNA's transcribed from one or more genes from the Locus may be qualitatively as well as quantitatively determined by hybridization to the DNA region of the invention or a subsequence thereof under conditions suitable for said hybridization.
  • genes belonging to the BCNR Locus family and present in an organism such as a plant may be identified and isolated by use of the DNA region of the invention, or a subsequence thereof, e.g. by screening a gene library of such an organism.
  • Various types of diagnosis using the DNA region of the present invention or a subsequence thereof or any of the DNA sequences SEQ ID NO.: 1-14 of the invention, or a subsequence thereof may be used to determine the presence in a plant of a DNA region of the invention or a DNA sequence of the invention by use of various techniques such as the hybridization technique or the polymerase chain reaction (PCR).
  • various techniques such as the hybridization technique or the polymerase chain reaction (PCR).
  • another important aspect of the present invention relates to a method for selecting, from a group of plants, plants containing a DNA region comprising the BCNR Locus of the invention or produced by the method according to the present invention as described above, comprising hybridizing with DNA sequence SEQ ID NO's:1-14 or with any DNA sequence showing a homology of at least 60% to a DNA sequence SEQ ID NO's:1-14, to DNA from the plant to be examined under the hybridization conditions of 2.0 ⁇ SSC, 0.1% SDS at 65°C and the washing conditions of 0.5 ⁇ SSC, 0.1% SDS at 50°C and identifying plants containing DNA capable of hybridizing with a DNA sequence SEQ ID NO's:1-14, or with any DNA sequence showing a homology of at least 60% to a DNA sequence SEQ ID NO's:1-14, to a degree of at least 60%.
  • the homology between a DNA sequence SEQ ID NO's: 1-14 and any DNA sequence is to a degree of preferably at least 70%, more preferably at least 75%, still more preferably at least 80% and still more preferably at least 85%, most preferably at least 90% using the hybridization technique under the conditions of 2.0 ⁇ SSC, 0.1% SDS and 65°C and under the washing conditions of 0.5 ⁇ SSC, 0.1% SDS at 50°C.
  • the hybridization between the DNA sequence and the DNA from the plant to be examined is to a degree of preferably at least 70%, more preferably at least 75%, still more preferably at least 80% and still more preferably at least 85%, most preferably at least 90% using the hybridization technique under the conditions of 2.0 ⁇ SSC, 0.1% SDS and 65°C and under the washing conditions of 0.5 ⁇ SSC, 0.1% SDS at 50°C.
  • Selection of plants containing a DNA region comprising the BCNR Locus may also be performed using another method of the invention, which comprises selecting, from a group of plants, plants containing a DNA region comprising the Beet Cyst Nematode Resistance Locus as defined herein, comprising using the Polymerase Chain Reaction and one or several primers determined from a DNA sequence SEQ ID NO's:1-14, or with any DNA sequence showing a homology of at least 60% to a DNA sequence SEQ ID NO's: 1-14, allowing any DNA sequence from a plant to be amplified whereby plants containing a DNA sequence complementary to the primer can by identified.
  • the homology between a DNA sequence SEQ ID NO's: 1-14 and any DNA sequence is to a degree of preferably at least 70%, more preferably at least 75%, still more preferably at least 80% and still more preferably at least 85%, most preferably at least 90% using the hybridization technique under the conditions of 2.0 ⁇ SSC, 0.1% SDS and 65°C and under the washing conditions of 0.5 ⁇ SSC, 0.1% SDS at 50°C.
  • a label which may be used for detection.
  • Useful labels are known in the art and are, e.g. a fluorophore, a radioactive isotope, an isotope or a complexing agent such as biotin.
  • DNA region of the invention comprising the BCNR Locus or any of the DNA sequences SEQ ID NO's: 1-14 may be used in a method of isolating a gene or messenger belonging to or derived from the family of genes present in the BCNR Locus from an organism, e.g.
  • a plant in particular a dicotyledon
  • the method comprising hybridizing a nucleic acid containing sample obtained from a gene library or cDNA library from the organism with the DNA region of the invention comprising the BCNR Locus, optionally in a labelled form, in a denatured form or an RNA copy thereof under conditions favorable to hybridization between the DNA region or RNA copy and the nucleic acid of the sample, and recovering the hybridized clone so as to obtain a gene or cDNA belonging to the BCNR Locus of the organism.
  • the DNA sequences of the invention being closely linked to the BCNR Locus may be used for the above-mentioned purposes.
  • the identification and isolation of a gene or cDNA clone in a sample belonging to the gene or genes present in the BCNR Locus by use of a DNA region of the invention or any of the DNA sequences SEQ ID NO's.: 1-14 of the invention, in particular a subsequence thereof, may be based on standard procedures, e.g. as described by Sambrook et al., 1989. For instance, to characterize related genes in other plants, it is preferred to employ standard Southern techniques.
  • the DNA region of the invention or any of the DNA sequences SEQ ID NO's: 1-14 of the invention may also be used in a method of quantifying the number of BCNR Loci or the amount of a BCNR Locus released messenger present in different tissues in an organism, e.g.
  • a plant comprising hybridizing a nucleic acid containing sample obtained from the organism with the DNA region comprising the BCNR Locus or DNA sequence of the invention comprising a nucleotide sequence substantially as shown in one of SEQ ID NO's: 1-14, especially a subsequence thereof, optionally in labelled form, in denatured form or an RNA copy thereof when using the BCNR Locus under conditions favorable to hybridization between the denatured DNA region or DNA sequence or RNA copy and the RNA of the sample and determining the amount of hybridized nucleic acid (Barkardottir et al., 1987).
  • the hybridization should be carried out in accordance with conventional hybridization methods under suitable conditions with respect to e.g. stringency, incubation time, temperature, the ratio between the DNA region comprising the BCNR Locus or any of the DNA sequences SEQ ID NO's:l-14 of the invention or a subsequence thereof to be used for the identification and the sample to be analyzed, buffer and salt concentration or other conditions of importance for the hybridization.
  • suitable conditions e.g. stringency, incubation time, temperature, the ratio between the DNA region comprising the BCNR Locus or any of the DNA sequences SEQ ID NO's:l-14 of the invention or a subsequence thereof to be used for the identification and the sample to be analyzed, buffer and salt concentration or other conditions of importance for the hybridization.
  • suitable conditions e.g. stringency, incubation time, temperature, the ratio between the DNA region comprising the BCNR Locus or any of the DNA sequences SEQ ID NO's:l-14 of the invention or a subseque
  • the support to which DNA or RNA regions of the sample to be analyzed are bound in denatured form is preferably a solid support and may be any of the supports conventionally used in DNA and RNA analysis.
  • the DNA sequence used for detecting the presence of a gene or genes from BCNR Locus or for detecting the presence of one of the markers closely linked to the BCNR Locus is preferably labelled, e.g. as explained above, and the presence of hybridized DNA is determined by autoradiography, scintillation counting, luminescence, or chemical reaction.
  • Another approach for detecting the presence of a specific BCNR Locus gene or genes, or the presence of a DNA sequence of the invention, e.g introduced by the genetic methods described previously, or a part thereof in an organism, e.g. a plant, in particular a dicotyledon, is to employ the principles of the well-known polymerase chain reaction, e.g. as described by Sambrock et al, 1989.
  • the sample to be analyzed for the presence of a BCNR Locus or gene or genes from the Locus in accordance with the methods outlined above may be taken from the group of plant parts consisting of fruits, leaves, stems, tubers, flowers, roots, sprouts, shoots and seeds.
  • the present invention relates to an anti-phytopathogenic composition, in the form of an anti-nematode composition comprising a gene product or gene products encoded by a DNA region of the invention comprising the BCNR Locus as defined above, or by a genetic construct of the invention as defined above and a suitable vehicle.
  • the present invention relates to an anti-phytopathogenic composition, in the form of an anti-nematode composition comprising a microorganism capable of expressing a gene product encoded by the DNA region comprising the BCNR Locus as defined above, or by a genetic construct of the invention defined above and a suitable vehicle.
  • Microorganisms suitable as constituents in an anti-phytopathogenic composition are mentioned above.
  • the anti-phytopathogenic composition in the form of an anti-nematode composition according to the present invention may be prepared by a method comprising culturing a microorganism harbouring and being capable of expressing a DNA region of the invention comprising the BCNR Locus or a genetic construct of the invention in an appropriate medium and under conditions which result in the expression of one or more anti- phytopathogenic gene products, especially anti-nematode resistant gene products encoded by the DNA regions, optionally rupturing the microorganisms so as to release their content of expressed anti-phytopathogenic gene product(s) into the medium, removing cell debris from the medium, and optionally subjecting the medium containing the gene product(s) to freeze-drying or spray-drying thereby obtaining an anti-phytopathogenic composition comprising the anti-nematode gene products(s).
  • the anti-phytopathogenic composition in the form of an anti-nematode composition according to the invention may be used in combating or inhibiting the hatching or germination and/or growth of a phytopathogenic organisms, in particular a nematode, in or on a plant or in any other material in which the presence of phytopathogenic nematode is undesirable. This will be further discussed below.
  • the anti-phytopathogenic composition of the invention preferably the anti-nematode composition showed, of course, be adapted to its intended purpose, both with respect to the vehicle to be used and with respect to the form, in which the anti-phytopathogenic agent is present
  • anti-phytopathogenic agent such as an anti-nematode agent
  • the active constituent of the anti-phytopathogenic composition f.x. the active constituent of the anti-nematode composition, responsible for or involved in providing the anti-phytopathogenic activity, in particular the anti-nematode activity.
  • anti-phytopathogenic gene product is meant a gene product encoded by the BCNR Locus of the invention or a genetic construct of the invention having anti-phytopathogenic activity, i.e. anti-nematode activity above.
  • the anti-phytopathogenic agent in particular the anti-nematode agent, is in itself a microorganism or will be prepared by a microorganism.
  • the most easy and inexpensive way of preparing the anti-phytopathogenic composition will be to use the microorganism as such or the medium in which it is grown as the antiphytopathogenic agent.
  • the anti-phytopathogenic gene produces) such as a polypeptide expressed from the microorganisms may be secreted into the medium, e.g. as a consequence of the action of a suitable signal peptide capable of directing the gene product out into the medium, or may be released from the microorganism by well known mechanical or chemical means. Before use, it may be advantageous to remove the microorganisms or any cell debris from the medium.
  • the medium may, in principle, serve as the vehicle for the anti-phytopathogenic agent, but it is preferred to add a further vehicle suited for the particular intended use.
  • a culture of the microorganisms expressing the anti-phytopathogenic gene product(s) such as polypeptides may be obtained as described above using methods known in the art. As mentioned above, it may be necessary or advantageous to subject the microorganism culture to a further treatment so as to release the content of the anti-phytopathogenic gene product(s) into the medium or to increase the amount released by secretion.
  • the medium comprising a substantial amount of the anti-phytopathogenic gene product(s), in particular the anti-nematode products may be directly applied to the soil in which the plants are present or in which the plants are to be grown, or to the plants or plant parts or to the irrigation water. Alternatively, seeds may be treated with the medium, optionally in combination with a conventional seed coating composition.
  • the microorganisms expressing the anti-phytopathogenic gene product(s) can be applied in various formulations containing agronomically acceptable vehicles, i.e. adjuvants or carriers, in dosages and concentrations chosen to maximize the beneficial effect of the microorganism.
  • agronomically acceptable vehicles i.e. adjuvants or carriers
  • the microorganisms may also be distributed as such under circumstances allowing the microorganisms to establish themselves in the material to be treated.
  • the microorganism is a microorganism conventionally found in the soil, e.g. a rhizobacterium, it will generally be desirable that the transformed microorganism establishes itself in the soil so that it continuously may secrete the anti-phytopathogenic gene product(s) out into the soil surrounding the plant.
  • the microorganisms or the medium comprising the anti-phytopathogenic gene product(s), in particular the anti-nematode gene product(s) may be added to premixes, e.g. artificial growth media or other soil mixes used in the cultivation of the plant in question.
  • premixes e.g. artificial growth media or other soil mixes used in the cultivation of the plant in question.
  • the microorganisms or the medium is in a solid form, e.g. in a powdery form or in the form of a granule.
  • the powdery form may be obtained by conventional means, e.g. by applying the microorganism on a particulate carrier by spray-drying or an equivalent method.
  • the microorganism expressing the anti-phytopathogenic gene product(s) When the microorganism expressing the anti-phytopathogenic gene product(s) is to be used in a humid state it may be in the form of a suspension or dispersion, e.g. as an aqueous suspension.
  • the anti-phytopathogenic activity of the transformed microorganism it may be advantageous to add a small amount of phytopathogenic organisms, such as nematodes to the medium in which the transformed microorganism is present. This should, however, naturally be done in such a way that the added phytopathogenic organisms are not transferred together with the microorganisms of to the composition.
  • the present invention further relates to a method of inhibiting or the hatching or germination and/or growth of a phytopathogen, such as nematode, in or on a plant, which method comprises
  • Fig. 1 shows the southern blot DNA hybridizations of probe CPRO101 (A), CPRO102 (B), CPRO103 (C), and CPRO104 (D) to EcoR1 restricted genomic DNA of AN5-90 (lane 1), AN5-109 (lane 2), AN5-203 (lane 4), B. vulgaris (lane 5) and B.patellaris (lane 6).
  • Fig. 2 shows the result of the southern blot DNA hybridization of probe 121-3 to EcoR1 restricted genomic DNA from the following sources beginning at the bottom of the figure with lane 1:
  • Lane 1 lambda E/H (EcoR1/HindIII), which is a size marker
  • Lane 17 B. webbiana
  • Lane 20 lambda E/H (EcoR1/HindIII), which is a size marker
  • Fig. 3 also shows DNA hybridization of probe 121-3 to Ncol digested genomic DNA from the plant lines AN5, AN5-203, AN5-109, AN5-90, AN1-89, B883, MS1-3 and AN1. Also in this hybridization only a few bands can be seen with the hybridization to B 883.
  • Fig. 4 shows the DNA hybridization of probe CPRO101 (A) and CPRO102 (B) to EcoR1 digested genomic DNA of B. vulgaris (lane 1), AN1-89 (lane 2), B. procumbens (lane 3), AN5 (lane 4), and B. patellaris (lane 5).
  • Fig. 5 shows the deduced localization of the specific probes CPRO101 and CPRO102 on the monosomic addition fragments of AN5-90, AN5-109 and AN5-203. ⁇ marks the BCNR Locus.
  • Fig. 6 shows the alignment of repetitive sequences from AN5-90, B883 and B. procumbens. The number in brackets indicates the localization of the repetitive sequences. Thus, 551 (2) indicates that the sequence is repeat No.2. pTSl and pTS2 are from a B. procumbens library and B883-1 is from the diploid introgression line B883 (Heijbroek et al., 1988). Compared to the DNA sequence SEQ ID NO:5, the DNA sequence B883-1 is the complementary sequence. The alignment also shows that parts of the repetitive sequences are highly conserved and thus may serve as primers for use in PCR experiments.
  • Fig. 7 shows the DNA sequence from the pTS 1 (Schmidt et al. 1990).
  • Fig. 8 shows the DNA sequence from the pTS2 (Schmidt et al. 1990).
  • Fig. 9 shows a RAPD/RFLP map of the BCN locus.
  • the beet cyst nematode resistant plant material selected using screening assay 1 described in Example 3 consisted of monosomic additions in B. vulgaris carrying an extra chromosome (2n+1) of either Beta patellaris or B. procumbens, and of fragment additions recovered from backcrossing the monosomic additions with B. vulgaris.
  • the original addition material from B. patellaris was AN5, a telosomic addition carrying the long arm of the chromosome named pat-1 (Speckmann et al., 1985; Lange et al., 1990a).
  • Backcrossing with diploid B. vulgaris (non-resistant) yielded three monosomic fragment additions: AN5-90, AN5-109 and AN5-203, all comprising the BCNR Locus.
  • the addition material from B. procumbens consisted of two monosomic additions carrying the chromosome named pro-1: AN1 and AN115, and one monosomic addition with the chromosome named pro-7: AN 101 (Speckmann et al., 1985; Van Geyt et al., 1988, Lange et al., 1988).
  • Backcrossing with B. vulgaris yielded the fragment addition AN1-89 (De Jong et al., 1986).
  • the diploid introgression line B883 Heijbroek et al., 1988 was used containing a chromosome segment of B. procumbens bearing the BCNR Locus, incorporated into the B. vulgaris genome.
  • Root tips were pre-treated in aqueous 8-hydroxyquinoline (0.002 M) for 5 hours, fixed in acetic acid-ethanol (1:3) and squashed in 45% acetic acid. The preparations were stained by carefully lifting the cover slip and adding a drop of 1% aqueous crystal violet. The size of the chromosome fragments was estimated through microscopical measurements on mitotic metaphase chromosomes (De Jong et al., 1986) as a percentage of the total genome.
  • telosomic addition AN5 carrying the long arm of chromosome 1 of B. patellaris (pat-1) in a background of 18 B. vulgaris chromosomes, produced three fragment additions containing the BCNR Locus (AN5-90, AN5-109 and AN5-203).
  • AN5 was morphological non-descript (Speckmann et al. 1985) and thus cannot easily be distinguished among susceptible sib plants (progeny plants which have lost the fragment and which are thus susceptible to nematode infection) in segregating populations. This is also the case for the three fragment additions, AN5-90, AN5-109 and AN5-203. On cytological examination the fragments were identified and were different in size.
  • Beta genome is only 1.57 pg (Arumuganathan and Earl, 1991).
  • a B. patellaris DNA library was constructed followed by hybridization of the DNA from the library to relevant strains of beets.
  • genomic subtraction according to Straus and Ausubel (1990) was used followed by screening for repetitive sequences present in B. patellaris but not in B. vulgaris and hybridization to genomic DNA from relevant strains of beet.
  • Total DNA was isolated from leaf tissue using an scaled-up version of the method of Dellaporta et al. (1983) yielding about 50 ⁇ g of DNA per gram of leaf tissue.
  • a Pstl genomic library of B. patellaris DNA was constructed in plasmid vector pGEM5Zf+ (Promega).
  • Total DNA was digested with the restriction enzyme Pstl (Boehringer Mannheim) and fractionated on a 0.8% agarose gel (Seakem GTG, FMC). The DNA fractions between 0.5 and 2 kbp were recovered from the agarose omitting strongly repetitive DNA fragments (present as a band in the total smear) ligated to the vector and cloned in E. coli JM101 (Maniatis et al. 1982).
  • Genomic subtraction was performed according to Straus and Ausubel (1990) on the total genomic DNA.
  • AN5-90 DNA was digested with Sau3A and biotinylated DNA from B. vulgaris without the fragment was used as driver.
  • the driver DNA concentration was increased to 15 ⁇ g/ ⁇ l and the hybridization time was increased to about one week for each round to overcome the problems with the large genome size of B. vulgaris.
  • the subtraction products were ligated to a BamHI/blunt end linker before amplification with the polymerase chain reaction (PCR, Sambrook et al., 1989).
  • An internal Kpnl site in the linker was used for cloning in the PUC19 vector (Sambrook et al., 1989).
  • the resulting library contained very few highly repetitive sequences from B. vulgaris. However, enrichment for specific low copy sequences was not conclusively proven.
  • DNA probes were labeled according to a non-radioactive method based on chemiluminescence, following the protocol of Kreike et al. (1990).
  • DNA labeling kit Boehringer Mannheim
  • digoxigenin 11-dUTP
  • Detection of the digoxigenin labeled probes was performed by using a anti-digoxigenin-alkaline phosphatase conjugate (Boehringer Mannheim) and AMPPD (Southern light kit, Tropix) as substrate for alkaline phosphatase. Light emission was visualised by autoluminescence on X-ray film (X-Omat, Kodak) (Allefs et al. 1990; Kreike et al. 1990).
  • Washing stringencies used after hybridization were 0.1 ⁇ SSC, 0.1% SDS at 65°C or 0.5 ⁇ SSC, 0.1% SDS at 50°C according to the size of the hybridizing probe. With smaller probes ( ⁇ 0.9 kbp) the lower stringency was used.
  • the Pstl genomic library of B. patellaris was screened for the presence of clones specifically hybridizing with chromosomal fragment addition DNA by Southern blot hybridization.
  • Southern blots contained EcoR1 digested DNA of 3-5 individual plants of each monosomic fragment addition (AN5, AN5-90 and AN5-109). The selected plants were checked twice for beet cyst nematode resistance using the screening assay 2 as described in Example 3, and for the presence of the chromosomal fragment addition by microscopy.
  • As control EcoR1 digested DNA of B. patellaris, and similar digested DNA from at least five susceptible sib plants containing the fragment additions were used.
  • Digoxigenin-labeled B. patellaris Pstl clones were used individually as probes or in combinations of two to four clones.
  • the nucleotide sequence of the markers was determined using the S angers chain termination method (Sambrook et al., 1989) using the Sequenase kit (United States Biochemical).
  • DNA markers linked to the BCNR Locus were isolated from a genomic Pstl library of B. patellaris DNA which was constructed as described above. Among 233 clones tested, the independent clones CPRO101 and CPRO102 hybridized specifically to B. patellaris DNA and to DNA of the telosomic fragment addition present in AN5 carrying the long arm of chromosome 1 from B. patellaris (Fig. 1). In addition, the clones hybridized to the addition fragment DNA of AN5-90 and AN5-109, respectively As mentioned above, these additions fragments are derived from AN5 and they all contain the BCNR Locus.
  • the sizes of the hybridizing EcoR1 restriction fragments were similar in AN5-90 and AN5 (probe CPRO101) and in AN5-109 and AN5 (probe CPRO102).
  • the two hybridizing Sacl fragments showing hybridization to clone CPRO101 were generated by the presence of an internal Sacl restriction site in this clone.
  • AN101 a monosomic addition carrying the BCNR Locus and chromosome pro-7 did not show any hybridization to clone CPRO101. Also clone CPRO102, which shows specific hybridization to AN5-109, did not detect homologous sequences in B. procumbens addition material AN1, AN101 or AN1-89 (Fig. 4).
  • the BCNR Locus comprised in the monosomic fragment additions AN5-90, AN5-109, AN5-203 and AN1-89 was transmitted to B. vulgaris by a small fragment of resp. approx. 8, 13, 19 and 13 Mbp of chromosome 1 of B. patellaris and B. procumbens.
  • CPRO101 also showed hybridization to the addition fragment DNA of AN5-90 while CPRO102 hybridized specifically to the addition fragment DNA of AN5-109.
  • Clones CPRO103 and CPRO104 hybridized exclusively to AN5 DNA and not to DNA of smaller addition fragments while CPRO105 in addition to the B. patellaris specific signal showed hybridization to B. vulgaris DNA.
  • the introgression line B883 contains an introgression of a segment of B. procumbens chromosome 1 containing a nematode resistance locus (Fig. 2). Based on the number of hybridizing bands in B883 DNA compared to DNA from the fragment addition ANl-89 it is appearent that the introgression material contains less than 13 Mbp B. procumbens DNA. Thus, the hybridizing DNA in the introgression line is contemplated to be closely linked to the BCNR from B. procumbens. As appears from Fig. 6, the alignment of the DNA sequences shows several conserved regions. These conserved regions may serve as primers in PCR.
  • a Pstl genomic library of B.patellaris was differentially screened for clones which are specific for the B.patellaris fragment present in the nematode resistant fragment addition AN5. This resulted in 5 RFLP markers (101-105 obtained from clones CPRO101 - CPR0105 respectively, see above) linked to the ben gene. (For details see Salentijn et al. (1992)).
  • RFLP markers 101-105 obtained from clones CPRO101 - CPR0105 respectively, see above
  • RAPD RAPD technology
  • Another 13 markers linked to the ben gene in AN5 were obtained. All of the markers were subcloned and partial sequences of the termini of five of the markers determined.
  • a long range physical map is useful to direct a chromosomal walk from the flanking molecular markers towards the ben gene.
  • the construction of such a map was initiated by a pulsed-field gel analysis of the DNA surrounding RFLP markers 101 and 102.
  • the high molecular weight restriction fragments hybridizing to these markers are listed in Table 2 below.
  • RFLP marker 121.3 a highly repetitive RFLP marker for the ben gene was analyzed. Southern analyses using DNA from members of the genus Beta, as well as DNA isolated from other hosts of the beet cyst nematode like X- Brassica napus, Brassicoraphanus, Sinapus alba and Raphanus sativus showed that hybridization of probe 121.3 is restricted to wild beets of the section Procumbentes only. Pulsed field gel analyses showed that this marker occurs in large clusters on the B. patellaris genome (see Table 2). Since the copy number of this repeat in the various B. patellaris fragment additions is dependent on the size of the contained fragment, the repeat clusters probably are dispersed over the genome.
  • a YAC library of the fragment addition AN5-203 is constructed.
  • AN5-203 harbors an approx. 19 Mb B.patellaris fragment containing the ben gene and the two flanking RFLP markers 101 and 102 (see map).
  • This library is used in a chromosomal walk from one RFLP marker to the other, meanwhile passing the ben gene.
  • DNA from AN5-90, AN5-109 and AN5-203 was restricted with either EcoRI, ClaI, MluI and HpaII and, after separation by pulsed field electrophoresis, hybridized to the repetitive marker 121.3. Also, double digestions with some of these enzymes were carried out. These experiments revealed RFLPs between the various fragment addition lines which are used to determine the amount of overlap between the various B. patellaris fragments.
  • markers for the BCN locus were isolated. These markers all are derived from lambda-clones isolated from the B883 genomic library upon hybridization of the library with the repetitive marker 121.3. These new markers may represent DNA regions flanking the 121.3 core repeat sequences. The nature of these new markers was investigated by Southern analysis using DNA from the various fragment additions and from B.patellaris and B .procumbens introgression lines. The markers 9.1 and 6.4 were found to be highly repetitive and gave hybridization patterns which were almost identical to the pattern obtained by marker 121.3. Apparently, these markers still contain (some) copies of the 121.3 repeat core sequence. Marker 9.2 also appeared to be highly repetitive but hybridized strongly with B. vulgaris.
  • markers 9.4 and 9.5 represent middle-repetitive DNA and give rise to approx. 5 major hybridizing fragments in the various fragment addition and introgressions. These markers also hybridize with B. vulgaris, but give rise to B.patellarislB. vulgaris RFLPs. The hybridization patterns of both markers with most of the plant DNAs are almost identical, but, interestingly, both markers cause different hybridization patterns upon hybridization with B883. In addition, both markers detect RFLPs between some of the fragment additions. Finally, also marker 9.11 appeared to be a middle-repetitive marker. This marker also detects RFLPs between some of the fragment additions but does not hybridize to B.vulgaris DNA.
  • RAPD markers are amplified by PCR using total B.patellaris DNA as template DNA under the following conditions:
  • Amount of template DNA 300 ng
  • Amount of primer DNA 50 ng
  • Taq-polymerase 0.1 U (Supertaq, Sphearo-Q)
  • Thermocycler profile 1 min. 92°C, 2 min. 35°C, 2 min. 72°C for 50 cycles. After the first 25 cycles the reaction is suspended, and a second amount of 0.1 U Taq polymerase is added.
  • RAPD markers are amplified by PCR using total B. patellaris DNA as template DNA under the following conditions:
  • Amount of template DNA 300 ng
  • Amount of primer DNA 50 ng
  • Taq-polymerase 0.1 U (Supertaq, Sphearo-Q)
  • Thermocycler profile 1 min. 92°C, 2 min. 35°C, 2 min. 72°C for 50 cycles. After the first 25 cycles the reaction is suspended, and a second amount of 0.1 U Taq polymerase is added.
  • Marker B10 Size approx. 1300 bp
  • the isolation and characterization of the BCNR Locus may be performed as follows below.
  • Markers are used as starting and finishing points of a chromosomal walk carried out using very large pieces of cloned DNA, for instance in yeast artificial chromosome (YAC) vectors as described by Schlesinger (1990) or using bacteriophage P1 vectors (Sternberg, 1990).
  • the cloned DNA is made from sugar beet fragment addition lines or introgression lines containing the BCNR Locus and one or more of the DNA markers.
  • the chromosomal walk is directed with the aid of genetic maps and long-range restriction maps which are constructed for the chromosomal region of interest.
  • the long-range restriction maps are made by digesting with restriction enzymes such as Mlul and Notl followed by the separation of the high molecular weight restriction fragments by pulsed field gel electrophoresis and final hybridization with the marker.
  • the final identification of the gene or genes giving raise to nematode resistance is performed by transferring the candidate gene or genes to sugar beet cells or Arabidopsis cells by the use of a suitable vector in order to produce transformed cells or plants.
  • the resulting transformed plants or cells are examined for nematode resistance using method 2 described in Example 3 and the gene or genes giving raise to plants having nematode resistance thus are the genes comprised in the BCNR Locus.
  • the DNA sequence of the gene or genes is/are obtained by the use of conventional methods such as in accordance with Sangers chain termination method (Maniatis et al., 1982).
  • nematode resistance of plants was performed according to one of the methods described below. Both methods are based on examination of the direct pathogenic activity of the nematodes on the roots of the plants. The methods may also be used to examine transformed cells or transgenic plants to which the BCNR Locus has been transferred.
  • Mature cysts were collected from oil-seed rape or sugar beet plants growing in sand in the greenhouse. The cysts were placed in a 1 mM ZnCl 2 solution in a Baermann funnel. Second stage larvae were collected every day for up to 2 months.
  • SEQ ID NO.1 The DNA sequence from clone 121-3 made from
  • Sau3A digested genomic DNA from leaf material from AN5-90 plants.
  • SEQ ID NO.2 The DNA sequence from clone 208 made from
  • Sau3A digested genomic DNA from leaf material from AN5-90 plants.
  • the sequence consists of 2 repeats.
  • SEQ ID NO.3 The DNA sequence from clone 342-1 made from
  • Sau3A digested genomic DNA from leaf material from AN5-90 plants.
  • the sequence consists of 3 repeats.
  • SEQ ID NO.4 The DNA sequence from clone 551 made from
  • Sau3A digested genomic DNA from leaf material from AN5-90 plants.
  • the sequence consists of 2 repeats.
  • SEQ ID NO.5 The DNA sequence from clone B883-1 from the
  • diploid introgression line B883 (Heijbroek et al., 1988).
  • SEQ ID NO .6 The initial and terminal DNA sequence from clone X2.1 made from amplified B patellaris DNA using RAPD primer X2.
  • SEQ ID NO .7 The initial and terminal DNA sequence from clone B11.4 made from amplified B patellaris DNA using RAPD primer Bll.
  • SEQ ID NO .8 The DNA sequence from clone U18.9 made from amplified B patellaris DNA using RAPD primer U18.
  • SEQ ID NO .9 The DNA sequence from clone Y10.7 made from amplified B patellaris
  • SEQ ID NO .10 The DNA sequence from clone A15.1 made from amplified B patellaris
  • SEQ ID NO .11 The DNA sequence from clone CPRO101 (Salentijn et al. (1992).
  • SEQ ID NO 12 The initial and terminal DNA sequence from clone 9-4 from the diploid introgression line B883 (Heijbroek et al (1988)).
  • SEQ ID NO 13 The initial and terminal DNA sequence from clone 9-5 from the diploid introgression line B883 (Heijbroek et al (1988)).
  • SEQ ID NO 14 The initial and terminal DNA sequence from clone 9-11 from the diploid introgression line B883 (Heijbroek et al (1988)).
  • Barkardottir et al. 1987, Developmental Genetics, Vol. 8, pp. 495-511.
  • Herrera-Estrella L. et al. Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector, 1983, Nature, Vol. 303, pp 209-213.
  • Herrera-Estrella L. et al. Use of reporter genes to study gene expression in plant cells, 1988, Plant Molecular Biology Manual B1, 1-22.
  • Velten J. et al. Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens, 1984, The EMBO Journal, Vol. 3, No. 12, pp. 2723-2730. Wing D. et. al., conserveed function in Nicotiana tabacum of a single Drosophila hsp 70 promoter heat shock element when fused to a minimal T-DNA promoter, 1989, Mol Gen Genet, Vol. 219, pp. 9-16.

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Abstract

Région d'ADN comportant le locus de résistance à l'anguillule de la betterave, ainsi qu'au moins une séquence d'ADN S présentant une homologie d'au moins 60 % avec une séquence d'ADN S(A) provenant d'une plante (A) ayant une résistance aux anguillules, ladite séquence étant découverte par soustraction génomique comprenant la soustraction à partir du génome de la plante résistante du génome d'une plante non résistante (B), éventuellement suivie de l'hybridation de l'ADN obtenu par soustraction respectivement avec l'ADN de la plante non résistante (B), et avec l'ADN d'une plante correspondante (C) dont on sait qu'elle est résistante aux anguillules, et de la sélection de clones contenant les séquences d'ADN de la plante (A) qui s'hybrident avec l'ADN de la plante (C) mais pas avec celui de la plante (B).
PCT/EP1993/000702 1992-03-25 1993-03-23 Matiere biologique WO1993019181A1 (fr)

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