WO1990006999A1 - Nouvelle souche de bacillus thuringiensis - Google Patents

Nouvelle souche de bacillus thuringiensis Download PDF

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Publication number
WO1990006999A1
WO1990006999A1 PCT/EP1989/001539 EP8901539W WO9006999A1 WO 1990006999 A1 WO1990006999 A1 WO 1990006999A1 EP 8901539 W EP8901539 W EP 8901539W WO 9006999 A1 WO9006999 A1 WO 9006999A1
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btpgsi387
strain
protoxins
toxins
crystal
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PCT/EP1989/001539
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English (en)
Inventor
Marnix Peferoen
Bart Lambert
Henk Joos
Jacques Mahillon
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E.I. Du Pont De Nemours And Company
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Priority to KR1019900701750A priority Critical patent/KR910700343A/ko
Publication of WO1990006999A1 publication Critical patent/WO1990006999A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/075—Bacillus thuringiensis

Definitions

  • This invention relates to a new strain of Bacillus thurinqiensis (the "BtPGSI387 strain") which produces crystallized proteins (the “BtPGSI387 crystal proteins”) which are packaged in crystals (the "BtPGSI387 crystals") during sporulation.
  • the BtPGSI387 strain was deposited under the provisions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen ("DSM") , Mascheroder Weg IB, Braunschweig, Federal Republic of Germany, under accession number 4783 on August 29, 1988.
  • This invention also relates to an insecticide composition that is active against Lepidoptera, particularly Noctuidae such as Spodoptera littoralis and that comprises the BtPGSI387 strain, as such, or preferably the BtPGSI387 crystals or the BtPGSI387 crystal proteins or the active component(s) thereof as an active ingredient.
  • This invention further relates to a transformed B. thurinqiensis ("Bt") strain obtained by electroporation of the BtPGSI387 strain with a vector carrying all or part of a gene which comes from another Bt strain and which encodes a foreign Bt toxin or protoxin having insecticidal activity against Lepidoptera, Coleoptera and/or Diptera.
  • Bt B. thurinqiensis
  • B. thurinqiensis is a gram-positive bacterium which produces endogenous crystals upon sporulation. The crystals are composed of proteins which are specifically toxic against insect larvae.
  • pathotype A that is active against Lepidoptera, e.g., caterpillars
  • pathotype B that is active against certain Diptera, e.g., mosquitos and black flies
  • pathotype C that is active against Coleoptera, e.g., beetles (Ellar et al, 1986). That conventional submerged fermentation techniques can be used to produce Bt spores on a large scale has made this bacterium commercially attractive as a source of insecticidal compositions.
  • a new B_. thurinqiensis strain of pathotype A i.e., the BtPGSI387 strain.
  • BtPGSI387 crystals and/or BtPGSI387 crystal proteins are particularly useful against the cotton leafworm, Spodoptera littoralis f and the tobacco budworm, Heliothis virescens. which are major pests of economically important crops.
  • the BtPGSI387 strain is transformed by electroporation with a foreign DNA sequence encoding a Bt protoxin or toxin.
  • the BtPGSI387 strain has been deposited at the DSM under accession number 4783. This strain produces crystals which are a mixture of two crystal proteins: a 130 kDa protein (the "first BtPGSI387 protoxin") that is particularly toxic to Manduca sp. and
  • Second BtPGSI387 protoxin a protein that is particularly toxic to Spodoptera sp. Trypsin digestion of the first and second BtPGSI387 protoxins produces, respectively: a 60 kDa protein (the "first BtPGSI387 toxin”) and a 63 kDa protein (the "second BtPGSI387 toxin”) .
  • the BtPGSI387 strain, its crystals, its protoxins and/or its toxins can be used as the active ingredient in an insecticide composition used to control insect pests belonging to the order of Lepidoptera.
  • the BtPGSI387 crystals can be isolated from sporulated cultures of the BtPGSI387 strain (Mahillon and Delcour, 1984) , and then, the respective protoxins and mixtures thereof, can be isolated from these crystals according to the method of Hofte et al (1986).
  • the BtPGSI387 strain can be transformed with one or more foreign Bt genes, such as the bt2 gene (U.S. patent application 821,582, filed January 22, 1986, an European patent application 86/300291.1 which are incorporated herein by reference) or another bt gene coding for a protoxin or toxin active against other Lepidoptera, and/or the bt!3 gene (European patent application 88/402115.5 which is also incorporated herein by reference) or another bt gene such as the btPGSI208 gene or btPGSI245 gene (European patent application 89/400428.2 which is also incorporated herein by reference) coding for a toxin active against Coleoptera.
  • the bt2 gene U.S. patent application 821,582, filed January 22, 1986, an European patent application 86/300291.1 which are incorporated herein by reference
  • another bt gene coding for a protoxin or toxin active against other Lepidoptera
  • a transformed Bt strain can be produced which is useful for combatting an even greater variety of insect pests, e.g., additional Lepidoptera and/or Coleoptera.
  • Transformation of the BtPGSI387 strain with a foreign Bt gene, incorporated in a conventional cloning vector, can be carried out in a well known manner, preferably using conventional electroporation techniques (Chassy et al, 1988).
  • the BtPGSI387 strain can be fermented by conventional methods (Dulmage, 1981) to provide high yields of cells. Under appropriate conditions which are well understood (Dulmage, 1981) , the BtPGSI387 strain sporulates within 24 hours to provide the BtPGSI387 crystal proteins in high yields.
  • An insecticide composition of this invention can be formulated in a conventional manner using the BtPGSI387 strain or preferably its crystals, crystal proteins, protoxins and/or toxins, together with suitable carriers, diluents, emulsifiers and/or dispersants.
  • This insecticide composition can be formulated as a wettable powder, pellets, granules or a dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc.
  • concentration of the BtPGSI387 strain, crystals, crystal proteins, protoxins or toxins in such a composition will depend upon the nature of the formulation and its intended mode of use.
  • an insecticide composition of this invention can be used to protect a cotton field for 2 to 4 weeks against Lepidoptera, with each application of the composition. For more extended protection (e.g., for the whole growing season) , additional amounts of the composition would have to be applied periodically.
  • a composition of this invention for combatting insects comprise an insecticidal amount of the BtPGSI387 crystals or preferably a mixture of the first and second BtPGSI387 protoxins, particularly a mixture of the first and second BtPGSI387 protoxins of the BtPGSI387 crystals.
  • a method for controlling Lepidoptera in accordance with this invention preferably comprises applying, to the locus (area) to be protected, an insecticidal amount of the BtPGSI387 crystals or preferably their mixture of the first and second BtPGSI387 protoxins.
  • the BtPGSI387 crystals or a mixture of the first and second BtPGSI387 protoxins or toxins, or the isolated first or second BtPGSI387 protoxin or toxin or intact cells of the BtPGSI387 strain containing the first and second BtPGSI387 protoxins can be applied to the locus to be protected.
  • the locus to be protected can include, for example, the habitat of the insect pests or growing vegetation or an area where vegetation is to be grown.
  • cells of the BtPGSI387 strain can be grown in a conventional manner on a suitable culture medium and then lysed using conventional means such as enzymatic degradation or detergents or the like.
  • the mixture of protoxins or the individual protoxins can then be separated and purified by standard techniques such as chromatography, extraction, electrophoresis, or the like.
  • the mixture of toxins or the individual toxins can then be obtained by trypsin digestion of their respective protoxin mixture or individual protoxins.
  • the BtPGSI387 cells can be harvested and then applied intact, either alive or dead, preferably dried, to the locus to be protected.
  • a purified BtPGSI387 strain (either alive or dead) be used, particularly a cell mass that is 90.0 to 99.9% BtPGSI387 strain.
  • BtPGSI387 crystals, a mixture of the first and second BtPGSI387 protoxins or toxins, individual BtPGSI387 protoxins or toxins, and/or the harvested cells of the protoxin-containing BtPGSI387 strain can be employed in an insecticidal composition.
  • Such an insecticidal composition can be formulated in a variety of ways, using any number of conventional additives, wet or dry, depending upon the particular use.
  • Additives can include wetting agents, detergents, stabilizers, adhering agents, spreading agents and extenders. Examples of compositions include pastes, dusting powders, wettable powders, granules, baits and aerosol compositions.
  • Bt protoxins and toxins insecticides, as well as fungicides, biocides, herbicides, and fertilizers, can also be employed along with the cells of the BtPGSI387 strain and/or the BtPGSI387 protoxins or toxins to provide additional advantages or benefits.
  • Such insecticidal compositions can be prepared in a conventional manner.
  • the amount of protoxins, toxins and/or cells of the protoxin-containing BtPGSI387 strain employed depends upon a variety of factors, such as the insect pest targeted, the composition used, the type of area to which the composition is to be applied and the prevailing weather conditions.
  • the concentration of the insecticidal protoxins and/or toxins will be at least about 0.1% of the weight of the formulation to about 100% by weight of the formulation, more often from about 0.15% to about 0.8% weight percent of the formulation.
  • insects can be fed the BtPGSI387 protoxins or toxins or mixtures thereof in the protected area, that is, the area where the protoxins and/or toxins have been applied.
  • some insects can be fed intact and alive cells of the BtPGSI387 strain or transformants thereof, so that the insects ingest some of the BtPGSI387 protoxins and suffer death or damage.
  • Figure 1 SDS-PAGE and immunoblot of trypsinized crystal proteins from the strains: 1) Bt HD-127, 2) BtPGSI387 and 3) BtSl.
  • Figure 2 a schematic drawing of the pWP37 plasmid of Example 3, showing its restriction sites.
  • the BtPGSI387 strain was isolated by the method of Travers et al (1987) from forest soil sampled in Kumba, Ca eroun, Africa and has been deposited at the Deutsche Sammlung von Mikroorganismen, accession No. 4783 on August 29, 1988.
  • the strain can be cultivated on conventional standard media, preferably LB medium (tryptone 10 g/1, yeast extract 5 g/1, NaCl 10 g/1 and agar 15 g/1), preferably at 28'C.
  • LB medium tryptone 10 g/1, yeast extract 5 g/1, NaCl 10 g/1 and agar 15 g/1
  • T3 medium tryptone 3 g/1, tryptose 2 g/1, yeast extract 1.5 g/1, 5 mg MnCl 2 , 0.05 M Na 2 P0 4 pH 6.8 and 1.5% agar
  • the BtPGSI387 strain can also grow under facultative anaerobic conditions, but sporulation only occurs under aerobic conditions.
  • Sterilization of the BtPGSI387 strain occurs by autoclave treatment at 120'C (1 bar pressure) for 20 minutes. Such treatment totally inactivates the spores and the crystalline BtPGSI387 toxins. UV radiation (254 nm) inactivates the spores but not the crystal proteins.
  • the morphological and biochemical characteristics of the BtPGSI387 strain are as follows.
  • the strain has a flat irregular colony with undulate to lobate margin, opaque and white appearance, the cells of which sporulate within 24 hours in Nutrient agar ("NA" from Difco Laboratories, Detroit, MI, USA) at 28*C while other Bt strains tested sporulate after 3 days under identical conditions.
  • BtPGSI387 the exponential growth phase of BtPGSI387 lasts longer in T3 medium, resulting in more biomass than Dipel Bt HD-1 strain.
  • the BtPGSI387 crystal proteins produced during sporulation are packaged in bipyra idal crystals. These properties together with the early initiation of sporulation of the BtPGSI387 strain, make this strain well suited for fermentation.
  • the BtPGSI387 strain forms gram-positive rods (1.7-2.4 x 4.4-7.0 urn) which form ellipsoidal spores centrally.
  • One-day old cells are weakly catalase-positive, but they shown no gelatinase and tryptophan deaminase activity (i.e., they are GEL- and TDA-) with API-20E test strips (API Systems S.A., Montalieu-Vercieu, France).
  • the cells also show no beta-galactosidase, lysine decarboxylase or ornithine decarboxylase activity (i.e., they are ONPG-, LDC- and 0DC-) with API-20E test strips, and they do not use citrate as their sole C-source (i.e. they are CIT-) according to API-20E test strips. Nor do the cells form
  • H 2 S or mdole with the API-20E test strips i.e., they are H S- and IND-
  • the BtPGSI387 strain showed arginine dehydrolase and urease activity (i.e., it is ADH+ and
  • API-20E test strips With API-20E test strips, it rapidly decomposed casein in skim-milk agar, and it weakly deaminated phenylalanine using the procedures of Sneath et al (1986) . Acid production from different sugars after 24 hours, as shown by API-50CHB test strips (API System SA) , is set forth in Table 1 below.
  • Bt tene Bacillus thurinqiensis subsp. tenebrionis from
  • Table 2 Diameter (in mm) of inhibition zones observed after 24 hours on agar seeded with different bacilli.
  • the enzyme spectra of the BtPGSI387 strain and other bacilli are shown in Table 3 below.
  • the results in Table 3 were obtained by using the extended API-ZYM test strips (API Systems S.A.). Esterase-, peptidase- (API, AP2, AP3, AP4, AP5 and AP6 test strips) and osidase-test strips were inoculated with 50 ul cell suspension (10 7 cfu/ml) .
  • the results of the osidase reaction were obtained after 4 hours incubation (28 ⁇ C) with 25 ul 0.1N NaOH, and the other results were obtained with 25 ul ZYM A and ZYM B reagent (API no. 7048) .
  • Table 3 only shows some of the results.
  • Table 3 The enzyme spectra of the BtPGSI387 strain and other bacilli.
  • BtSl Bacillus thurinqiensis from DSM under accession no. 4288
  • the BtPGSI387 crystals were isolated from the BtPGSI387 strain by density gradient centrifugation (H ⁇ fte et al.,1986). Alternatively, the crystals could have been solubilized selectively from the spore crystal mixture in 50mM Na 2 C0 3 and 5mM dithiotreitol ("DTT") at pH 10, and this suspension could then have been filtered through a filter with 0.45 urn diameter pore size.
  • DTT dithiotreitol
  • the antigenic properties of the BtPGSl387 crystal proteins are similar to those of the crystal proteins from the well-known Bt HD-127 strain (H ⁇ fte et al, 1988), pathotype A toxin (Fig. 1) , when tested against monoclonal and polyclonal antibodies, and distinct from those of the crystal proteins from the well-known Bt HD-1 (Yamamoto et al, 1983), Bt berliner (H ⁇ fte et al, 1986), Bt tenebrionis and Bt israelensis (Chungjatupornchai et al, 1988) strains.
  • Antibodies 82.1, 2H7, 54.1, 1B12, 5D11, 4F11 and 9H11 are monoclonal antibodies raised against Bt berliner crystals.
  • Antibodies 2D11, 4B10 and 4A2 are monoclonal antibodies raised against the purified 63 kDa toxin of Bt HD-127.
  • Antibody 2A10 is a monoclonal antibody raised against Bt tenebrionis.
  • Antibody ABt2 is a polyclonal antibody raised against purified Bt2 toxin.
  • Antibody ABtten is a polyclonal antibody raised against purified Bt tenebrionis toxin.
  • Antibody ABtl5 is a polyclonal antibody raised against purified Btl5 toxin.
  • the pattern of the trypsinized crystal proteins from the BtPGSI387 strain was also compared to the patterns of the crystals from Bt strains HD-127 and BtSl as shown in
  • Fig. 1 The crystal proteins from each Bt strain were trypsin-digested by: dissolving the crystals overnight at
  • a preculture of the BtPGSI387 strain was grown in 10 ml LB medium in a 100 ml baffled flask shaken gently (200 rpm) overnight at 28*C. 125 ml of T3 medium was then inoculated with enough of the preculture to provide a culture with an optical density of 12.5. The culture was then grown for 24 h at 28 * C with gently agitation (120 rpm) in a 1 liter baffled flask. After sporulation the spore-crystal mixture was recovered by centrifugation and dried with acetone according to the method of Dulmage (1981) . The spore-crystal mixture was suspended in water and the suspension was coated onto the surface of the artificial diet and allowed to dry for two hours.
  • the insecticidal activity of the coating containing the BtPGSI387 spore-crystal mixture was evaluated against Lepidoptera larvae.
  • the larvae were placed on the artificial diet coated with the BtPGSI387 spore-crystal mixture and on the artificial diet coated with aqueous suspension containing other Bt spore-crystal mixtures.
  • the LC50 of the BtPGSI387 crystals was significantly lower than the LC50s of the other Bt spore- crystal mixtures, tested. The results are summarized in Table 5 below.
  • Table 5 Toxicity of spore-crystal mixture from BtPGSI387, Bt HD-127 and Bt HD-1 strains against larvae of Spodoptera littoralis and Heliothis virescens .
  • LC50 ug/cm of spore-crystal mixtures causing 50% mortality
  • the BtPGSI387 crystal proteins of the BtPGSI387 spore-crystal mixture caused larvae to stop feeding after a few hours and die within a few days, and the death rate was a function of the concentration of crystal proteins on the artificial diet.
  • the crystals dissolved in the alkaline conditions of the insect's midgut and released the respective BtPGSI387 protoxins. These protoxins were proteolytically processed by the insect's midgut proteases to produce the BtPGSI387 toxins.
  • Example 3 Electroporation of the BtPGSI387 strain
  • pWP3713 is derived from plas id pWP37 shown in Fig. 2.
  • pWP3713 contains the bt!3 gene (European patent application 88/402115.5) which encodes a crystal protoxin having insecticidal activity against Coleoptera, particularly the Colorado potato beetle.
  • pWP37 is constructed by inserting the EcoRI/Pstl polylinker fragment from pLK37 (Botter an and Zabeau, 1987) into the EcoRI/Pstl site of the EcoRI/bovine alkaline phosphatase ("BAP)-treated pGKV2 plasmid (Van der Vossen et al, 1985).
  • BAP EcoRI/bovine alkaline phosphatase
  • the bt!3 gene is cloned into the Hindlll restriction site of the BAP-treated pWP37 to produce pWP3713.
  • the BtPGSI387 strain transformed with pWP3713 has insecticidal activity against Lepidoptera, as well as against Coleoptera.
  • the transformed BtPGSI387 strain or mixtures of its protoxins and/or toxins can therefore be used as the active ingredient in an insecticidal composition against Lepidopteran and Coleopteran insect pests.
  • flanking sequences in pWP37 which show homology with chromosomal DNA of the BtPGSI387 strain or with plasmid DNA of the BtPGSI387 strain, allows homologous recombination resulting in a transformed Bt strain having the new gene as a chromosomal insert or as an insert in the host plasmid.
  • this invention is not limited to the BtPGSI387 strain, deposited at the DSM under accession number 4783. Rather the invention also includes any mutant or variant of the BtPGSI387 strain which produces crystal proteins, protoxins or toxins having substantially the same properties as the BtPGSI387 crystal proteins, protoxins or toxins.
  • variants of the BtPGSI387 strain include variants which: 1) produce acid on a glycerol substrate and/or 2) do produce acid on mannose and saccharose substrates.
  • variants of the BtPGSI387 strain also include variants whose trypsinized crystal protein pattern differs somewhat from that of the BtPGSI387 strain as shown in Fig. 1.

Abstract

L'invention concerne une nouvelle souche de Bacillus thuringiensis produisant des protéines cristallines pendant la sporulation, toxiques pour les lépidoptères. Les protéines cristallines comprennent des protoxines de 130 kDa et de 135 kDa pouvant produire respectivement des toxines de 60 et de 63 kDa, en tant que produits de digestion de trypsine. On peut utiliser la souche elle-même, ou ses cristaux ou ses mélanges de protoxines ou de toxines, comme constituant actif dans une composition insecticide pour combattre les lépidoptères.
PCT/EP1989/001539 1988-12-12 1989-12-11 Nouvelle souche de bacillus thuringiensis WO1990006999A1 (fr)

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KR1019900701750A KR910700343A (ko) 1988-12-12 1989-12-11 바실러스 써린젠시스의 신규 균주

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GB88403154.3 1988-12-12

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WO1990006999A1 true WO1990006999A1 (fr) 1990-06-28

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WO1991016433A1 (fr) * 1990-04-26 1991-10-31 Plant Genetic Systems N.V. Nouvelles souches de bacillus thuringiensis et leurs genes de codage de toxines insecticides
WO1994005771A2 (fr) * 1992-08-27 1994-03-17 Plant Genetic Systems N.V. Nouvelles souches de bacillus thuringiensis et leurs proteines insecticides
WO1994012642A1 (fr) * 1992-11-24 1994-06-09 Novo Nordisk Entotech, Inc. Souches de bacillus thuringiensis efficaces contre des lepidopteres nuisibles
WO1994024264A1 (fr) 1993-04-09 1994-10-27 Plant Genetic Systems N.V. Nouvelles souches de bacille thuringiensis et leurs proteines insecticides
WO1999000407A2 (fr) * 1997-06-27 1999-01-07 Plant Genetic Systems N.V. Toxine de bacillus thuringiensis amelioree
US6043415A (en) * 1996-10-07 2000-03-28 Ramot Univ. Auth. For Applied Research And Industrial Development Ltd. Synthetic Bacillus thuringiensis cryic gene encoding insect toxin
WO2007007147A2 (fr) 2005-07-08 2007-01-18 Universidad Nacional Autonoma De Mexico Instituto De Biotecnologia Nouvelles proteines bacteriennes avec activite pesticide
US7265269B2 (en) 2001-01-09 2007-09-04 Bayer Bioscience N.V. Nucleic acids encoding a novel Cry2Ae bacillus thuringiensis insecticidal protein
EP2045262A1 (fr) 1999-12-28 2009-04-08 Bayer BioScience N.V. Protéines insecticides provenant de Bacillus thuringiensis
EP2213681A1 (fr) 2002-03-22 2010-08-04 Bayer BioScience N.V. Nouvelles protéines insecticides à base de Bacillus thuringiensis
WO2012165961A1 (fr) 2011-05-31 2012-12-06 Keygene N.V. Plantes résistantes aux nuisibles
EP2554674A1 (fr) 2007-12-21 2013-02-06 Keygene N.V. Promoteurs spécifiques trichomes
WO2013058654A2 (fr) 2011-10-19 2013-04-25 Keygene N.V. Procédés d'obtention de cinnamolide et/ou de drimendiol
WO2017039452A1 (fr) 2015-09-04 2017-03-09 Keygene N.V. Gène de diplosporie
US9994621B2 (en) 2007-06-01 2018-06-12 Bayer Cropscience N.V. Genes encoding insecticidal proteins
WO2020239984A1 (fr) 2019-05-29 2020-12-03 Keygene N.V. Gène pour parthénogenèse

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KR910700343A (ko) 1991-03-14
AU4949090A (en) 1990-07-10

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