EP0090033A1 - Procede de modification genetique de cereales avec des vecteurs de transformation - Google Patents

Procede de modification genetique de cereales avec des vecteurs de transformation

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Publication number
EP0090033A1
EP0090033A1 EP82903287A EP82903287A EP0090033A1 EP 0090033 A1 EP0090033 A1 EP 0090033A1 EP 82903287 A EP82903287 A EP 82903287A EP 82903287 A EP82903287 A EP 82903287A EP 0090033 A1 EP0090033 A1 EP 0090033A1
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EP
European Patent Office
Prior art keywords
plant
transformation vector
organ
accordance
tissue
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP82903287A
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German (de)
English (en)
Inventor
Robert L. Erwin
Ernest T. Hubbard
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International Plant Research Institute
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International Plant Research Institute
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Publication date
Application filed by International Plant Research Institute filed Critical International Plant Research Institute
Publication of EP0090033A1 publication Critical patent/EP0090033A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • This invention relates to the use of genetic engineering techniques in the modification of plants, particularly cereals, such as wheat, rice, barley and maize. More particularly, it concerns the construction and use of transformation vectors which include at least one gene associated with the expression of a desired agronomic property or the production of a desired product, at least one gene associated with a selectable or identi fiable plant trait and a DNA sequence derived from the chromosomal DNA of a plant.
  • DNA preparations reported in these experiments contained only DNA from the recipient species, and as such, were devoid of foreign genes. Moreover, these DNA preparations had no specific sequence or size homogeneity.
  • Transformation vectors useful in the genetic modification of photosynthetic plant cells, plant tissues and plant organs have been prepared. They are double-stranded DNA molecules which include a DNA sequence corresponding to a gene foreign to the plant cell, tissue or organ to be transformed and associated with a selectable or identifiable trait when present therein and at least one DNA sequence substantially identical to a DNA sequence located in the plant cell, plant tissue or plant organ and capable of homologous recombination with the DNA present in the plant cell, plant tissue or plant organ when the transformation vector is introduced therein.
  • the transformation vector also includes a DNA sequence containing a gene associated with expression of a desired agronomic property or production of a desired product.
  • genes include those associated with production of proteins possessing improved nutritional quality such as the zein genes of maize, the hordein genes of barley and the gliadin genes of wheat and those associated with resistance to environmental stress such as drought resistance, salt tolerance and herbicide resistance.
  • This invention also concerns methods of preparing the transformation vectors, methods of introducing desired genes into plant cells, tissues or organs and the resuiting plant cells, tissues or organs and plants derived therefrom.
  • Fig. 1 illustrates a transformation vector which includes a foreign gene associated with expression of a selectable or identifiable plant trait and a DNA sequence substantially identical to a portion of a plant's DNA.
  • Fig. 2 illustrates a transformation vector which includes a foreign gene . associated with expression of a desired property or production of a desired product and a DNA sequence substantially identical to a portion of a plant's DNA.
  • Fig. 3 illustrates a transformation vector as in Fig. 1 which additionally includes a DNA sequence associated with expression of a desired agronomic property or production of a desired product.
  • Fig. 4 illustrates a transformation vector which includes a foreign gene associated with expression of a selectable or identifiable plant trait, a foreign DNA sequence associated with expression of a desired agronomic property or production of a desired product, and two DNA sequences each substantially identical to portions of a plant's DNA.
  • the two DNA sequences are located at opposite ends of both the DNA sequence associated with the desired property or product and the DNA sequence containing the foreign gene associated with a selectable or identifiable trait.
  • Fig. 5 illustrates a transformation vector which includes a foreign gene for ampicillin resistance flanked at both ends by plant DNA expressed in wheat in vitro. It also includes a DNA sequence containing codons for increased lysine levels flanked at both ends by wheat storage protein genes. Finally, it includes a sequence associated with replication and selection in E. coli.
  • Fig. 6 illustrates a transformation vector derived from the E. coli plasmid pBR322.
  • the selectable or identifiable trait is ampicillin resistance.
  • the flanking DNA sequences are substantially identical to portions of maize chromosomal DNA expressed when the maize is in the germinating embryo state.
  • the final DNA sequence is derived from a chicken and is associated with production of ovalbumin.
  • TRANSFORMATION VECTOR a double-stranded DNA molecule which is capable of mediating the introduction of genes into cells and includes at least one selectable or identifiable trait.
  • PLANT any photosynthetic member of the kingdom Planta which is characterized by a membrane-bound nucleus, genetic material organized into chromosomes, membranebound cytoplasmic organelles, and the ability to undergo meiosis.
  • PLANT CELL the structural and physiological unit of plants, consisting of a protoplast and cell wall.
  • PLANT TISSUE - a group of plant cells organized into a structural and functional unit.
  • PLANT ORGAN a distinct and visibly differentiated part of a plant such as root, stem, leaf or Gmb-yro-r
  • SELECTABLE OR IDENTIFIABLE TRAIT - a feature or quality encoded by one or more genes present in a plant cell, plant tissue or plant organ which distinguishes the cell, tissue or organ from others lacking the feature or quality.
  • PLANT CHROMOSOME - a DNA molecule present in the nucleus of a plant cell which is associated with proteins and RNA and undergoes reductive division during meiosis.
  • HOMOLOGOUS RECOMBINATION the exchange of nucleic acid sequences between two distinct DNA molecules based upon the interaction of substantially identical base sequences.
  • SUBSTANTIALLY IDENTICAL DNA SEQUENCES - two DNA sequences which are sufficiently similar in nucleic acid base composition and sequence to facilitate homologous recombination with one another including different mutational forms of a gene or alleles.
  • REPETITTVE SEQUENCES - DNA sequences which are present in more than 10 copies per haploid genome.
  • ENVIRONMENTAL STRESS any condition which results in altered metabolic activity, reduced growth, damage to cell, tissue or organ when imposed upon living cells and which is capable of causing cell death. Examples of environmental stresses are heat, drought, saline soils and pathogens.
  • INJECTION the process of introducing molecules into cells, tissues and organs by positive pressure through a syringe. Macroinjection is accomplished without the aid of a microscope; microinjection is accomplished with the aid of a microscope.
  • TRANSFORMATION the process of introducing at least one gene into a cell which results in stable maintenance of the introduced gene.
  • CO-TRANSFORMATION the process of introducing at least two genes located on different molecules into the same cell which results in the stable maintenance of the introduced genes.
  • FOREIGN GENE a sequence of DNA encoding a specific product, products, or biological function which is obtained from a different species than that species into which the gene is introduced by a transformation vector.
  • FOREIGN DNA - a sequence of DNA which is obtained from a different species than that species into which it is introduced on a transformation vector.
  • Transformation vectors useful for the genetic modification of photosynthetic plant cells, plant tissues and plant organs may be prepared. They are double-stranded DNA molecules which include a gene foreign to the plant cell, tissue or organ to be altered and which are associated with the expression of a selectable or identifiable trait when the transformation vector is present in the plant cell, tissue or organ or in a plant derived therefrom. They also include at least one DNA sequence which is substantially identical to a portion of the DNA present in the plant cell, tissue or organ and which is capable of homologous recombination with the DNA present in the plant cell, tissue or organ after introduction of the transformation vector therein.
  • Foreign DNA sequences which include a gene associated with a selectable or identifiable trait are available from a wide variety of sources including bacterial plasmids, plant viruses and plants.
  • One group of such genes are those associated with resistance to antibiotics which are capable of inhibiting the growth of cereal cell cultures when present in the growth medium. These genes are generally Obtained from bacterial plasmids or bacterial transposons. Examples include genes for ampicillin resistance which may be obtained from pBR322 or any Tn3-containing DNA sequence, genes for kanamycin or G418 resistance which can be obtained from Tn5 or any Tn5-containing DNA sequence and genes for chloramphenicol resistance.
  • Another group of suitable foreign genes are those involved in the utilization of various substrates by plant cell cultures or the prevention of substrate inhibition. These genes may be obtained from organisms capable or incapable of utilizing a particular substrate. Examples include genes for galactose utilization, e.g. the gene for the enzyme UDP-4-galactose epimerase, which can be obtained from E. coli capable of utilizing galactose as a sole carbon source or from an E. coli F-prime plasmid containing genes for the galactose utilization pathway. Other examples are genes for lactose utilizaton, including the gene for ⁇ -galactosidase and genes involved in preventing amino acid inhibition of plant cell culture growth.
  • Examples of the latter are genes for asparto kinase, homoserine dehydrogenase and dihydropicolinic acid synthetase.
  • substrate utilization genes are obtained by subcloning DNA fragments into E. coli and selecting for growth on an appropriate medium.
  • genes associated with traits such as herbicide resistance, disease resistance or salt tolerance in one plant species may be employed as genes for selectable or identifiable traits in another.
  • a transformation vector employing this principle is illustrated in Fig. 2.
  • DNA sequences substantially identical to DNA sequences present in plant cells, tissues or organs are obtainable by various methods including the following: (1) total plant DNA is cut by restriction endonucleases, ligated to plasmid DNA, and cloned in E. coli. Plasmids containing plant DNA inserts are chosen following selection for insertional inactivation of an antibiotic resistance gene, size screening by agarose gel electrophoresis and DNA-DNA hybridization; (2) plant DNA is sheared, denatured, allowed to partially reassociate and repeated sequences are separated from unique sequences by hydroxylapatite chromatography. The repeated sequence fraction is then cloned in E.
  • coli and plasmids are chosen as described in method (1); (3) DNA sequences containing genes expressed during specific developmental stages or in specific tissues are obtained by isolating messenger. RNA from the chosen stage or tissue, synthesizing DNA complementary to the messenger RNA and cloning the resulting DNA in E. coli. Plasmids chosen as described in method (1) are subjected to characterization by hybrid-selected in vitro translation of mRNA or by frequency of occurrence of the sequence in the total given messenger RNA pool; and (4) sequences are obtained as described in method (3) followed by identification using DNA-DNA hybridization of the native gene sequence in a genomic DNA library prepared in Charon phages. Native genes isolated from gene libraries are characterized by restriction mapping and heteroduplex analysis to determine the number and location of intervening sequences. Structural gene sequences are then isolated by subcloning in E. coli for use in vector construction.
  • Transformation vectors of the type shown in Fig. 1 contain a foreign gene for a selectable or identifiable trait and a plant-derived DNA sequence substantially identical to DNA present in a plant cell, tissue or organ to be transformed. They are obtained by ligating the two sequences together or by ligating them to the same plasmid and cloning in E. coli using methods well known to those skilled in the art. Recombinant plasmids are identified by selection for insertional inactivation of resistance genes, colony hybridization, screening for insert size, selection for biological function of the foreign gene or any combination of these methods. Depending upon the method used, the resulting transformation vector may be linear or circular.
  • the plant-derived DNA portion of the transformation vector may vary widely, it is preferred that this DNA sequence be expressed during a specific devel opmental stage of the life cycle of the plant cell, tissue or organ or that this DNA sequence be a repetitive sequence which is naturally present in the plant cell, tissue or organ. Also, it is preferred that this DNA sequence be substantially identical to a portion of the chromosomal DNA of the plant cell, tissue or organ to be transformed.
  • the transformation vector contain two plant-derived DNA sequences located at flanking positions relative to the foreign gene for the selectable or identifiable trait. These sequences may be the same as or different from one another provided only that they be substantially identical to DNA sequences located in the plant cell, tissue or organ to be transformed and be capable of homologous recombination with DNA present therein when the transformation vector is introduced into the plant cell, tissue or organ.
  • the transformation vector In order to introduce a desired agronomic property or permit production of a desired product in a plant cell, plant tissue, plant organ or in a plant derived therefrom, the transformation vector also includes a DNA sequence coding for the desired property or product.
  • a desired agronomic property or permit production of a desired product in a plant cell, plant tissue, plant organ or in a plant derived therefrom, the transformation vector also includes a DNA sequence coding for the desired property or product.
  • a DNA sequence coding for the desired property or product Of particular interest are non-selectable genes whose presence would be desirable in cereals.
  • Suitable agronomic genes include storage protein genes such as the zein genes of maize, albumin and globulin genes of maize, gliadin genes of wheat, albumin and globulin genes of wheat, hordein genes of barley, animal protein genes such as chicken ovalbumin or collagen genes, non-cereal plant protein genes such as soybean storage protein genes, and synthetic genes.
  • genes associated with disease resistance which cannot be selected for in vitro, including rust resistance in wheat, fungal smut resistance or stalk rot resistance in maize, powdery mildew resistance in barley, and rice blast resistance in rice;
  • genes associated with stress tolerance including tolerance to heat, drought, salt, cold and minerals such as aluminum or boron;
  • genes associated with herbicide resistance particularly systemic herbicides that are not selectable in vitro;
  • genes associated with plant architecture including genes associated with leaf shape, floral morphology, tiller number, and height and root length; and
  • genes associated with plant yield, vigor and the like are also included.
  • the DNA sequences containing these genes are obtained by combining specific plant genes with genes or gene fragments from a plant or a foreign source.
  • a gene for production of the corn storage protein, zein, having increased lysine content is obtained by first obtaining a purified zein gene from corn endosperm using the approach described in methods (3) and (4) hereinabove for obtaining plant DNA. This is followedby insertion into the zein gene of a DNA sequence coding for increased lysine production such as a segment of the chicken ovalbumin gene or a synthetic DNA sequence.
  • this DNA sequence contains genes completely foreign to the recipient plant such as genes for herbicide resistance or disease resistance rather than foreign sequences serving to modify pre-existing genes such as zein with increased lysine content
  • the DNA sequence is constructed in such a way that the foreign sequence is joined to or flanked by a plant DNA sequence expressed during the desired developmental stage or in the desired tissue. Transformation vectors containing foreign genes associated with selectable or identifiable plant traits, genes associated with expression of a desired agronomic property or production of a desired product, and one or two plant-derived DNA sequences, are shown in Figs. 3 and 4, respectively. In the preferred embodiment shown in Fig. 4, the two sequences flank both the gene for a foreign selectable or identifiable trait and the agronomic gene.
  • transformation vectors are prepared using the methods described previously for transformation vectors which do not include the agronomic gene. They are characterized by analysis of protein products encoded by the genes contained in the vectors and by DNA sequence and restriction endonuclease analysis.
  • the transformation vectors of this invention may be introduced in plant cells, tissues or organs or into plants derived therefrom by various well known methods, including injection, both marcrdinjection and microinjection, and by DNA-mediated transformation or cotransformation.
  • the introduction of the transformation vectors is carried out under suitable conditions permitting transformation, including homologous recombination between vector DNA and naturally-occurring plant DNA, to occur and permitting the subsequent expression of introduced genes.
  • transformed cells or tissues may be selected or identified using the introduced trait.
  • the transformed cells may be grown in culture to produce desired products or plants having desired properties may be regenerated using methods known in the art. In the former case, product is recovered after cell growth using known methods. In the latter, regenerated plantlets are transplanted to greenhouses when plants are grown using known methods.
  • a transformation vector such as that shown in Fig. 1 which includes a gene associated with ampicillin resistance renders cereal embryos resistant to levels of ampicillin which would otherwise prevent plant growth.
  • the transformed embryos are then grown until plantlets are produced.
  • the plantlets are then transplanted and grown to produce a plant. Additional examples are shown in Figs. 5 and 6 which illustrate specific transformation vectors useful in the genetic modification of cereals to produce desired properties.
  • the transformation vectors may be introduced into growing plants or organs under suitable conditions such as the following.
  • Vectors are injected into the free nuclear (milk-stage) endosperm of the karyopses using a syringe. Approximately 0.5 ⁇ l of solution per karyopsis is used. The solution typically has a vector DNA concentration of about 1 mg/ml in a standard TRIS-EDTA/water solution.
  • Microinjection into cells or tissues is accomplished using a micromanipulator manufactured by
  • MNlB 4 mM n-octyl alcohol 2 percent gum arabic (acacia)
  • Aceto-carmin 0.5 percent w/v in 45 percent acetic acid
  • Triton solution 0.1 percent Triton X 100 [Sigma], 0.05 M Tris [pH 8.5], and 0.05 M Na 2 EDTA. Incubate at room temperature for 10-20 minutes. If lysis does not occur and the suspension does not become very viscous, incubate at 37°C for 30 minutes.
  • Refractive index should be between 1.390 and 1.396 or a density of 1.59 g/ml. The density is the most reliable and can be determined easily by weighing a known volume. This mixture will just fill one SW 50.1 rotor tube. 9. Centrifuge at 35,000 rpm for 48 hours at 20°C.
  • the lower band contains the covalently closed circular DNA. Collect the DNA by puncturing the side of the tube with a 20- to 21-gauge syringe needle.
  • Restriction endonucleases were obtained from New England BioLabs. The reaction conditions for each enzyme were essentially as described by the suppliers. Restriction enzymes were added to a concentration of approximately one unit/ ⁇ g of DNA and incubated from 1 to 3 hours at 37°C.
  • the reaction buffer for Eco RI contained 50 mM NaCl, 100 mM tris-HCl, pH 7.4, 6 mM MgCl 2 , and 6 mM 2mercaptoethanol.
  • the buffer for Hae III contained 6 mM tris-HCl, pH 7.4, 6 mM MgCl 2 , and 6 mM 2-mercaptoethanol.
  • the buffer for Hind III, Bam HI and PstI contained 50 mM NaCl, 6 mM tris-HCl, pH 7.4, 6 mM MgCl 2 . and 6 mM 2-mercaptoethanol. The reactions were stopped by heating the solution at 65°C for 5 minutes.
  • ALKALINE PHOSPHATASE BAP
  • Tetracycline plates can be stored at 10°C for a few weeks before use.
  • Ampicillin plates can be stored at 10°C for a few days before use. VII. SLAB GEL ELECTROPHORESIS
  • DNA samples were analyzed by electrophoresis in slab gels of either agarose or polyacrylamide, depending on the expected size of the DNA molecules.
  • circular DNA and linear DNA molecules greater than 1200 base pairs in length were separated on 1 percent to 1.4 percent agarose slab gels (vertical or horizontal).
  • DNA samples containing molecules smaller, than 1200 base pairs in length were separated on 5 percent to 8 percent vertical polyacrylamide slab gels.
  • Vertical 4garose and polyacrylamide gels were 20 cm long and 1.5 mm thick; horizontal agarose gels were approximately 3 mm thick and of varying lengths.
  • Electrophoresis 'buffer in all cases contained 40 mM tris base, 5 mM sodium acetate, and 1 mM
  • Polyacrylamid gels were prepared from a 30 percent stock acrylamide solution (29:1::acrylamide:bisacrylamide) using 65 mg ammonium persulfate and 50 ul TEMED per 100 mis of gel. Samples were mixed with bromphenol blue and either sucrose or glycerol before layering on the gel. Electrophoresis was carried out at 150 volts or less for one to four, hours at room temperature without buffer circulation. After electrophoresis, the gels were stained with 0.4 ⁇ g/ml ethidium bromide in water for 30 minutes and the DNA bands visualized with a short wavelength ultraviolet light.
  • Cultures are A188 corn and "Chinese Spring" wheat (48.6g FR. Wt.); grind with mortar and teflon pestle (2 ml extraction buffer/lg tissue - on ice; pellet 500g, 5 min., JA-17 (low speed spin); pellet 30,000g, 10 min., JA-17 (medium speed spin); layer supernatant on cushion buffer (10 ml supernatant/5 ml buffer); pellet 130,000g, 2 hours, TI 70; and freeze pellet at -70°C.
  • Each 0.4-cm slot can be loaded with up to 5 ⁇ g of restriction-endonuclease-cleaved DNA. Use about 1 ⁇ g of cleaved bacterial DNA.
  • Ethidium bromide (0.5 ⁇ g/ml) is included in the electrophoresis buffer, and the gel is photographed under shortwave UV light.
  • a nitrocellulose filter sheet (S8S B85 or HAWP Millipore) cut to the size of the gel, is placed on top of the gel.
  • a stack of about 6 cm of paper towels cut to size are placed on top of the 3MM paper.
  • the stack is uniformly compressed with a 1-kg weight on a thick sheet of plexiglass.
  • the filter is turned over with the shrunken gel attached, and the gel lanes and edges are marked with a soft-lead pencil.
  • the nitrocellulose filter is rinsed in 2X SSPE for 10 minutes and dried in a vacuum oven at 80°C for 2 hours.
  • Hybridization is conducted as described in XII.
  • the filter can be identified and oriented by including within the plastic wrap a piece of paper marked with radioactive ink. (See XIII hereinafter.)
  • 100X BFP 2 percent w/v bovine serum albumin, Ficoll, and polyvinyl pyrrolidone. 1 liter of 20X SSPE
  • An intensifying screen is attached to the lid of the film holder (Dupont Cronex Lightning-Plus ZC; 224-156 without blockers, 8 X 10 in.).
  • Cronex 4 film is about one-quarter to one-half as fast as the X-Omat R film, but it is of higher resolution.
  • MICRONUTRIENTS (stock solution) 40 ml.
  • Immature embryos ( ⁇ 1.0-1.5 mm in length) are isolated, asceptically from sterilized seeds. Embryos are placed polar axis down, (scutellum up) on culture initiation medium (basal medium and 1 mg/l 2,4-D) and incubated at 28oC (in presence or absence of light).
  • Selection is accomplished by transferring cultures to basal media supplemented with 1 ⁇ g/ral 2,4-D and 50 ⁇ g/ml ampicillin. These cultures are transferred every 102 weeks to media adjusted with 2,4-D and ampicillin as judged by the viability of the cultures. Surviving cultures are regenerated by step-down transfer to hormonefree media. Regenerants are grown to maturity in the greenhouse, where self or cross pollinations are made and phenotypic data is recorded. DNA can be isolated from leaves of these plants and used for DNA/DNA hybridization analysis.
  • Tris-EdTA + pM7 (ca. 1 mg/ml).
  • Day 39 62 plants regenerated from pM7 injected, ampi cillin-selected culture by transferring cultures to basal media minus hormone.

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Abstract

De nouveaux vecteurs de transformation peuvent être utilisés pour la modification génétique de cellules végétales, de tissus végétaux ou d'organes végétaux. Ces vecteurs de transformation sont des molécules d'ADN à double brin qui comprennent un gène étranger à cette cellule végétale, à ces tissus végétaux ou à cet organe végétal et il est associé à un caractère sélectionnable ou identifiable lorsqu'il est présent dans ces molécules et au moins une séquence ADN sensiblement identique à une séquence ADN qui est présente dans cette cellule végétale, ces tissus végétaux ou cet organe végétal et est capable d'une recombinaison homologue avec cette séquence ADN végétale lorsque le vecteur de transformation est introduit dans la cellule végétale, les tissus végétaux ou l'organe végétal. Des molécules d'ADN à double brin comprennent de plus un gène associé à l'expression d'une propriété agronomique désirée ou à la production d'un produit désiré dans des cellules végétales, des tissus végétaux, des organes végétaux ou dans des plantes dérivées à partir de ceux-ci, lesquelles molécules peuvent être utilisée pour créer des plantes ayant des propriétés souhaitées ou obtenir des produits ayant une valeur commerciale. Ces vecteurs de transformation sont particulièrement utiles pour modifier la constitution génétique de céréales, y compris l'orge, le blé, le riz et le maïs.
EP82903287A 1981-10-01 1982-09-30 Procede de modification genetique de cereales avec des vecteurs de transformation Withdrawn EP0090033A1 (fr)

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US307595 1981-10-01

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