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WO1996040949A1 - Induction de la sterilite male dans des plantes par expression de niveaux eleves d'avidine - Google Patents

Induction de la sterilite male dans des plantes par expression de niveaux eleves d'avidine Download PDF

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WO1996040949A1
WO1996040949A1 PCT/US1996/008583 US9608583W WO9640949A1 WO 1996040949 A1 WO1996040949 A1 WO 1996040949A1 US 9608583 W US9608583 W US 9608583W WO 9640949 A1 WO9640949 A1 WO 9640949A1
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plant
promoter
gene
avidin
nucleotide sequence
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PCT/US1996/008583
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WO1996040949B1 (fr
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John A. Howard
Marc C. Albertson
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Pioneer Hi-Bred International, Inc.
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Priority to RO97-02278A priority Critical patent/RO120270B1/ro
Priority to PL96323745A priority patent/PL323745A1/xx
Priority to EP96919051A priority patent/EP0832261A2/fr
Priority to AU61493/96A priority patent/AU708618B2/en
Priority to CA002223460A priority patent/CA2223460C/fr
Priority to JP9501127A priority patent/JPH11512922A/ja
Priority to BR9609069A priority patent/BR9609069A/pt
Publication of WO1996040949A1 publication Critical patent/WO1996040949A1/fr
Publication of WO1996040949B1 publication Critical patent/WO1996040949B1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the present invention relates to a method for controlling fertility of plants using DNA molecules that encode avidin.
  • this invention is directed to methods for producing transgenic, male-sterile plants which express avidin.
  • this invention is directed to methods for restoring male fertility in the progeny of male-sterile plants.
  • Control of pollen fertility is essential in hybrid crop production. See, for example, J. M. Poehlman, BREEDING FIELD CROPS, 3rd ed. (Van Nostrand and Reinhold, New York, NY, 1987) , which is herein incorporated by reference.
  • control of pollen fertility is typically accomplished by physically removing the male inflorescence, or tassel, prior to pollen shed.
  • Manual detasseling is highly labor intensive. Although mechanical detasseling is less labor intensive than manual detasseling, it is less reliable and requires subsequent examination of the crop and possibly, remedial manual detasseling. Both methods of detasseling cause a loss of female parent yield.
  • Pollen fertility also can be controlled by applying a foliar spray having male gametocidal properties .
  • Cross et al . , Sex Plant Reprod. 4 : 235 (1991) the application of ethrel to anthers results in additional pollen mitoses in wheat, degeneration of tapetal cells in barley, and malformed or aborted pollen in Eragrostis .
  • Bennet et al . Nature 240 : 566 (1972), Colhoun et al . , Plant Cell Environ . 6: 21 (1983) ; Berthe et al . , Crop Sci . 18 : 35 (1978) .
  • Plants that are used as females either fail to shed pollen, produce pollen that is biochemically unable to effect self-fertilization, or fail to make pollen. Plants that are unable to self- fertilize are said to be "self-incompatible. " Difficulties associated with the use of a self- incompatibility system include availability and propagation of the self-incompatible female line, and stability of the self-compatibility. In some instances, self-incompatibility can be overcome chemically, or immature buds can be pollinated by hand before the bio ⁇ chemical mechanism that blocks pollen is activated. Self-incompatible systems that can be deactivated are often very vulnerable to stressful climatic conditions that break or reduce the effectiveness of the biochemical block to self-pollination.
  • CMS cytoplasmic male sterility
  • Nuclear sterility can be either dominant or recessive. Dominant sterility can only be used for hybrid seed formation if vegetative or clonal propagation of the female line is possible. Recessive sterility can be used if sterile and fertile plants are easily discriminated. Commercial utility of dominant and recessive sterility systems is limited, however, by the expense of clonal propagation and roguing the female rows of self-fertile plants, respectively. Although there are hybridization schemes involving the use of CMS, there are limitations to its commercial value. One example of a CMS system is a specific mutation in the cytoplasmically located mitochondria which can, when in the proper nuclear background, lead to the failure of mature pollen formation.
  • the nuclear background can compensate for the cytoplasmic mutation and normal pollen formation occurs.
  • Specific nuclear "restorer genes" allow pollen formation in plants with CMS mitochondria.
  • CMS for commercial seed production involves the use of three breeding lines: a male-sterile line (female parent) , a maintainer line which is isogeneic to the male-sterile line but contains fully functional mitochondria, and a male parent line.
  • the male parent line may or may not carry the specific restorer genes in the cytoplasm.
  • a CMS system can be used without restoration.
  • crops for which the fruit or seed of the hybrid is the commercial product the fertility of the hybrid seed must be restored by specific restorer genes in the male parent or the male-sterile hybrid must be pollinated. Pollination of non-restored hybrids can be achieved by including a small percentage of male fertile plants to effect pollination.
  • the CMS trait is inherited maternally, since all cytoplasmic organelles are inherited from the egg cell only.
  • CMS systems possess limitations that can preclude them as a sole solution to production of male sterile plants.
  • one particular CMS type in maize (T-cytoplasm) confers sensitivity to the toxin produced by infection by a particular fungus.
  • CMS systems may break down under certain environmental conditions.
  • an isolated DNA molecule comprising a nucleotide sequence encoding avidin operably linked to a plant promoter sequence.
  • the isolated DNA sequence is contained in an expression vector.
  • the plant promoter sequence is a constitutive promoter, and in another preferred embodiment the constitutive promoter is a ubiquitin promoter.
  • a transgenic plant in which the plant comprises a heterologous nucleotide sequence comprising an avidin gene, and wherein the heterologous nucleotide sequence increases the concentration of avidin in the tissues of the plant, causing male sterility.
  • the transgenic plant is a corn plant, a soybean plant, or a sunflower plant.
  • the heterologous DNA molecule further comprises a constitutive promoter sequence, which in a preferred embodiment is a ubiquitin promoter sequence.
  • a method of producing a transgenic male-sterile plant comprising introducing into plant cells an expression vector comprising a promoter and an avidin gene, wherein the promoter controls the expression of the avidin gene, and where the avidin gene expression causes male sterility.
  • a transgenic plant is regenerated from the plant cells.
  • the promoter comprises a ubiquitin promoter or an inducible promoter.
  • a method of using an avidin gene to produce a male-fertile hybrid plant comprising producing a first parent male-sterile plant comprising a DNA molecule as described above, in which the expression of avidin causes male sterility, producing a second transgenic parent plant expressing a second foreign gene, and cross-fertilizing the first parent with the second parent to produce a hybrid plant that expresses the second foreign gene, and in which the product of the second foreign gene reduces expression of avidin in the hybrid plant, thereby producing a male-fertile hybrid plant.
  • the second foreign gene is selected from the group consisting of an antisense gene, a ribozyme gene and an external guide sequence gene.
  • the antisense gene comprises avidin mRNA sequences, which in a further preferred embodiment is under the control of an inducible promoter.
  • the ribozyme gene comprises avidin mRNA sequences.
  • the external guide sequence gene comprises avidin mRNA sequences.
  • the DNA molecule of the first parent plant further comprises a LexA operator which is operatively linked to said promoter, wherein the second foreign gene is the LexA repressor gene.
  • the promoter sequence is an anther-specific promoter sequence comprising an anther box selected from the group consisting of the nucleotide sequence of SEQ ID NO:l; the nucleotide sequence of SEQ ID NO:2; the nucleotide sequence of SEQ ID NO:3; and a functional fragment thereof.
  • the anther box has the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and the anther-specific promoter further comprises a core promoter selected from the group consisting of: CaMV 35S core promoter; the nucleotide sequence of SEQ ID NO:4; the nucleotide sequence of SEQ ID NO:5; the nucleotide sequence of SEQ ID NO:6; and functional fragments thereof.
  • a method for restoring fertility in a plant rendered male sterile by expression of avidin comprising spraying the plant with a solution of biotin.
  • Figure 1 shows the avidin-encoding plasmid PHI5168.
  • Figure 2 shows the plasmid PHI610 encoding the Jbar gene.
  • a structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • mRNA messenger RNA
  • a foreign gene refers in the present description to a DNA sequence that is operably linked to at least one heterologous regulatory element. For example, any gene other than the maize 5126 structural gene is considered to be a foreign gene if the expression of that gene is controlled by an anther-specific regulatory element of the 5126 gene.
  • a promoter is a DNA sequence that directs the transcription of a gene, such as a structural gene, an antisense gene, a ribozyme gene or an external guide sequence gene.
  • a promoter is located in the 5' region of a gene, proximal to the transcriptional start site. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
  • a plant promoter is a promoter sequence that will direct the transcription of a gene in plant tissue.
  • a core promoter contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription.
  • a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
  • the SGB6 core promoter consists of about 38 nucleotides 5 ' -ward of the transcriptional start site of the SGB6 gene, while the Cauliflower Mosaic Virus (CaMV) 35S core promoter consists of about 33 nucleotides 5'- ward of the transcriptional start site of the 35S genome.
  • CaMV Cauliflower Mosaic Virus
  • a tissue-specific promoter is a DNA sequence that, when operably linked to a gene, directs a higher level of transcription of that gene in a specific tissue than in some or all other tissues in an organism.
  • an anther-specific promoter is a DNA sequence that directs a higher level of transcription of an associated gene in plant anther tissue.
  • the SGB6 anther-specific promoter for example, can direct the expression of a foreign gene in anther tissue, but not in root tissue or coleoptile tissue.
  • an "anther-specific promoter” comprises two functional elements: an "anther box” and a core promoter.
  • a particular anther box may possess the functional characteristics of a classic enhancer.
  • the combination of an anther box and a core promoter can stimulate gene expression to a greater extent than a core promoter alone. This is true even in the case of a chimeric anther-specific promoter containing an anther box and a core promoter derived from different genes. Such chimeric anther-specific regulatory elements are described below.
  • An operator is a DNA molecule that is located 5 ' -ward of a gene and that contains a nucleotide sequence which is recognized and bound by a repressor protein.
  • the binding of a repressor protein with its cognate operator results in the inhibition of the transcription of the gene.
  • the LexA gene encodes a repressor protein that binds to the LexA operator.
  • An isolated DNA molecule is a fragment of DNA that has been separated from the DNA of an organism.
  • a cloned DNA molecule encoding an avidin gene is an isolated DNA molecule.
  • Another example of an isolated DNA molecule is a chemically-synthesized DNA molecule, or enzymatically-produced cDNA, that is not integrated in the genomic DNA of an organism.
  • An enhancer is a DNA regulatory element that can increase the efficiency of transcription.
  • cDNA Complementary DNA
  • cDNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA refers to a double- stranded DNA molecule consisting of such a single- stranded DNA molecule and its complementary DNA strand.
  • expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • a cloning vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • An expression vector is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked to" the regulatory elements.
  • a recombinant host may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • a transgenic plant is a plant having one or more plant cells that contain a foreign gene.
  • RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA.
  • a DNA molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA.
  • the RNA transcript is termed an antisense RNA and a DNA sequence that encodes the antisense RNA is termed an antisense gene.
  • Antisense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
  • a ribozyme is an RNA molecule that contains a catalytic center. The term includes RNA enzymes, self- splicing RNAs, and self-cleaving RNAs.
  • a DNA sequence that encodes a ribozyme is termed a ribozyme gene.
  • An external guide sequence is an RNA molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P.
  • a DNA sequence that encodes an external guide sequence is termed an external guide sequence gene.
  • the present invention provides methods for generating male sterile plants by preparing transgenic plants that express avidin.
  • the avidin can be expressed constitutively, in a non-tissue specific manner, or can be expressed in an anther-specific manner. Methods for restoring male fertility are also provided.
  • An avidin gene is isolated and inserted into an expression vector suitable for introduction into plant tissue.
  • the expression vector further comprises a promoter which may be a constitutive promoter, a non- tissue specific promoter, such as the ubiquitin promoter, a tissue specific promoter such as an anther-specific promoter, or an inducible promoter.
  • the expression vector is introduced into plant tissue by standard methods and transgenic plants are selected and propagated.
  • the transgenic plants that express avidin are male sterile. Male fertility can be restored by coexpression in the transgenic plants of a second gene that inhibits transcription of the avidin gene, or inhibits translation of avidin mRNA. Alternatively, male fertility can be restored by spraying developing plants with solutions of biotin. 3. Isolation of DNA Molecules that Encode Avidin
  • Nucleotide sequences encoding avidin can be isolated by known methods. For example, a cDNA encoding chicken egg white avidin can be isolated from a chicken oviduct cDNA library by the methods described by Gope et al . , Nucleic Acids Res . , 15:3595 (1987), which is incorporated herein by reference.
  • genomic clones of the avidin gene can be obtained from genomic DNA of organisms that are known to produce avidin.
  • a chicken avidin gene can be cloned from chicken genomic DNA by the methods described in Keinanen et al . , Eur. J. Biochem. 220 : 615 (1994) .
  • Avidin genes can also be isolated using the polymerase chain reaction (PCR) using standard methods. See Erlich, PCR TECHNOLOGY: PRINCIPLES AND APPLICATIONS FOR DNA AMPLIFICATION (Stockton Press, NY, 1989) and Innis et al . , PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Academic Press, San Diego, 1990) . Methods for PCR amplification of long nucleotide sequences, such as the ELONGASETM system (Life Technologies, Inc., Gaithersburg, MD) can also be used for obtaining longer nucleotide sequences, such as genomic clones encoding avidin.
  • PCR polymerase chain reaction
  • PCR primers complementary to the 5' and 3' termini of known avidin gene sequences can be synthesized using commercial oligonucleotide synthesizers, such as those supplied by Applied Biosystems (Foster City, CA) .
  • the primers include additional nucleotide sequences containing restriction endonuclease cleavage sites. The presence of such sites allows for the directional cloning of PCR products into suitable cloning vectors after treatment with an appropriate restriction enzyme. See Finney, "Molecular Cloning of PCR Products" in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al . Eds. (John Wiley & Sons, New York, 1987) p. 15.7.1.
  • Template DNA for the PCR can either be cDNA or genomic DNA, and can be prepared from an appropriate organism using methods well known in the art. Sambrook et al . , MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989) . Genomic DNA can be directly prepared from avian, reptilian, or amphibian tissue using commercials reagents, such as TriazolTM (Life Technologies, Inc., Gaithersburg, MD) . Alternatively, cDNA encoding avidin can be prepared using mRNA extracted from chicken oviduct tissue using a commercially available kit (Pharmacia, Piscataway, NJ) .
  • mRNA preparation is used as a template for cDNA synthesis using poly(dT) or random hexamer primers by standard techniques. See Sambrook et al . , supra . cDNA synthesis is then carried out using a commercially available kit
  • Genomic DNA or cDNA fragments isolated by the methods described above can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases.
  • a vector such as a bacteriophage or cosmid vector
  • Techniques for manipulation of such vectors are disclosed by Ausubel et al . (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 3.0.5-3.17.5 (1990) ["Ausubel”] .
  • nucleotide sequences encoding avidin can be obtained by synthesizing DNA molecules encoding avidin using mutually priming long oligonucleotides. See, for example, Ausubel at pages 8.2.8 to 8.2.13. Also, see Wosnick et al . , Gene 6O:115 (1987) . Current techniques using the polymerase chain reaction provide the ability to synthesize genes as large as 1.8 kilobases in length. Adang et al . , Plant Molec. Biol . 21 : 1131 (1993); Bambot et al . , PCR Methods and Applications 2 : 266 (1993) .
  • the nucleotide sequence of a synthesized avidin gene can be based upon any known avidin-encoding DNA molecule. See, for example, Gope et al . , supra . These clones can be analyzed using a variety of standard techniques such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis. Primer extension analysis or SI nuclease protection analysis, for example, can be used to localize the putative start site of transcription of the cloned gene. Ausubel at pages 4.8.1-4.8.5; Walmsley et al . , "Quantitative and Qualitative Analysis of Exogenous Gene Expression by the SI Nuclease Protection Assay, " in METHODS IN MOLECULAR BIOLOGY, VOL. 7: GENE TRANSFER AND EXPRESSION PROTOCOLS, Murray (ed.), pages 271-281 (Humana
  • an expression vector contains: (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance gene to provide for the growth and selection of the expression vector in the bacterial host; (2) a cloning site for insertion of an exogenous DNA sequence, for example an avidin gene; (3) eukaryotic DNA elements that control initiation of transcription of the exogenous gene, such as a promoter;
  • DNA elements that control the processing of transcripts such as a transcription termination/polyadenylation sequence; and (5) a gene encoding a marker protein (e . g. , a reporter gene) , wherein the gene is operably linked to the DNA elements that control transcription initiation.
  • a marker protein e . g. , a reporter gene
  • General descriptions of plant expression vectors and reporter genes can be found in Gruber et al . , "Vectors for Plant Transformation, " in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al. (eds.), pages 89-119 (CRC Press, 1993) .
  • a plant ubiquitin promoter system is used. Plant ubiquitin promoters are well known in the art. See, for example, European Patent Application 0 342 926, which is incorporated herein by reference. The ubiquitin promoter is a constitutive promoter.
  • an inducible promoter is used to allow inducible regulation of the expression of avidin.
  • An example of such an inducible promoter is the glutathione S-transferase system in maize. See Wiegand et al . , Plant Mol . Biol . 7:235 (1986) . This method also allows for reversible induction of male sterility. Thus, expression of avidin and concomitant male sterility, can be controlled as desired by activation of the promoter. When the promoter is not activated, avidin is not expressed, and the plants are male fertile.
  • Expression vectors containing an avidin gene can be introduced into protoplasts, or into intact tissues, such as immature embryos and meristems, or into callus cultures, or into isolated cells. Preferably, expression vectors are introduced into intact tissues.
  • General methods of culturing plant tissues are provided, for example, by Miki et al . , "Procedures for Introducing Foreign DNA into Plants,” in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al . (eds.) , pages 67- 88 (CRC Press, 1993), and by Phillips et al .
  • Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens . Horsch et al . , Science 227: 1229 (1985) .
  • a disarmed Ti- plasmid is used as a vector for foreign DNA sequences. Transformation can be performed using procedures described, for example, in European patent application Nos. 116 718 (1984) and 270 822 (1988) .
  • Preferred Ti- plasmid vectors contain the foreign DNA sequence between the border sequences, or at least located upstream of the right border sequence.
  • vectors can be used for transforming plant cells using procedures such as direct gene transfer (see, for example, PCT application WO 85/01856 and European application 275 069) , in vi tro protoplast transformation (for example, U.S. patent No. 4,684,611), plant virus-mediated transformation (for example, European application No. 067 553 and U.S. patent No. 4,407,956), and liposome-mediated transformation (for example, U.S. patent No. 4,536,475) .
  • Suitable methods for corn transformation are provided by Fromm et al . , Bio/Technology 8 : 833 (1990), and by Gordon-Kamm et al . , The Plant Cell 2 : 603 (1990) .
  • direct transfer methods are preferred for the transformation of a monocotyledonous plant, particularly a cereal such as rice, corn, sorghum, barley or wheat.
  • Suitable direct transfer methods include microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Gruber et al . , supra, Miki et al . , supra, and Klein et al . , Bio/Technology 10 : 268 (1992) . More preferably, expression vectors are introduced into tissues of a monocotyledonous plant using microprojectile-mediated delivery with a biolistic device. 5. Regulation of Male Fertility
  • an expression vector is constructed in which a DNA sequence encoding avidin is operably linked to DNA sequences that regulate gene transcription in plant tissue.
  • the general requirements of an expression vector are described above.
  • avidin be not merely expressed in a transient manner, but that the expression vector is introduced into plant embryonic tissue in such a manner that avidin will be expressed at a later stage of development in the adult plant.
  • mitotic stability can be achieved using plant viral vectors that provide epichromosomal replication.
  • a preferred method of obtaining mitotic stability is provided by the integration of expression vector sequences into the host chromosome.
  • mitotic stability can be provided by the microprojectile delivery of an expression vector to plant tissue, or by using other standard methods as described above. See, for example, Fromm et al . , Bio/Technology 8 : 833 (1990), Gordon-Kamm et al . , The Plant Cell 2 : 603 (1990) , and Walters et al . , Plant Molec . Biol . 18 : 189 (1992) .
  • Transcription of the avidin gene after introduction into plant tissue is preferably controlled by a constitutive promoter that non-specifically stimulates gene expression in plant tissue.
  • a constitutive promoter suitable for this purpose is the ubiquitin promoter, as described supra .
  • Transcription of the avidin gene may also be controlled by a promoter which stimulates gene expression in a tissue-specific manner.
  • a particularly preferred anther-specific promoter is the 5126 promoter which was isolated from the maize inbred B73 line. The 5126 promoter stimulates the expression of a foreign gene from quartet to early uninucleate microspore-stage anthers.
  • SGB6 promoter Another suitable anther-specific promoter is the SGB6 promoter, which also was isolated from the B73 line.
  • the SGB6 promoter can induce expression of a foreign gene in anther tapetal cells from the quartet stage to the mid- uninucleate stage of microspore development.
  • anther-specific gene expression can be provided using a combination of an anther box and a core promoter.
  • a particularly preferred anther box is a
  • 5126 anther box which comprises the following nucleotide sequence:
  • Another suitable anther box resides within a 94 base pair DNA fragment defined by nucleotides 583 to 490 upstream of the SGB6 start site of transcription.
  • the nucleotide sequence of the 94 base pair SGB6 anther box is:
  • a suitable anther box is obtained from the maize G9 promoter, which stimulates gene expression during the meiotic to quartet stages of development.
  • G9 anther box comprises the following nucleotide sequence:
  • the 5126, SGB6 and G9 anther boxes can be obtained by synthesizing oligonucleotides, as described above.
  • deletion analysis can be performed to localize one or more additional anther-specific regulatory sequences within the disclosed anther boxes.
  • Such "functional fragments" of the anther boxes also can be used to regulate avidin gene expression in an anther-specific manner.
  • Preferred core promoters are derived from the 5126 core promoter, the SGB6 core promoter, the G9 core promoter and the Cauliflower Mosaic Virus 35S core promoter.
  • a particularly preferred core promoter is the 5126 core promoter which comprises the following nucleotide sequence:
  • the SGB6 core promoter consists of about 38 nucleotides upstream of the transcriptional start site of the SGB6 gene.
  • a suitable SGB6 core promoter has the following nucleotide sequence:
  • a suitable core promoter derived from the G9 gene comprises the nucleotide sequence:
  • Suitable core promoters also are provided by functional fragments of 5126, SGB6 and G9 core promoters. Methods for obtaining such functional fragments are described above.
  • the developmental window of avidin gene expression can be extended by using a chimeric regulatory element comprising an anther box from one gene and an anther- specific promoter from a second gene.
  • SGB6 regulatory sequences stimulate gene expression from quartet through mid-uninucleate stages of development
  • G9 regulatory sequences stimulate gene expression during meiosis and through the quartet stage of development.
  • the combination of an SGB6 anther box and a G9 promoter stimulates transcription of a foreign gene during meiosis and through the mid-uninucleate stage of development.
  • various combinations of anther boxes and anther-specific promoters are particularly useful for the present invention.
  • a viral core promoter also may be used.
  • suitable viral core promoters include a Cauliflower Mosaic Virus (CaMV) core promoter, a Figwort Mosaic Virus core promoter, and the like. Gruber et al . , supra .
  • the viral core promoter is the CaMV 35S core promoter, or a variant thereof.
  • the expression vector contains a selectable marker gene, such as a herbicide resistance gene.
  • a selectable marker gene such as a herbicide resistance gene.
  • such genes may confer resistance to phosphinothricine, glyphosate, sulfonylureas, atrazine, or imidazolinone.
  • the selectable marker gene is the J ar gene or pat gene which encodes phosphinothricin acetyltransferase.
  • the nucleotide sequences of bar genes can be found in Leemans et al . , European patent application No. 0-242-246 (1987) , and in White et al . , Nucleic Acids Res . 18 : 1062 (1990) .
  • Wohlleben et al . , Gene 70 : 25 (1988) disclose the nucleotide sequence of the pat gene.
  • Bar or pat gene expression confers resistance to herbicides such as glufosinate (sold as Basta ® and Ignite ® , among others) and bialaphos (sold as Herbi-ace ® and Liberty ® ) .
  • herbicides such as glufosinate (sold as Basta ® and Ignite ® , among others) and bialaphos (sold as Herbi-ace ® and Liberty ® ) .
  • the expression vector can contain DNA sequences encoding avidin under the control of a constitutive promoter or an anther-specific promoter, as well as the selectable marker gene under control of a constitutive promoter.
  • the selectable marker gene can be delivered to host cells in a separate selection expression vector by "co-transformation" of embryonic tissue.
  • transgenic male-sterile plants require the production of a second "restorer" line of transgenic plants.
  • a transgenic plant that is male- sterile due to barnase expression would be crossed with a male-fertile plant that expresses the barnase inhibitor, as discussed above.
  • Mariani et al . Nature 357 : 384 (1992) .
  • ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule.
  • Steinecke et al . EMBO J. 11:1525 (1992)
  • Perriman et al . Antisense Res . & Devel .
  • RNA molecules In a similar approach, fertility can be restored by the use of an expression vector containing a nucleotide sequence that encodes an antisense RNA.
  • the binding of antisense RNA molecules to target mRNA molecules results in hybridization arrest of translation. Paterson, et al . , Proc . Na tl . Acad. Sci . USA, 74:4370 (1987) .
  • a suitable antisense RNA molecule would have a sequence that is complementary to avidin mRNA.
  • the antisense RNA is under the control of an inducible promoter. Activation of this promoter then allows restoration of male fertility.
  • expression vectors can be constructed in which an expression vector encodes RNA transcripts capable of promoting RNase P-mediated cleavage of avidin mRNA molecules.
  • an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to avidin mRNA, which is subsequently cleaved by the cellular ribozyme.
  • the external guide sequence comprises a ten to fifteen nucleotide sequence complementary to avidin mRNA, and a 3 ' -NCCA nucleotide sequence, wherein N is preferably a purine.
  • the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 '-side of the base- paired region. Id.
  • transgenic male-sterile plants contain an expression vector that, in addition to a promoter sequence operably linked to an avidin gene, also contains a prokaryotic regulatory element.
  • Transgenic male- fertile plants are produced that express a prokaryotic polypeptide under the control of the promoter.
  • the prokaryotic polypeptide binds to the prokaryotic regulatory sequence and represses the expression of avidin.
  • LexA gene/LexA operator system can be used to regulate gene expression pursuant to the present invention. See U.S. patent No. 4,833,080 ("the first amino acid sequence of the LexA gene/LexA operator system")
  • the expression vector of the male-sterile plant would contain the LexA operator sequence, while the expression vector of the male-fertile plant would contain the coding sequences of the LexA repressor.
  • the LexA repressor would bind to the LexA operator sequence and inhibit transcription of the avidin gene.
  • LexA operator DNA molecules can be obtained, for example, by synthesizing DNA fragments that contain the well-known LexA operator sequence. See, for example, the '080 patent and Garriga et al . , Mol . Gen . Genet . 236 : 125 (1992) .
  • the LexA gene may be obtained by synthesizing a DNA molecule encoding the LexA repressor. Gene synthesis techniques are discussed above and LexA gene sequences are described, for example, by Garriga et al . , supra .
  • DNA molecules encoding the LexA repressor may be obtained from plasmid pRB500, American Type Culture Collection accession No. 67758.
  • Still another method for restoring fertility utilizes the high affinity of avidin for biotin, by spraying developing plants with a solution of biotin.
  • the biotin solution also comprises a minimum amount of an organic cosolvent such as DMSO to ensure complete solubility of the biotin.
  • Spraying is commenced during the meiotic phase of pollen development, and is repeated at regular intervals until pollen shed is observed. The intervals between spraying will vary between 1 and 7 days. In a preferred embodiment of the invention, spraying is repeated every 3 to 5 days.
  • prokaryotic regulatory systems such as the lac repressor/ lac operon system or the trp repressor/ trp operon system.
  • PHI5168 a vector carrying the avidin gene under control of the ubiquitin promoter and also containing a PINII terminator sequence, was used to form transgenic corn plants.
  • the structure of PHI5168 is shown in Figure 1.
  • PHI610 an expression vector carrying the Jbar gene under control of the double 35S promoter, and also carrying a PINII terminator sequence, was cotransformed along with the avidin construct, allowing the selection of transgenic plants by treatment of the transformation mixture with bialophos.
  • the structure of PHI610 is shown in Figure 2.
  • the two expression vectors were transformed into embryogenic suspension cultures be derived from type II embryogenic culture according to the method of Green, et al .
  • MS Murashige and Skoog
  • the cells were dispersed in 0.5 mL fresh culture medium to form a thin layer of cells.
  • the uncovered petri plate was placed in the sample chamber of a particle gun device manufactured by Biolistics Inc.,
  • Cultures of transformed plant cells containing the foreign genes were cultivated for 4-8 weeks in 56OR (medium) (N6-based medium with 1 mg/ml bialaphos) . This medium selects for cells that express the bar gene. Embryo formation was then induced in the embryogenic cultures and the cells germinated into plants. A two culture medium sequence was used to germinate somatic embryos observed on callus maintenance medium. Callus was transferred first to a culture medium (maturation medium) containing 5.0 mg/L indoleacetic acid (IAA) for 10 to 14 days while callus proliferation continued. Callus loading was at 50 mg per plate to optimize recovery per unit mass of material.
  • medium medium
  • IAA indoleacetic acid
  • Germinating somatic embryos were characterized by a green shoot elongating with a connecting root access. Somatic embryos were then transferred to medium in a culture tube
  • plants were about 7-10 cm tall, and were of sufficient size and vigor to be hardened to greenhouse conditions.
  • plants to be transferred to the growth chamber were removed from the sterile containers and solidified agar medium was rinsed off the roots.
  • the plantlets were placed in a commercial potting mix in a growth chamber with a misting device for maintaining the relative humidity near 100% without excessively wetting the plant roots. After 3-4 weeks in the misting chamber the plants were robust enough for transplantation into field conditions.
  • Plants in the field were analyzed by observing male sterility. Selected plants were then further analyzed for presence of the avidin gene by PCR and Southern blotting, and for expression of avidin by ELISA, by standard methods.
  • Soybean (Glycine max) seed of Pioneer variety 9341, is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Gas is produced by adding 3.5 ml hydrochloric acid (34 to 37% w/w) to 100 ml sodium hypochlorite (5.25% w/w) . Exposure is for 16 to 20 hours in a container approximately 1 cubic ft in volume. Surface sterilized seed is stored in petri dishes at room temperature.
  • Seed is germinated by plating an 1/10 strength agar solidified medium according to Gambourg (B5 basal medium with minimal organics, Sigma Chemical Catalog No. G5893, 0.32 gm/L sucrose; 0.2% weight/volume 2- (N-morpholino) ethanesulfonic acid (MES) , (3.0 mM) without plant growth regulators and culturing at 28° with a 16-hour day length and cool white fluorescent illumination of approximately 20 ⁇ Em ⁇ 2 S "1 . After 3 or 4 days, seed is prepared for co-cultivation. The seed coat is removed and the elongating radical is removed 3 to 4 mm below the cotyledons.
  • Gambourg B5 basal medium with minimal organics, Sigma Chemical Catalog No. G5893, 0.32 gm/L sucrose; 0.2% weight/volume 2- (N-morpholino) ethanesulfonic acid (MES) , (3.0 mM) without plant growth regulators and culturing at 28° with a
  • Inoculations are conducted in batches such that each plate of seed is treated with a newly resuspended pellet of Agrobacterium.
  • Bacterial pellets are resuspended individually in 20 ml inoculation medium, containing B5 salts (G5893) , 3.2 g/L; sucrose, 2.0% w/v; 6- benzylaminopurine (BAP) , 45 ⁇ m; indolebutyric-acid (IBA) , 0.5 ⁇ M; acetosyringone (AS) , 100 ⁇ M; buffered to pH 5.5 with MES 10 mM. Resuspension is achieved by vortexing.
  • the inoculum is then poured into a petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. This is accomplished by dividing seed in half by longitudinal section through the shoot apex, preserving the 2 whole cotyledons. The two halves of the shoot apex are then broken off their respective cotyledons by prying them away with a surgical blade. The cotyledonary node is then macerated with a surgical blade by repeated scoring along the axis of symmetry. Care is taken not to cut entirely through the explant to the axial side. Explants are prepared in roughly 5 minutes and then incubated for 30 minutes at room temperature with bacteria but without agitation.
  • the explants are transferred into plates of the same medium solidified with Gelrite (Merck & Company Inc.) , 0.2% w/v. Explants are embedded with adaxial side up and leveled with the surface of the medium and cultured at 22°C for 3 days under cool white fluorescent light, approximately 20 ⁇ Em "2 S "1 .
  • the explants are moved to liquid counterselection medium containing B5 salts (G5893), 3.2 g/1; sucrose, 2% w/v; BAP, 5 ⁇ M; IBA, 0.5 ⁇ M; vancomycin, 200 ⁇ g/ml; cefotaxime, 500 ⁇ g/ml, buffered to pH 5.7 with MES, 3 mM. Explants are washed in each petri dish with constant slow gyratory agitation at room temperature for 4 days. Counterselection medium is replaced 4 times.
  • B5 salts G5893
  • sucrose 2% w/v
  • BAP 5 ⁇ M
  • IBA 0.5 ⁇ M
  • vancomycin 200 ⁇ g/ml
  • cefotaxime 500 ⁇ g/ml
  • the explants are then picked to agarose-solidified selection medium containing B5 salts (G5893) , 3.2 g/1; sucrose, 2% w/v; BAP, 5.0 ⁇ M; IBA, 0.5 ⁇ M; kanamycin sulfate, 50 ⁇ g/ml; vancomycin, 100 ⁇ g/ml; cefotaxime, 30 ⁇ g/ml; timentin, 30 ⁇ g/ml, buffered to pH 5.7 with MES, 3mM.
  • Selection medium is solidified with Seakem Agarose, 0.3% w/v.
  • the explants are embedded in the medium, adaxial side down and cultured at 28° with a 16 hour day length in cool white fluorescent illumination of 60 to 80 ⁇ Em "2 S "1 .
  • explants are again washed with liquid medium on the gyratory shaker.
  • the wash is conducted overnight in counterselection medium containing kanamycin sulfate, 50 ⁇ g/ml.
  • explants are picked to agarose/solidified selection medium. They are embedded in the medium adaxial side down and cultured for another 2 week period. After 1 month on selection medium, transformed tissue is visible as green sectors of regenerating tissue against a background of bleached nonhealthy tissue.
  • Explants without green sectors are discarded and explants with green sectors are transferred to elongation medium containing B5 salts (G5893), 3.2 g/1; sucrose, 2% w/v; IBA, 3.3 ⁇ M; gibberellic acid, 1.7 ⁇ M; vancomycin, 100 ⁇ g/ml; cefotaxime, 30 ⁇ g/ml; and timentin, 30 ⁇ g/ml, buffered to pH 5.7 with MES, 3 mM.
  • Elongation medium is solidified with Gelrite, 0.2% w/v.
  • the green sectors are embedded adaxial side up and cultured as before. Culture is continued on this medium with transfers to fresh plates every two weeks.
  • Rooting medium consists of B5 salts (G5893) , 3.2 g/1; sucrose, 15 g/1; nicotinic acid, 20 ⁇ M; pyroglutamic acid (PGA) , 900 mg/L and IBA, 10 ⁇ M.
  • the rooting medium is buffered to pH 5.7 with MES, 3 mM and solidified with Gelrite at 0.2% w/v. After 10 days, the shoots are transferred to the same medium without IBA or PGA. Shoots are rooted and held in these tubes under the same environmental conditions as before.
  • An expression cassette encoding avidin is used to generate transgenic sunflower plants and seeds.
  • the DNA sequence coding for avidin is inserted into an expression cassette under control of the ubiquitin promoter.
  • This expression cassette is then subcloned into a binary vector such as PHI 5765 using the EcoRl site.
  • the binary vector is then transferred into an Agrobacterium tumefaciens helper strain.
  • Sunflower plants are transformed with Agrobacterium strain LBA4404 after microparticle bombardment as described by Bidney, et al . , Plant Mol . Bio. ; Vol. 18; p. 301; 1992. Briefly, seeds of Pioneer Sunflower Line SMF- 3 are dehulled and surface sterilized. The seeds are imbibed in the dark at 26°C for 18 hours on filter paper moistened with water.
  • the cotyledons and root radical are removed and meristem explants cultured on 374BGA medium (MS salts, Shephard vitamins, 40 mg/L adenine sulfate, 3% sucrose, 0.8% phytagar pH 5.6 plus 0.5 mg/L of BAP, 0.25 mg/L, IAA and 0.1 mg/L GA) . Twenty-four hours later, the primary leaves are removed to expose the apical meristem and the explants are placed with the apical dome facing upward in a 2 cm circle in the center of a 60 mm by 20 mm petri plate containing water agar.
  • the explants are bombarded twice with tungsten particles suspended in TE buffer or with particles associated with an expression plasmid containing the avidin gene.
  • the meristem explants are co-cultured on 374BGA medium in the light at 26°C for an additional 72 hours of co-culture.
  • Agrobacterium treated meristems are transferred following the 72 hour co-culture period to medium 374 (374BGA with 1% sucrose and no BAP, IAA or GA 3 ) and supplemented with 250 ⁇ g/ml cefotaxime.
  • the plantlets are allowed to develop for an additional 2 weeks under 16 hour day and 26°C incubation conditions to green or bleach. Plantlets are transferred to medium containing kanamycin and allowed to grow.
  • the presence of avidin in the plants is confirmed and quantitated as described in Example 2. The presence of male sterility is found to correlate with expression of avidin by the plants.
  • NAME BENT, Stephen A.
  • GATTGATCTC TGGTATTTAC CACTCTTTCC TTCCTTCCTT CCTTCAATTC TAAATACCAC 120

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Abstract

Il est possible de produire des plantes mâles stériles en accroissant la concentration endogène d'avidine dans les tissus desdites plantes. On parvient à ce résultat en produisant des plantes transgéniques contenant un vecteur d'expression dans lequel un promoteur est lié fonctionnellement à une séquence d'ADN codant l'avidine. L'invention concerne également des procédés permettant de rétablir la fécondité mâle.
PCT/US1996/008583 1995-06-07 1996-06-06 Induction de la sterilite male dans des plantes par expression de niveaux eleves d'avidine WO1996040949A1 (fr)

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RO97-02278A RO120270B1 (ro) 1995-06-07 1996-06-06 Inducerea sterilităţii masculine la plante, prin expresia la nivele ridicate a avidinei
PL96323745A PL323745A1 (en) 1995-06-07 1996-06-06 Method of causing male sterility with plants by high avidin expression
EP96919051A EP0832261A2 (fr) 1995-06-07 1996-06-06 Induction de la sterilite male dans des plantes par expression de niveaux eleves d'avidine
AU61493/96A AU708618B2 (en) 1995-06-07 1996-06-06 Induction of male sterility in plants by expression of high levels of avidin
CA002223460A CA2223460C (fr) 1995-06-07 1996-06-06 Induction de la sterilite male dans des plantes par expression de niveaux eleves d'avidine
JP9501127A JPH11512922A (ja) 1995-06-07 1996-06-06 高レベルのアビジン発現による植物の雄性不稔の誘発
BR9609069A BR9609069A (pt) 1995-06-07 1996-06-06 Indução de esterelidade masculina em plantas por expressão de níveis elevados de avidina

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WO1997017455A3 (fr) * 1995-11-06 1997-09-12 John Howard Production commerciale d'avidine dans des plantes
WO1998039461A1 (fr) * 1997-03-20 1998-09-11 Prodigene Inc. Methode de production et d'extraction commerciales de proteines a partir de graines
WO1999004023A1 (fr) * 1997-07-14 1999-01-28 Pioneer Hi-Bred International, Inc. Induction de la sterilite male dans des plantes par l'expression de niveaux eleves d'avidine
WO1999018772A1 (fr) * 1997-10-09 1999-04-22 Midwest Oilseeds, Inc. Gene de sterilite male mutant de soja
WO2000007427A3 (fr) * 1998-08-03 2000-09-21 Agricultural Res Org Procede de degenerescence selective et eventuellement reversible d'un tissu vegetal somatique
WO2002012305A1 (fr) * 2000-06-19 2002-02-14 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, avidine 9, et polynucleotide codant ce polypeptide
US6504085B1 (en) 1997-03-07 2003-01-07 Prodigene, Inc. Methods of commercial production and extraction of protein from seed
WO2002071834A3 (fr) * 2001-03-09 2003-03-27 Pioneer Hi Bred Int Composes de liaison de la biotine pour induire la sterilite chez les plantes
EP1347678A2 (fr) * 2001-01-04 2003-10-01 Yeda Research And Development Co. Ltd. Procede permettant de maintenir une ligne parentale genique male-femelle sterile pour la production de ble hybride
US6972350B1 (en) 1998-07-15 2005-12-06 The Horticulture And Food Research Institute Of New Zealand Pest-resistant plants comprising a construct encoding a vacuole targeting sequence and avidin or streptavidin
US7071384B2 (en) 1997-03-07 2006-07-04 Genencor International, Inc. Methods for commercial production of heterologous laccase in plant tissue and extraction of the laccase from plant seed
WO2007116394A3 (fr) * 2006-04-11 2007-12-06 Israel State Promoteur de gènes végétaux et son utilisation
EP1865059A1 (fr) 2001-03-12 2007-12-12 Japan Tobacco, Inc. Nouvelle protéine, son codage génétique et son procédé d'utilisation
WO2010046423A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides sulfonylurées sur des plantes cultivées
WO2010046422A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides de type auxine sur des plantes cultivées
WO2014053395A1 (fr) 2012-10-01 2014-04-10 Basf Se Utilisation de composés de n-thio-anthranilamide sur des plantes cultivées
WO2014079820A1 (fr) 2012-11-22 2014-05-30 Basf Se Utilisation de composés d'anthranilamides pour réduire les infections virales véhiculées par les insectes
WO2015161744A1 (fr) * 2014-04-22 2015-10-29 未名兴旺系统作物设计前沿实验室（北京）有限公司 Identification et utilisation du promoteur ptaasg048 spécifiquement exprimé par l'anthère d'une plante
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WO2016091674A1 (fr) 2014-12-12 2016-06-16 Basf Se Utilisation de cyclaniliprole sur des plantes cultivées
WO2016162371A1 (fr) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Utilisation d'un composé de carboxamide insecticide contre les nuisibles sur des plantes cultivées
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US5962769A (en) * 1995-06-07 1999-10-05 Pioneer Hi-Bred International, Inc. Induction of male sterility in plants by expression of high levels of avidin
WO1996040925A3 (fr) * 1995-06-07 1997-05-01 Pioneer Hi Bred Int Elements regulateurs conferant une specificite au tapetum
WO1997017455A3 (fr) * 1995-11-06 1997-09-12 John Howard Production commerciale d'avidine dans des plantes
US5767379A (en) * 1995-11-06 1998-06-16 John Howard Commercial production of avidin in plants
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CN1192784A (zh) 1998-09-09
BR9609069A (pt) 1999-04-06
AU6149396A (en) 1996-12-30
PL323745A1 (en) 1998-04-14
JPH11512922A (ja) 1999-11-09
RO120270B1 (ro) 2005-11-30
AU708618B2 (en) 1999-08-05
AR002352A1 (es) 1998-03-11
KR19990022728A (ko) 1999-03-25
CA2223460A1 (fr) 1996-12-19
HUP9900885A2 (hu) 1999-07-28
CA2223460C (fr) 2000-02-15
EP0832261A2 (fr) 1998-04-01
HUP9900885A3 (en) 2001-02-28

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