WO2021076346A1 - Événement de maïs dp-202216-6 et empilement dp-023211-2 - Google Patents

Événement de maïs dp-202216-6 et empilement dp-023211-2 Download PDF

Info

Publication number
WO2021076346A1
WO2021076346A1 PCT/US2020/053947 US2020053947W WO2021076346A1 WO 2021076346 A1 WO2021076346 A1 WO 2021076346A1 US 2020053947 W US2020053947 W US 2020053947W WO 2021076346 A1 WO2021076346 A1 WO 2021076346A1
Authority
WO
WIPO (PCT)
Prior art keywords
event
plant
sequence
dna
maize
Prior art date
Application number
PCT/US2020/053947
Other languages
English (en)
Inventor
Jeffrey Habben
Kristen Denise RINEHART KREBS
Benjamin P Weers
Original Assignee
Pioneer Hi-Bred International, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pioneer Hi-Bred International, Inc. filed Critical Pioneer Hi-Bred International, Inc.
Publication of WO2021076346A1 publication Critical patent/WO2021076346A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4684—Zea mays [maize]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Definitions

  • Embodiments disclosed herein relate to the field of plant molecular biology, including to DNA constructs for conferring insect resistance to a plant and increasing yield of a plant.
  • Embodiments disclosed herein also include insect resistant corn plant containing event DP-023211-2 and maize event DP-202216-6 and methods and compositions thereof.
  • Corn is an agriculturally important crop and serves as a food and feed source for animal, human, and industrial uses. Increased grain yield may be achieved in maize plants by a variety of ways, including expression of a transgene to increase grain yield in addition to improved breeding. Performance of a transgene in a plant including the agronomic parameters, may be impacted by a variety of factors such as the use of expression elements including promoter/regulatory elements, the genomic location of the insert sequence, copy number of the inserted transgene and genetic (germplasm) and environmental factors such as soil, temperature, light, insects, and moisture.
  • the identification of constructs, testing of orthologs and transformation events that result in increased insect resistance and increased grain yield of a maize plant at a commercially relevant level in the field are the result of a substantial and significant developmental effort towards product advancement.
  • the embodiments relate to the insect resistant corn ( Zea mays) plant event DP- 023211-2, also referred to as “maize line DP-023211-2,” “maize event DP-023211-2,” and “DP-023211-2 maize,” to the DNA plant expression construct of corn plant event DP- 023211-2, and to methods and compositions for the detection of the transgene construct, flanking, and insertion (the target locus) regions in corn plant event DP-023211-2 and progeny thereof.
  • a com seed may also include Event DP-202216-6, wherein a representative sample of corn event DP-202216-6 seed of has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-124653. In some embodiments, a corn plant, or part thereof, grown from the seed of PTA-124653 is described herein. As described herein, Event DP-202216-6 is also referred to as “Event 16”, “El 6” “event 16” or “Event 16-6” and they all refer to the same maize event DP-202216-6.
  • the protein encoded by the Maize MADS box ZmM28 gene in the plasmid PHP40099 or the Event DP-202216-6 is also referred to as AG099 protein and the corresponding DNA sequence as AG099 gene or AG099 DNA.
  • a maize plant stably transformed with a recombinant polynucleotide sequence encoding a polypeptide comprising an amino sequence that is at least 90%, 93% 95%, 97%, 98% or 99% identical to SEQ ID NO: 1 of PCT/US2019/027599 (herein incorporated by reference), wherein the maize plant exhibits increased grain yield compared to a control maize plant not containing the recombinant polynucleotide.
  • the recombinant polynucleotide is operably linked to a weak heterologous constitutive regulatory element.
  • the grain yield is at least about three bushels/acre when compared to the control maize plant, wherein the maize plant and the control maize plant are grown in a field under normal crop growing conditions. In some embodiments, the grain yield in the field range from about 2 to about 8 bu/acre when compared to the control population of maize plants grown in a population density of about 20,000 to about 50,000 plants per acre.
  • the weak heterologous constitutive regulatory element is a maize GOS2 promoter.
  • the amino acid sequence is at least 95% identical to SEQ ID NO: 1 of PCT/US2019/027599 and the maize plant comprises a polynucleotide encoding a polypeptide that provides herbicide tolerance and a polynucleotide that encodes a polypeptide or an RNA sequence that provides resistance to one or more insect pests.
  • Maize seed produced from the maize plant described herein exhibit yield improvement characteristics.
  • the regulatory element comprises a heterologous intron element.
  • compositions and methods relate to methods for producing and selecting an insect resistant monocot crop plant.
  • Compositions include a DNA construct that when expressed in plant cells and plants confers resistance to insects.
  • a DNA construct, capable of introduction into and replication in a host cell is provided that when expressed in plant cells and plants confers insect resistance to the plant cells and plants.
  • the DP-023211-2 event includes the DvSSJl (SEQ ID NO: 6 of PCT/US 19/28485, herein incorporated by reference) and IPD072 (polynucleotide SEQ ID NO: 4 of PCT/US 19/28485 and amino acid SEQ ID NO: 5 of PCT/US 19/28485, each incorporated herein by reference) cassettes, which confer resistance to certain Coleopteran plant pests.
  • the insect control components have demonstrated efficacy against western corn rootworm (WCR), northern corn rootworm (NCR), and southern corn rootworm (SCR).
  • DNA molecules are provided that comprise at least one junction sequence of each of DP-023211-2 and DP-202216-6; wherein a junction sequence spans the junction located between heterologous DNA inserted into the genome and the DNA from the maize cell flanking the insertion site, and may be diagnostic for the DP-023211-2 event and DP- 202216-6 event.
  • methods of producing a com plant comprising a stack of DP-023211-2 event and DP-202216-6 event comprises the steps of: (a) sexually crossing a first parental com line comprising the expression cassettes disclosed herein, and a second parental corn line that lacks such expression cassettes, thereby producing a plurality of progeny plants; and (b) selecting a progeny plant that is insect resistant.
  • Such methods may optionally comprise the further step of back-crossing the progeny plant to the second parental com line to produce a true-breeding corn plant that is insect resistant.
  • a further embodiment relates to the stack of DP-023211-2 and DP-202216-6 event maize plant or plant parts, including, but not limited to, pollen, ovules, vegetative cells, the nuclei of pollen cells, and the nuclei of egg cells of the corn plant and the progeny derived thereof.
  • compositions of this disclosure include seed deposited as ATCC Patent Deposit No. PTA-124722 and plants, plant cells, and seed derived therefrom. Applicant(s) deposited at least 2500 seeds of maize event DP-023211-2 (Patent Deposit No. PTA-124722) with the American Type Culture Collection (ATCC), Manassas, VA 20110-2209 USA, on January 18, 2018. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The seeds deposited with the ATCC on January 18, 2018 were taken from the deposit maintained by Pioneer Hi-Bred International, Inc., 7250 NW 62 nd Avenue, Johnston, Iowa 50131-1000.
  • Applicant(s) have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicant(s) do not waive any infringement of their rights granted under this patent or rights applicable to event DP-023211-2 under the Plant Variety Protection Act (7 USC 2321 et seq.). Unauthorized seed multiplication is prohibited. The seed may be regulated.
  • compositions of this disclosure also include a representative sample of seeds which was deposited as Patent Deposit No. PTA-124653 and plants, plant cells, and seed derived therefrom.
  • Applicant(s) have made a deposit of at least 2500 seeds of maize event DP- 202216-6 (Patent Deposit No. PTA-124653) with the American Type Culture Collection (ATCC), Manassas, VA 20110-2209 USA, on January 12, 2018. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
  • corn means Zea mays or maize and includes all plant varieties that can be bred with corn, including wild maize species.
  • insect resistant and “impacting insect pests” refers to effecting changes in insect feeding, growth, and/or behavior at any stage of development, including but not limited to: killing the insect; retarding growth; reducing reproductive capability; inhibiting feeding; and the like.
  • the terms “pesticidal activity” and “insecticidal activity” are used synonymously to refer to activity of an organism or a substance (such as, for example, a protein) that can be measured by numerous parameters including, but not limited to, pest mortality, pest weight loss, pest attraction, pest repellency, and other behavioral and physical changes of a pest after feeding on and/or exposure to the organism or substance for an appropriate length of time.
  • pesticidal proteins are proteins that display pesticidal activity by themselves or in combination with other proteins.
  • insert DNA refers to the heterologous DNA within the expression cassettes used to transform the plant material while “flanking DNA” can exist of either genomic DNA naturally present in an organism such as a plant, or foreign (heterologous) DNA introduced via the transformation process which is extraneous to the original insert DNA molecule, e.g. fragments associated with the transformation event.
  • a “flanking region” or “flanking sequence” as used herein refers to a sequence of at least 20 bp (in some narrower embodiments, at least 50 bp, and up to at least 5000 bp), which is located either immediately upstream of and contiguous with and/or immediately downstream of and contiguous with the original non-native insert DNA molecule.
  • Transformation procedures of the foreign DNA may result in transformants containing different flanking regions characteristic and unique for each transformant.
  • flanking regions When recombinant DNA is introduced into a plant through traditional crossing, its flanking regions will generally not be changed. It may be possible for single nucleotide changes to occur in the flanking regions through generations of plant breeding and traditional crossing.
  • Transformants will also contain unique junctions between a piece of heterologous insert DNA and genomic DNA, or two (2) pieces of genomic DNA, or two (2) pieces of heterologous DNA.
  • a "junction" is a point where two (2) specific DNA fragments join. For example, a junction exists where insert DNA joins flanking DNA.
  • a junction point also exists in a transformed organism where two (2) DNA fragments join together in a manner that is modified from that found in the native organism.
  • Junction DNA refers to DNA that comprises a junction point.
  • Junction sequences set forth in this disclosure include a junction point located between the maize genomic DNA and the 5’ end of the insert, which range from at least -5 to +5 nucleotides of the junction point, from at least -10 to +10 nucleotides of the junction point, from at least -15 to +15 nucleotides of the junction point, and from at least -20 to +20 nucleotides of the junction point; and a junction point located between the 3’ end of the insert and maize genomic DNA, which range from at least -5 to +5 nucleotides of the junction point, from at least -10 to +10 nucleotides of the junction point, from at least -15 to +15 nucleotides of the junction point, and from at least -20 to +20 nucleotides of the junction point.
  • Junction sequences also include a junction point located between the target locus and the 5’ end of the insert.
  • Com plant containing event DP-202216-6 and event DP-023211-2 may be bred by first sexually crossing a first parental corn plant consisting of a corn plant grown from event DP-202216-6 and event DP-023211-2 com plant and progeny thereof derived from transformation with the expression cassettes of the embodiments that increase yield when compared to a control plant, and a second parental corn plant that does not have such constructs, thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that demonstrates yield increase; and selfing the first progeny plant, thereby producing a plurality of second progeny plants; and then selecting from the second progeny plants plant with yield increase.
  • com plants containing event DP-202216-6 and event DP- 023211-2 events may be crossed with com plants containing other com events or combination thereof and the resulting properties of the progeny plants are evaluated.
  • corn plants containing event DP-202216-6 and event DP-023211-2 may be crossed or combined with com plants including one or more combinations, of the following: MON810; DAS-59122-7; MIR604; MON89034; MON863; MON87411; MON87403; MON87427; MON-00603-6 (NK603); MON-87460-4; MON-88017-3; LY038; TC1507; 5307; DAS-06275-8; BT176; BT11; MIR162; GA21; MZDT09Y; SYN- 05307-1; DP-004114-3; MON-95379; and DAS-40278-9.
  • Some embodiments comprise a seed produced from a corn plant wherein a progeny from the seed exhibits increased yield and reduced stature when compared to the control plant.
  • a corn plant, seed, cell or part thereof comprising event DP- 202216-6 and exhibiting reduced stature, wherein a representative sample of seed of said corn event has been deposited with American Type Culture Collection (ATCC) with Accession No. PTA-124653.
  • ATCC American Type Culture Collection
  • heterologous in reference to a nucleic acid sequence is a nucleic acid sequence that originates from a different non-sexually compatible species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous nucleotide sequence can be from a species different from that from which the nucleotide sequence was derived, or, if from the same species, the promoter is not naturally found operably linked to the nucleotide sequence.
  • a heterologous protein may originate from a foreign species, or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • regulatory element refers to a nucleic acid molecule having gene regulatory activity, i.e. one that has the ability to affect the transcriptional and/or translational expression pattern of an operably linked transcribable polynucleotide.
  • gene regulatory activity thus refers to the ability to affect the expression of an operably linked transcribable polynucleotide molecule by affecting the transcription and/or translation of that operably linked transcribable polynucleotide molecule.
  • Gene regulatory activity may be positive and/or negative and the effect may be characterized by its temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and/or chemically responsive qualities as well as by quantitative or qualitative indications.
  • Promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence comprises proximal and more distal upstream elements, the latter elements are often referred to as enhancers.
  • an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments.
  • the “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
  • the translation leader sequence may affect numerous parameters including, processing of the primary transcript to mRNA, mRNA stability and/or translation efficiency.
  • the “3’ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3’ end of the mRNA precursor.
  • a DNA construct is an assembly of DNA molecules linked together that provide one or more expression cassettes.
  • the DNA construct may be a plasmid that is enabled for self replication in a bacterial cell and contains various endonuclease enzyme restriction sites that are useful for introducing DNA molecules that provide functional genetic elements, i.e., promoters, introns, leaders, coding sequences, 3’ termination regions, among others; or a DNA construct may be a linear assembly of DNA molecules, such as an expression cassette.
  • the expression cassette contained within a DNA construct comprises the necessary genetic elements to provide transcription of a messenger RNA.
  • the expression cassette can be designed to express in prokaryotic cells or eukaryotic cells. Expression cassettes of the embodiments are designed to express in plant cells.
  • the DNA molecules disclosed herein are provided in expression cassettes for expression in an organism of interest.
  • the cassette includes 5’ and 3’ regulatory sequences operably linked to a coding sequence.
  • “Operably linked” means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. Operably linked is intended to indicate a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • the cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes or multiple DNA constructs.
  • the expression cassette may include in the 5’ to 3’ direction of transcription: a transcriptional and translational initiation region, a coding region, and a transcriptional and translational termination region functional in the organism serving as a host.
  • the transcriptional initiation region e.g ., the promoter
  • the expression cassettes may additionally contain 5’ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation.
  • transgenic generally includes any cell, cell line, callus, tissue, plant part, or plant, the genotype of which has been altered by the presence of a heterologous nucleic acid including those initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic event and retains such heterologous nucleic acids.
  • a transgenic “event” is produced by transformation of plant cells with a heterologous DNA construct s), including a nucleic acid expression cassette that comprises a transgene of interest, the regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location.
  • An event is characterized phenotypically by the expression of the transgene.
  • an event is part of the genetic makeup of a plant.
  • the term “event” also refers to progeny produced by a sexual outcross between the transformant and another variety, wherein the progeny includes the heterologous DNA.
  • the inserted DNA and the linked flanking genomic DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location.
  • a progeny plant may contain sequence changes to the insert arising as a result of conventional breeding techniques.
  • the term “event” also refers to DNA from the original transformant comprising the inserted DNA and flanking sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.
  • the term "plant” includes reference to whole plants, parts of plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same.
  • parts of transgenic plants comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, leaves, and roots originating in transgenic plants or their progeny previously transformed with a DNA molecule disclosed herein, and therefore consisting at least in part of transgenic cells.
  • plant cell includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • the class of plants that may be used is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
  • Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host plants containing the transformed nucleic acid fragments are referred to as “transgenic” plants.
  • progeny in the context of a stack of event DP-023211-2 and event DP-202216-6, denotes an offspring of any generation of a parent plant which comprises corn event DP-023211-2 and com event DP-202216-6.
  • Isolated polynucleotides disclosed herein may be incorporated into recombinant constructs, typically DNA constructs, which are capable of introduction into and replication in a host cell.
  • a construct may be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • a number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels el al., (1985; Supp.
  • plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5’ and 3’ regulatory sequences and a dominant selectable marker.
  • Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression
  • Identity to the sequence of the present disclosure may be a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence exemplified or described herein.
  • Hybridization and hybridization conditions as provided herein can also be used to define such plants and polynucleotide sequences of the subject disclosure. A sequence comprising the flanking sequences plus the full insert sequence can be confirmed with reference to the deposited seed.
  • two different transgenic plants can also be crossed to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation.
  • the disclosed compositions can be introduced into the genome of a plant using genome editing technologies, or previously introduced polynucleotides in the genome of a plant may be edited using genome editing technologies.
  • the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like.
  • the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion.
  • the desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest.
  • Existing traits of interest could be either an endogenous trait or a previously introduced trait.
  • genome editing technologies may be used to alter or modify the introduced polynucleotide sequence.
  • Site specific modifications that can be introduced into the disclosed polynucleotide compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides (e.g. US Publication 2013/0019349), or through the use of double- stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like.
  • Such technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion or substitution of nucleotides within the introduced polynucleotide.
  • double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, additional expression elements, such as enhancer and promoter sequences.
  • genome editing technologies may be used to position additional insecticidally-active proteins in close proximity to a disclosed polynucleotide compositions disclosed herein within the genome of a plant, to generate molecular stacks of insecticidally-active proteins.
  • altered target site refers to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence.
  • alterations include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).
  • a “probe” is an isolated nucleic acid to which is attached a conventional, synthetic detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid, for example, to a strand of isolated DNA from a corn event whether from a com plant or from a sample that includes DNA from the event. Probes may include not only deoxyribonucleic or ribonucleic acids but also polyamides and other modified nucleotides that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
  • Primer pairs are isolated nucleic acids that anneal to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase.
  • Primer pairs refer to their use for amplification of a target nucleic acid sequence, e.g., by PCR or other conventional nucleic-acid amplification methods.
  • PCR or “polymerase chain reaction” is a technique used for the amplification of specific DNA segments (see, U.S. Patent Nos. 4,683,195 and 4,800,159; herein incorporated by reference).
  • Probes and primers are of sufficient nucleotide length to bind to the target DNA sequence specifically in the hybridization conditions or reaction conditions determined by the operator. This length may be of any length that is of sufficient length to be useful in a detection method of choice. Generally, 11 nucleotides or more in length, 18 nucleotides or more, and 22 nucleotides or more, are used. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Probes and primers according to embodiments may have complete DNA sequence similarity of contiguous nucleotides with the target sequence, although probes differing from the target DNA sequence and that retain the ability to hybridize to target DNA sequences may be designed by conventional methods. Probes can be used as primers, but are generally designed to bind to the target DNA or RNA and are not used in an amplification process.
  • Specific primers may be used to amplify an integration fragment to produce an amplicon that can be used as a “specific probe” for identifying an event in biological samples.
  • the probe is hybridized with the nucleic acids of a biological sample under conditions which allow for the binding of the probe to the sample, this binding can be detected and thus allow for an indication of the presence of an event in the biological sample.
  • the specific probe is a sequence which, under appropriate conditions, hybridizes specifically to a region within the 5’ or 3’ flanking region of the event and also comprises a part of the foreign DNA contiguous therewith.
  • the specific probe may comprise a sequence of at least 80%, from 80 and 85%, from 85 and 90%, from 90 and 95%, and from 95 and 100% identical (or complementary) to a specific region of the event.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as the PCR primer analysis tool in Vector NTI version 6 (Informax Inc., Bethesda MD); PrimerSelect (DNASTAR Inc., Madison, WI); and Primer (Version 0.5 ® , 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, the sequence can be visually scanned and primers manually identified using guidelines known to one of skill in the art.
  • kits refers to a set of reagents, and optionally instructions, for the purpose of performing method embodiments of the disclosure, more particularly, the identification of an event in biological samples.
  • a kit may be used, and its components can be specifically adjusted, for purposes of quality control (e.g. purity of seed lots), detection of an event in plant material, or material comprising or derived from plant material, such as but not limited to food or feed products.
  • Plant material as used herein refers to material which is obtained or derived from a plant.
  • Primers and probes based on the flanking DNA and insert sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed sequences by conventional methods, e.g., by re-cloning and sequencing such sequences.
  • the nucleic acid probes and primers hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method may be used to identify the presence of DNA from a transgenic event in a sample.
  • a nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity or minimal complementarity.
  • molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other.
  • Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low- stringency” conditions.
  • the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions.
  • Conventional stringency conditions are described by Sambrook et al.
  • nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.
  • T m The thermal melting point
  • T m 81.5 °C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • M is the molarity of monovalent cations
  • %GC is the percentage of guanosine and cytosine nucleotides in the DNA
  • % form is the percentage of formamide in the hybridization solution
  • L is the length of the hybrid in base pairs.
  • Tm is reduced by about 1 °C for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10 °C.
  • stringent conditions are selected to be about 5 °C lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH.
  • other stringency conditions can be applied, including severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 °C lower than the T m ; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 °C lower than the T m ; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20 °C lower than the T m.
  • a complementary sequence has the same length as the nucleic acid molecule to which it hybridizes. In some embodiments, the complementary sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, the complementary sequence is 1%, 2%, 3%, 4%, or 5% longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, a complementary sequence is complementary on a nucleotide-for-nucleotide basis, meaning that there are no mismatched nucleotides (each A pairs with a T and each G pairs with a C).
  • a complementary sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or less mismatches. In some embodiments, the complementary sequence comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or less mismatches.
  • stringent conditions permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild- type sequence (or its complement) would bind and optionally to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
  • amplified DNA refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template.
  • DNA extracted from a tissue sample of a corn plant may be subjected to a nucleic acid amplification method using a DNA primer pair that includes a first primer derived from flanking sequence adjacent to the insertion site of inserted heterologous DNA, and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the event DNA.
  • the second primer may be derived from the flanking sequence.
  • the amplicon is of a length and has a sequence that is also diagnostic for the event.
  • the amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol.
  • primer pairs can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert nucleotide sequence of the PHP74643 expression construct as well as a portion of the sequence flanking the transgenic insert.
  • a member of a primer pair derived from the flanking sequence may be located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to the limits of the amplification reaction.
  • the use of the term “amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
  • Nucleic acid amplification can be accomplished by any of the various nucleic acid amplification methods known in the art, including PCR.
  • a variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in Innis et al, (1990) supra.
  • PCR amplification methods have been developed to amplify up to 22 Kb of genomic DNA and up to 42 Kb of bacteriophage DNA (Cheng et al, Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as other methods known in the art of DNA amplification may be used in the practice of the embodiments of the present disclosure. It is understood that a number of parameters in a specific PCR protocol may need to be adjusted to specific laboratory conditions and may be slightly modified and yet allow for the collection of similar results. These adjustments will be apparent to a person skilled in the art.
  • the amplicon produced by these methods may be detected by a plurality of techniques, including, but not limited to, Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide is designed which overlaps both the adjacent flanking DNA sequence and the inserted DNA sequence.
  • the oligonucleotide is immobilized in wells of a microwell plate.
  • a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled ddNTPs specific for the expected next base.
  • Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking sequence due to successful amplification, hybridization, and single base extension.
  • Another detection method is the pyrosequencing technique as described by Winge (2000) Innov. Pharma. Tech. 00: 18-24.
  • an oligonucleotide is designed that overlaps the adjacent DNA and insert DNA junction.
  • the oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (for example, one primer in the inserted sequence and one in the flanking sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5’ phosphosulfate and luciferin.
  • dNTPs are added individually and the incorporation results in a light signal which is measured.
  • a light signal indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single or multi-base extension.
  • Fluorescence polarization as described by Chen et al ., (1999) Genome Res. 9:492- 498 is also a method that can be used to detect an amplicon. Using this method an oligonucleotide is designed which overlaps the flanking and inserted DNA junction. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (for example, one primer in the inserted DNA and one in the flanking DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP.
  • Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single base extension.
  • Quantitative PCR is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by commercially available manufacturers. Briefly, in one such qPCR method, a FRET oligonucleotide probe is designed which overlaps the flanking and insert DNA junction.
  • the FRET probe and PCR primers are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
  • Molecular beacons have been described for use in sequence detection as described in Tyangi etal. (1996) Nature Biotech. 14:303-308. Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity.
  • the FRET probe and PCR primers (for example, one primer in the insert DNA sequence and one in the flanking sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.
  • insects include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.
  • larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae, and Curculionidae including, but not limited to: Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (Diaprepes root weevil); Hypera punctata Fabricius (clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevil); M.
  • Anthonomus grandis Boheman boll weevil
  • Cylindrocopturus adspersus LeConte unsunflower stem weevil
  • Diaprepes abbreviatus Linnaeus Diaprepes root weevil
  • Hypera punctata Fabricius
  • hemipterus sericeus Olivier (silky cane weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug); S.
  • livis Vaurie sucgarcane weevil
  • Rhabdoscelus obscurus Boisduval New Guinea sugarcane weevil
  • flea beetles cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae including, but not limited to: Chaetocnema ectypa Horn (desert corn flea beetle); C.
  • latifrons LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp.
  • M communis Gyllenhal (wireworm); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae such as, but not limited to, Migdolus jryanus Westwood (longhorn beetle); and beetles from the Buprestidae family including, but not limited to, Aphanisticus cochinchinae seminulum Obenberger (leaf mining buprestid beetle).
  • the stack maize event may further comprise a stack of additional traits.
  • Plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene and co- transformation of genes into a single plant cell.
  • the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid).
  • the stack maize event disclosed herein alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, insect resistance, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like).
  • additional input traits e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, insect resistance, and the like
  • output traits e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like.
  • Genes encoding insectidal proteins may also be stacked including but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et ah, (2010) J. Agric.
  • Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386
  • Examples of d-endotoxins also include but are not limited to Cryl A proteins of US Patent Numbers 5,880,275 and 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of a-helix 1 and/or a-helix 2 variants of Cry proteins such as Cryl A) of US Patent Numbers 8,304,604 and 8.304,605, CrylB of US Patent Application Serial Number 10/525,318; CrylC of US Patent Number 6,033,874; CrylF of US Patent Numbers 5,188,960,
  • Cry proteins are well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature” (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the "www" prefix).
  • the insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16).
  • Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry -transgenic plants including but not limited to Cryl Ac, CrylAc+Cry2Ab, CrylAb, CrylA.105, Cry IF, CrylFa2, CrylF+CrylAc, Cry2Ab, Cry3A, mCry3A, Cry3Bbl, Cry34Abl, Cry35Abl, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C.
  • More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & CrylFa (US2012/0317682), CrylBE & CrylF (US2012/0311746), Cry 1CA & Cryl AB (US2012/0311745), Cry IF & CryCa (US2012/0317681), Cry IDA & CrylBE (US2012/0331590), CrylDA & CrylFa (US2012/0331589), CrylAB & CrylBE (US2012/0324606), and CrylFa & Cry2Aa, Cryll or CrylE (US2012/0324605).
  • Vip3Ab & CrylFa US2012/0317682
  • CrylBE & CrylF US2012/0311746
  • Cry 1CA & Cryl AB US2012/0311745
  • Cry IF & CryCa US2012/0317681
  • Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the like.
  • VIP vegetable insecticidal proteins
  • Pesticidal proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can be accessed on the world wide web using the "www" prefix).
  • Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491,698 and 8,084,418).
  • Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism.
  • TC protein from Photorhabdus, Xenorhabdus or Paenibacillus, for example
  • TC protein TC protein “potentiators” derived from a source organism of a different genus.
  • TC protein “potentiators” derived from a source organism of a different genus.
  • Class C proteins are TccC, XptClXb and XptBIWi.
  • Pesticidal proteins also include spider, snake and scorpion venom proteins.
  • spider venom peptides include but are not limited to ly cotoxin- 1 peptides and mutants thereof (US Patent Number 8,334,366).
  • the maize event may be stacked with one or more additional Bt insecticidal toxins, including, but not limited to, a Cry3B toxin disclosed in US Patent Numbers 8,101,826, 6,551,962, 6,586,365, 6,593,273, and PCT Publication WO 2000/011185; a mCry3B toxin disclosed in US Patent Numbers 8,269,069, and 8,513,492; a mCry3 A toxin disclosed in US Patent Numbers 8,269,069, 7,276,583 and 8,759,620; or a Cry34/35 toxin disclosed in US Patent Numbers 7,309,785, 7,524,810, 7,985,893,
  • the stacked maize event may be stacked with one or more additional transgenic events containing these Bt insecticidal toxins and other Coleopteran active Bt insecticidal traits for example, event MON863 disclosed in US Patent Number 7,705,216; event MIR604 disclosed in US Patent Number 8,884,102; event 5307 disclosed in US Patent Number 9,133,474; event DAS-59122 disclosed in US Patent Number 7,875,429; event DP-4114 disclosed in US Patent Number 8,575,434; event MON 87411 disclosed in US Patent Number 9,441,240; and event MON88017 disclosed in US Patent Number 8,686,230 all of which are incorporated herein by reference.
  • event MON863 disclosed in US Patent Number 7,705,216
  • event MIR604 disclosed in US Patent Number 8,884,102
  • event DAS-59122 disclosed in US Patent Number 7,875,429
  • event DP-4114 disclosed in US Patent Number 8,575,434
  • the stacked maize event may further be stacked with MON87427; MON- 00603-6 (NK603); MON-87460-4; LY038; DAS-06275-8; BT176; BT11; MIR162; GA21; MZDT09Y; SYN-05307-1; MON-95379; and DAS-40278-9.
  • a com plant may be treated with a seed treatment.
  • the seed treatment may be a fungicide, an insecticide, or a herbicide.
  • a series of grain yield trials were conducted from Years 1 through 4 in elite corn hybrids across multiple testing locations in order to assess the yield in elite maize hybrids having AG099 events.
  • about eighty-six locations containing approximately thirty unique hybrids with maturities ranging from 105-112 days were used to evaluate the performance of AG099 events relative to a wild type control.
  • Testing sites were established across locations such as Iowa, Illinois, Missouri, Kansas, Texas, California, Wisconsin, South Dakota and Minnesota. Locations were managed to achieve various yield levels ranging from highly drought stressed 70 bu/acre to optimal growing conditions 250 bu/acre.
  • Soil types consisted of a variety of high sand, sandy loam, silty loam, loam and some clay.
  • Table 1 Comparison of two AG099 Events in multiple hybrid background to Wild- Type control averaged across multiple locations in Year 4 of the multi-year trial.
  • Event 16 and Event 18 demonstrated an average increase of 4.1 bu/acre and 3.5 bu/acre respectively. Moreover, when averaged across all hybrids in an environment, Event 16 improved yield over the wild type control in 83% of those environments ( ⁇ 5.7 bu/ac) and Event 18 improved yield in 78% of the environments.
  • Table 3 Yield level of corn Event DP-202216-6 (E16) at various population densities.
  • Example 2 Expression of Endogenous Transcription Factor by Genome Editing
  • expression of an endogenous maize MADS transcription factor ZmM28 (SEQ ID NO: 1 of PCT/US2019/027599) is modulated by a heterologous promoter element.
  • heterologous ZmGOS2 promoter from maize is used to replace an endogenous regulatory region of the genomic locus that encodes SEQ ID NO: 1 of PCT/US2019/027599 polypeptide or a sequence that is substantially similar to SEQ ID NO: 1 of PCT/US2019/027599.
  • the heterologous regulatory elements e.g., promoters and introns
  • guide RNAs were designed to target the upstream regulatory regions of the genomic locus that drives the expression of the polynucleotide encoding SEQ ID NO: 1 of PCT/US2019/027599.
  • Cas9 genome editing technique was used to replace the endogenous promoter region of the genomic loci that encodes SEQ ID NO: 1 of PCT/US2019/027599 with promoters to increase one or more agronomic parameters.
  • a moderatively-constitutive promoter promoter ZM-GOS2
  • OS-ACTIN a different moderately expressed constitutive promoter
  • TO plants were generated using particle gun bombardment through co-bombardment of the insert plasmid, Cas9 plasmid, gRNA plasmid(s) and three helper plasmids. Genomic DNA was isolated from the TO plants and copy number analysis of plasmids were performed. HR1 and HR2 junction PCRs were performed to select the TOs for further evaluation. A subsequent, more detailed screening was done in which long-range PCRs (HR1 & HR2 PCRs) was performed to cover the expanse of the intended insertion region as well as some anchoring native flanking region on each side.
  • HR1 & HR2 PCRs long-range PCRs
  • Enhancer elements such as those described for example in Table 1 of WO2018183878A1, incorporated herein by reference.
  • Suitable expression modulating elements include for example one or more copies (1X-4X) of SEQ ID NOS: 1-10 from Table 1 of WO2018183878A1.
  • EME sequences can be inserted as discrete heterologous sequences or the regulatory region can be genome edited (either using a template directed double strand-break repair or through base editing deaminases) to create heterologous enhancer sequences that modulate the expression level of zmm28.
  • Maize transgenic plants expressing a recombinant maize polynucleotide sequence encoding the polypeptide (SEQ ID NO: 1 of PCT/US2019/027599) were field tested.
  • the transgenic maize plants demonstrated efficacy for increased yield and yield stability.
  • Transformed maize plants containing the recombinant polynucleotide encoding SEQ ID NO: 1 of PCT/US2019/027599 were then converted into elite inbreds and top-crossed for a series of grain yield trials for three years.
  • Several hybrid platforms were evaluated in multiple unique environments that included various levels of drought and nitrogen stress and well as environments targeted for optimal yield levels, where yield levels ranged from 80 bu/acre to 250 bu/acre.
  • Maize transgenic plants containing the recombinant polynucleotide encoding SEQ ID NO: 1 of PCT/US2019/027599 demonstrated about 3.4 bu/acre increase over control maize plants not containing the recombinant AG099 gene, based on multi-year, multi-hybrid, multi-location field trials at P ⁇ 0.05.
  • a mixed model analysis of variance was conducted using ASREML.
  • Experimental designs were split plots with hybrid background as the main plot and event or the control as the sub plot. Two to three replicates were established at each testing site with main plots randomized within replication and sub plots randomized within main plot. Experimental entries were grown in four-row plots that ranged from 4.4 m to 5.3 m in length with a 0.5 m alley in between. Whole testing sites and individual plots of poor quality were removed from data collection procedures and analysis per a standardized procedure. Grain weights and moistures for each experimental entry were measured by harvesting the center two rows of the four-row plot using a small-plot research combine. Yield was standardized within the experiment by adjusting the harvested grain weight of each plot to fifteen percent moisture.
  • Plants at approximately the V2 growth stage were manually infested with approximately 375-750 (varied by location) WCRW eggs applied into the soil on each side of the plant (-750-1,500 eggs/plant total). Additionally, plots were planted in fields that had a high probability of containing a natural infestation of WCRW. Plant roots were evaluated at approximately the R2 growth stage. Two plants per plot were tagged with unique identifiers and removed from the plot and washed with pressurized water. The root damage was rated using the 0-3 node injury scale (CRWNIS) (Oleson, et al. (2005) J Econ.
  • CRWNIS 0-3 node injury scale
  • Agronomic field trials containing the five molecular stack construct designs as used in Example 5 containing both DvSSJl and IPD072, were executed in the summer of 2016 to generate yield data and to evaluate other agronomic characteristics. Multiple events were tested for each construct design (Table 8). All inbred and hybrid materials tested for an event were generated from a single TO plant.
  • Hybrid trials were planted at 16 locations with a single replicate of the entry list at each location. Grain was harvested from 10 of the 16 locations. Each entry in a common background was crossed to three testers to generate hybrid seed for testing. Experiments were nested by testers, with the entries randomized within each nest. Various observations and data were collected at each planted location throughout the growing season. The following agronomic characteristics were analyzed for comparison to a wild type entry (WT), or an entry with the same genetics but without the molecular stacks of DvSSJl and IPD072, also referred to as base comparator (Tables 9-10): 1.) Growing degree units to silk (GDUSLK): Measurement records the total accumulated growing degree units when 50% of the plants in the plot have fully emerged silks. A single day equivalent is approximately 2.5 growing degrees units for this data set.
  • Growing degree units to shed Measurement records the total accumulated growing degree units when 50% of the plants in the plot have tassels that are shedding pollen. A single day equivalent is approximately 2.5 growing degrees units for this data set.
  • Ear height Measurement from the ground to the attachment point of the highest developed ear on the plant. Ear height is measured in inches.
  • Plant height Measurement from the ground to the base of the flag leaf.
  • Plant height is measured in inches.
  • Moisture Measurement of the percent grain moisture at harvest.
  • Inbred trials were planted at eight locations with two replicates of the entry list at each location. One replicate at each location was nested by construct design; the other replicate was planted as a randomized complete block. Agronomic data and observations were collected for the inbred trials and analyzed for comparison to a wild type entry (WT), or untraited version of the same genotype. Data generated for the inbred trials included the following agronomic traits:
  • Growing degree units to shed Measurement records the total accumulated growing degree units when 50% of the plants in the plot have tassels that are shedding pollen. A single day equivalent is approximately 2.5 growing degrees units for this data set.
  • Ear height Measurement from the ground to the attachment point of the highest developed ear on the plant. Ear height is measured in inches.
  • Plant height Measurement from the ground to the base of the flag leaf.
  • Plant height is measured in inches.
  • Ear photometry yield (PHTYLD): Calculated yield estimates from images of harvested ears from each plot. Units for the values shown are bu/acre. Trial Results
  • a mixed model framework was used to perform multi location analysis.
  • main effect construct design is considered as fixed effect.
  • Factors for location, background, tester, event, background by construct design, tester by construct design, tester by event, location by background, location by construct design, location by tester, location by background by construct design, location by tester by construct design, location by event, location by tester by event are considered as random effects.
  • the spatial effects including range and plot within locations were considered as random effects to remove the extraneous spatial noise.
  • the heterogeneous residual was assumed with autoregressive correlation as AR1 * AR1 for each location.
  • the estimate of construct design and prediction of event for each background were generated.
  • the P-tests were conducted to compare construct design/event with WT.
  • a mixed model framework was used to perform multi location analysis.
  • main effect construct design is considered as fixed effect.
  • Factors for location, background, event, background by construct design, location by background, location by construct design, location by background by construct design, location by event and rep within location are considered as random effects.
  • the spatial effects including range and plot within locations were considered as random effects to remove the extraneous spatial noise.
  • the heterogeneous residual was assumed with autoregressive correlation as AR1 * ARl for each location.
  • the estimate of construct design and prediction of event for each background were generated.
  • the P-tests were conducted to compare construct design/event with WT. A difference was considered statistically significant if the .P-value of the difference was less than 0.05.
  • the IPD072Aa ELISA method utilized a kit developed by produced by Pioneer Hi-Bred International, Inc. to determine the concentration of the IPD072Aa protein in samples.
  • Standards typically analyzed in triplicate wells
  • diluted samples typically analyzed in duplicate wells
  • IPD072Aa-specific antibody was incubated in a plate pre-coated with a IPD072Aa-specific antibody. Following incubation, unbound substances were washed from the plate.
  • HRP horseradish peroxidase
  • Detection of the bound IPD072Aa-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the optical density (OD) of each well was determined using a plate reader.
  • the PAT ELISA method utilized an ELISA kit produced by EnviroLogixTM Inc. to determine the concentration of PAT protein in samples. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were co-incubated with a PAT-specific antibody conjugated to the enzyme HRP in a plate pre-coated with a different PAT-specific antibody. Following incubation, unbound substances were washed from the plate. Detection of the bound PAT-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the OD of each well was determined using a plate reader.
  • the PMI ELISA method utilized a kit developed by produced by Pioneer Hi-Bred International, Inc. to determine the concentration of the PMI protein in samples. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated in a plate pre-coated with a PMI-specific antibody. Following incubation, unbound substances were washed from the plate. A different PMI-specific antibody, conjugated to the enzyme HRP, was added to the plate and incubated. Unbound substances were washed from the plate. Detection of the bound PMI-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the OD of each well was determined using a plate reader.
  • SoftMax Pro GxP (Molecular Devices) microplate data software was used to perform the calculations required to convert the OD values obtained for each set of sample wells to a protein concentration value.
  • a standard curve was included on each ELISA plate.
  • the equation for the standard curve was derived by the software, which used a quadratic fit to relate the OD values obtained for each set of standard wells to the respective standard concentration (ng/ml).
  • sample concentration values were adjusted for a dilution factor expressed as 1 :N by multiplying the interpolated concentration by N.
  • Adjusted Concentration Interpolated Sample Concentration x Dilution Factor For example, given an interpolated concentration of 3.6 ng/ml and a dilution factor of 1:20
  • sample concentration 72 ng/ml
  • extraction buffer volume 0.60 ml
  • sample target weight 10 mg
  • reportable assay LLOQ 4.5 ng/ml
  • extraction buffer volume 0.60 ml
  • sample target weight 10 mg
  • IPD072Aa protein in V9 root tissue in two generations of DP-023211-2 maize Means, standard deviations, and ranges for IPD072Aa protein in V9 root tissue in two generations of DP-023211-2 maize are provided in Table 11 and means, standard deviations, and ranges for PAT and PMI proteins in V9 leaf tissue in two generations of DP-023211-2 maize are provided in Table 12.
  • Table 11 Expressed IPD072Aa Protein Concentrations in V9 Root Samples of DP- 023211-2 maize
  • BC1F1 and BC2F1 Separate generations (BC1F1 and BC2F1) of DP-023211-2 maize were grown in 4- inch pots, organized in flats containing 15 pots, using typical greenhouse production conditions in 2017 in Johnston, Iowa, USA. Root samples were collected from 10 plants at approximately the V9 growth stages
  • RNA samples were quantified using a NanoDrop-8000 and stored in a -80 °C freezer.
  • the reference standard of DvSSJl hairpin RNA was produced by in vitro transcription.
  • the total RNA was extracted from the transgenic plants and used to synthesize the cDNA of the full-length DvSSJl by reverse transcription using 5’ and 3’ rapid amplification of cDNA ends (RACE).
  • RACE rapid amplification of cDNA ends
  • the resulting cDNA was cloned into a pUC57 vector under the T7 promoter.
  • Plasmid DNA of the DvSSJl full-length construct was isolated from a bacterial culture and used for in vitro transcription of DvSSJl hpRNA by SunScript RT RNaseH- kit (Sygnis, Heidelberg, Germany).
  • the working concentration of DvSSJl hpRNA was 10 ng/m ⁇ .
  • Nine-point concentrations ranging from 0.0105 to 16 pg per 40 m ⁇ were used for generating the standard curve. The measurements of each point of the standard curve were generated and averaged.
  • RNA from non-GM HC69 maize plants was used as negative control.
  • the QuantiGene assay was conducted according to the manufacturer’s instructions with a modification.
  • the test samples, negative control samples, and DvSSJl hpRNA reference standards were assayed in triplicated wells in a volume of 100 m ⁇ in a 96-well hybridization plate.
  • 250 ng of total RNA was mixed with a quarter strength of the probe set and heated at 95 °C. After heating for 3 minutes, the samples were cooled and maintained at 54 °C until use.
  • a mixture of 40 m ⁇ of the RNA sample and 5 m ⁇ of probe set was transferred to a hybridization plate containing 55 m ⁇ of bead mix for overnight hybridization.
  • the assay plates were read for florescence intensity and by a MagPix analyzer (Luminex. Corp., Austin, TX) according to the manufacturer’s instructions. The net median florescence intensity (MFI) from each assay well was reported. Root tissue sample from five plants per generation was collected to obtain the fresh- weight to dry-weight ratios. Fresh weights were recorded for each sample. Samples were then placed on dry ice, lyophilized, and the dry weights were recorded.
  • MFI median florescence intensity
  • the mean, standard deviation, and coefficient of variation were calculated for each set of triplicate samples using the net MFI value. Standard curves were generated on the QuantiGene Assay plates and used to interpolate DvSSJl dsRNA concentrations based on the net MFI values. The concentration of DvSSJl RNA from each test sample was further converted to a pg/mg fresh weight (fw) value. All fresh weight values were further converted to pg/mg of dry weight (dw) values. The mean, standard deviation, and range of the DvSSJl RNA levels were determined on both fw and dw basis for each of 5 plants in 2 generations.
  • the lowest standard concentration was 0.0105 pg/rxn, and the minimum dilution used was 0.574 rxn/mg.
  • the DvSSJl dsRNA expression results for root samples of DP-023211-2 maize were averaged from the five plants analyzed per generation, and the means, standard deviations, and ranges are summarized in Table 13.
  • Table 13 Summary of DvSSJl RNA Expression Levels in V9 Root Tissue of DP- 023211-2 maize Example 9. Stacking of event DP-202216-6 and event DP-023211-2

Abstract

Des modes de réalisation de la présente invention concernent le domaine de la biologie moléculaire végétale, dont des constructions d'ADN pour conférer à une plante une résistance aux insectes et augmenter le rendement d'une plante. Des modes de réalisation de l'invention comprennent également une plante de maïs résistant aux insectes contenant un événement DP-023211-2 et un événement de maïs DP-202216-6, et des procédés et des compositions associés.
PCT/US2020/053947 2019-10-18 2020-10-02 Événement de maïs dp-202216-6 et empilement dp-023211-2 WO2021076346A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962916856P 2019-10-18 2019-10-18
US62/916,856 2019-10-18

Publications (1)

Publication Number Publication Date
WO2021076346A1 true WO2021076346A1 (fr) 2021-04-22

Family

ID=72964840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/053947 WO2021076346A1 (fr) 2019-10-18 2020-10-02 Événement de maïs dp-202216-6 et empilement dp-023211-2

Country Status (1)

Country Link
WO (1) WO2021076346A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949773A (zh) * 2017-05-18 2018-12-07 萧郁芸 兰花胚珠发育调控基因及其方法
WO2023091884A1 (fr) * 2021-11-16 2023-05-25 Pioneer Hi-Bred International, Inc. Événement de maïs das-01131-3 et procédés de détection de celui-ci

Citations (106)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US5188960A (en) 1989-06-27 1993-02-23 Mycogen Corporation Bacillus thuringiensis isolate active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins
US5877012A (en) 1993-03-25 1999-03-02 Novartis Finance Corporation Class of proteins for the control of plant pests
US5880275A (en) 1989-02-24 1999-03-09 Monsanto Company Synthetic plant genes from BT kurstaki and method for preparation
WO2000011185A2 (fr) 1998-08-19 2000-03-02 Monsanto Co. Expression amelioree de la proteine insecticide cry3bb dans des plantes
US6033874A (en) 1996-11-27 2000-03-07 Ecogen, Inc. CRY1C polypeptides having improved toxicity to lepidopteran insects
US6048838A (en) 1997-05-05 2000-04-11 Dow Agrosciences Llc Insecticidal protein toxins from xenorhabdus
US6083499A (en) 1996-04-19 2000-07-04 Mycogen Corporation Pesticidal toxins
US6218188B1 (en) 1997-11-12 2001-04-17 Mycogen Corporation Plant-optimized genes encoding pesticidal toxins
US6248535B1 (en) 1999-12-20 2001-06-19 University Of Southern California Method for isolation of RNA from formalin-fixed paraffin-embedded tissue specimens
US6326351B1 (en) 1996-09-24 2001-12-04 Monsanto Technology Llc Bacillus thuringiensis CryET33 and CryET34 compositions and uses therefor
US6340593B1 (en) 1998-10-23 2002-01-22 Mycogen Corporation Plant-optimized polynucleotides encoding approximately 15 kDa and approximately 45 kDa pesticidal proteins
US6551962B1 (en) 2000-10-06 2003-04-22 Monsanto Technology Llc Method for deploying a transgenic refuge
US6586365B2 (en) 2000-10-06 2003-07-01 Monsanto Technology, Llc Method for reducing pest damage to corn by treating transgenic corn seeds with clothianidin pesticide
US6593273B2 (en) 2000-10-06 2003-07-15 Monsanto Technology Llc Method for reducing pest damage to corn by treating transgenic corn seeds with pesticide
US6713063B1 (en) 1996-11-20 2004-03-30 Monsanto Technology, Llc Broad-spectrum δ-endotoxins
WO2004074462A2 (fr) 2003-02-20 2004-09-02 Athenix Corporation Genes de delta-endotoxines et leurs methodes d'utilisation
US20040197917A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-014, delta-endotoxin gene and methods for its use
US20040197916A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-004, a delta-endotoxin gene and methods for its use
US20040210965A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-007, a delta-endotoxin gene and methods for its use
US20040210964A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-009, a delta-endotoxin gene and methods for its use
US20040216186A1 (en) 2003-02-20 2004-10-28 Athenix Corporation AXMI-006, a delta-endotoxin gene and methods for its use
US20040250311A1 (en) 2003-02-20 2004-12-09 Athenix Corporation AXMI-008, a delta-endotoxin gene and methods for its use
WO2005021585A2 (fr) 2003-08-28 2005-03-10 Athenix Corporation Axmi-003, un gene de $g(d)-endotoxine, et procede d'utilisation correspondant
WO2005038032A1 (fr) 2003-10-14 2005-04-28 Athenix Corporation Axmi-010, gene de delta-endotoxine et ses procedes d'utilisation
US6962705B2 (en) 1996-11-20 2005-11-08 Monsanto Technolgy Llc Hybrid Bacillus thuringiensis δ-endotoxins with novel broad-spectrum insecticidal activity
US7064249B2 (en) 1998-11-04 2006-06-20 Monsanto Technology Llc Plants transformed to express Cry2A δ-endotoxins
WO2006083891A2 (fr) 2005-01-31 2006-08-10 Athenix Corporation Axmi-018, axmi-020, et axmi-021, famille de genes de delta-endotoxine et leurs methodes d'utilisation
US20060191034A1 (en) 2003-07-07 2006-08-24 Baum James A Insecticidal proteins secreted from bacillus thuringiensis and uses therefor
US7105332B2 (en) 2002-06-26 2006-09-12 E.I. Du Pont De Nemours And Company Genes encoding proteins with pesticidal activity
WO2006119457A1 (fr) 2005-05-02 2006-11-09 Athenix Corporation Axmi-028 et axmi-029, famille de nouveaux genes de delta-endotoxine et procedes d'utilisation associes
US7244820B2 (en) 2001-03-30 2007-07-17 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US7276583B2 (en) 2001-08-31 2007-10-02 Syngenta Participations Ag Modified Cry3A toxins
US7309785B1 (en) 2003-10-03 2007-12-18 Dow Agrosciences Llc Modified chimeric Cry35 proteins
US7329736B2 (en) 2006-04-14 2008-02-12 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US7449552B2 (en) 2006-04-14 2008-11-11 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US20080295207A1 (en) 2007-04-27 2008-11-27 Monsanto Technology Llc Hemipteran-and Coleopteran Active Toxin Proteins from Bacillus Thuringiensis
US7462760B2 (en) 2002-06-26 2008-12-09 Pioneer Hi-Bred International, Inc. Genes encoding plant protease-resistant pesticidal proteins and method of their use
WO2008148872A1 (fr) * 2007-06-06 2008-12-11 Cropdesign N.V. Augmentation du rendement de plantes par modulation du facteur de transcription zmm28 de la boîte mads du maïs
US7491698B2 (en) 2003-01-21 2009-02-17 Dow Agrosciences Llc Mixing and matching TC proteins for pest control
US7491869B2 (en) 2004-02-20 2009-02-17 Pioneer Hi-Bred International, Inc. Methods of using non-plant insecticidal lipase encoding nucleic acids in transgenic plants
US7524810B1 (en) 2003-10-03 2009-04-28 Dow Agrosciences Llc Modified Cry34 proteins
US20090144852A1 (en) 2007-10-16 2009-06-04 Athenix Corporation Axmi-066 and axmi-076: delta-endotoxin proteins and methods for their use
US20100005543A1 (en) 2008-06-25 2010-01-07 Athenix Corporation Toxin genes and methods for their use
US20100017914A1 (en) 2007-03-28 2010-01-21 Syngenta Participations Ag Insecticidal proteins
US7705216B2 (en) 2002-07-29 2010-04-27 Monsanto Technology Llc Corn event PV-ZMIR13 (MON863) plants and compositions and methods for detection thereof
US20100197592A1 (en) 2009-02-05 2010-08-05 Athenix Corporation Variant axmi-r1 delta endotoxin genes and methods for their use
US7803943B2 (en) 2005-03-17 2010-09-28 Biotium, Inc. Methods of using dyes in association with nucleic acid staining or detection and associated technology
US20100298211A1 (en) 2009-03-11 2010-11-25 Athenix Corporation Axmi-001, axmi-002, axmi-030, axmi-035, and axmi-045: toxin genes and methods for their use
US7858849B2 (en) 2006-12-08 2010-12-28 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
US7875429B2 (en) 2004-09-29 2011-01-25 Pioneer Hi-Bred International, Inc. Corn event DAS-59122-7 and methods for detection thereof
US20110023184A1 (en) 2009-07-02 2011-01-27 Nalini Manoj Desai Axmi-205 pesticidal gene and methods for its use
US20110064710A1 (en) 2009-09-11 2011-03-17 Benson Terry A Novel bacillus thuringiensis isolate
US7923602B2 (en) 2006-06-14 2011-04-12 Athenix Corp. AXMI-031, AXMI-039, AXMI-040 and AXMI-049, a family of novel delta endotoxin genes and methods for their use
US7985893B2 (en) 2005-06-09 2011-07-26 Pioneer Hi Bred International Inc Sclerotinia-resistant Brassica
WO2011103248A2 (fr) 2010-02-18 2011-08-25 Athenix Corp. Gènes de delta-endotoxine axmi221z, axmi222z, axmi223z, axmi224z, et axmi225z et leurs procédés d'utilisation
WO2011103247A2 (fr) 2010-02-18 2011-08-25 Athenix Corp. Gènes de delta-endotoxine axmi218, axmi219, axmi220, axmi226, axmi227, axmi228, axmi229, axmi230, et axmi231 et leurs procédés d'utilisation
US20110263488A1 (en) 2006-06-15 2011-10-27 Athenix Corp. Family of pesticidal proteins and methods for their use
US8084416B2 (en) 2008-12-23 2011-12-27 Athenix Corp. AXMI-150 delta-endotoxin gene and methods for its use
US8101826B2 (en) 1999-08-19 2012-01-24 Monsanto Technology Llc Expression of Cry3B insecticidal protein in plants
US8236757B2 (en) 2005-04-01 2012-08-07 Athenix Corp AXMI-027, AXMI-036 and AXMI-038, a family of delta-endotoxin genes and methods for their use
US8269069B1 (en) 2008-07-30 2012-09-18 Dow Agrosciences, Llc Modified Bacillus thuringiensis cry proteins that inhibit coleopterans
WO2012139004A2 (fr) 2011-04-07 2012-10-11 Monsanto Technology Llc Famille de toxines inhibitrices d'insectes actives à l'encontre des insectes hemiptères et/ou lépidoptères
US20120278954A1 (en) 2011-02-11 2012-11-01 Bowen David J Pesticidal Nucleic Acids and Proteins and Uses Thereof
US8304604B2 (en) 2009-04-17 2012-11-06 Dow Agrosciences, Llc. DIG-3 insecticidal Cry toxins
US8304605B2 (en) 2009-06-16 2012-11-06 Dow Agrosciences, Llc. DIG-11 insecticidal cry toxins
US8319019B2 (en) 2009-01-23 2012-11-27 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
US8318900B2 (en) 2009-02-27 2012-11-27 Athenix Corp. Pesticidal proteins and methods for their use
US20120311745A1 (en) 2009-12-16 2012-12-06 Dow Agrosciences Llc Combined use of cry1ca and cry1ab proteins for insect resistance management
US20120311746A1 (en) 2009-12-16 2012-12-06 Dow Agroscience Llc Insect resistance management with combinations of cry1be and cry1f proteins
US20120317681A1 (en) 2009-12-16 2012-12-13 Thomas Meade COMBINED USE OF CRY1Ca AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US20120317682A1 (en) 2009-12-16 2012-12-13 Dow Agrosciences Llc Combined use of vip3ab and cry1fa for management of resistant insects
US8334431B2 (en) 2008-07-02 2012-12-18 Athenix Corporation AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184: insecticidal proteins and methods for their use
US8334366B1 (en) 2009-04-29 2012-12-18 The United States Of America, As Represented By The Secretary Of Agriculture Mutant lycotoxin-1 peptide sequences for insecticidal and cell membrane altering properties
US20120324605A1 (en) 2009-12-16 2012-12-20 Dow Agrosciences Llc Insectcidal protein combinations for controlling fall armyworm and european corn borer, and methods for insect resistance management
US20120331590A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc Use of cry1da in combination with cry1be for management of resistant insects
US20120331589A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc COMBINED USE OF CRY1Da AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US20130019349A1 (en) 2006-01-12 2013-01-17 Incima Us Llc Epsps mutants
US8513492B2 (en) 1997-12-18 2013-08-20 Monsanto Technology Llc Coleopteran-resistant transgenic plants and methods of their production
US8575434B2 (en) 2009-12-17 2013-11-05 Pioneer Hi Bred International Inc Maize event DP-004114-3 and methods for detection thereof
US8686230B2 (en) 2003-12-15 2014-04-01 Monsanto Technology Llc Corn plant MON88017 and compositions and methods for detection thereof
US8884102B2 (en) 2004-03-25 2014-11-11 Syngenta Participations Ag Corn event MIR604
US9133474B2 (en) 2008-12-16 2015-09-15 Syngenta Participations Ag Corn event 5307
US20150257389A1 (en) * 2014-03-14 2015-09-17 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
US20160186204A1 (en) 2013-08-16 2016-06-30 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2016114973A1 (fr) 2015-01-15 2016-07-21 Pioneer Hi Bred International, Inc Protéines insecticides et leurs procédés d'utilisation
US9441240B2 (en) 2012-05-08 2016-09-13 Monsanto Technology Llc Corn event MON 87411
US9475847B2 (en) 2012-07-26 2016-10-25 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US20160347799A1 (en) 2014-02-07 2016-12-01 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
US20160366891A1 (en) 2013-09-13 2016-12-22 Pioneer Hi Bred Int Insecticidal proteins and methods for their use
WO2017023486A1 (fr) 2015-08-06 2017-02-09 Pioneer Hi-Bred International, Inc. Protéines insecticides d'origine végétale et leurs méthodes d'utilisation
US20170166921A1 (en) 2014-02-07 2017-06-15 Pioneer Hi-Bred International, Inc. Novel insecticidal proteins from plants
WO2017105987A1 (fr) 2015-12-18 2017-06-22 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
US9688730B2 (en) 2012-07-02 2017-06-27 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US20170233440A1 (en) 2014-10-16 2017-08-17 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2017192560A1 (fr) 2016-05-04 2017-11-09 Pioneer Hi-Bred International, Inc. Protéines insecticides et procédés pour les utiliser
WO2018005411A1 (fr) 2016-07-01 2018-01-04 Pioneer Hi-Bred International, Inc. Protéines insecticides issues de plantes et procédés pour leur utilisation
WO2018075350A1 (fr) 2016-10-21 2018-04-26 Pioneer Hi-Bred International, Inc. Protéines insecticides issues de plantes et procédés pour leur utilisation
WO2018084936A1 (fr) 2016-11-01 2018-05-11 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018111551A1 (fr) 2016-12-14 2018-06-21 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018118811A1 (fr) 2016-12-22 2018-06-28 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018183878A1 (fr) 2017-03-31 2018-10-04 Pioneer Hi-Bred International, Inc. Éléments de modulation d'expression et leur utilisation
WO2018208882A1 (fr) 2017-05-11 2018-11-15 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018232072A1 (fr) 2017-06-16 2018-12-20 Pioneer Hi-Bred International, Inc. Protéines insecticides issues de plantes et procédés pour leur utilisation

Patent Citations (127)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US5880275A (en) 1989-02-24 1999-03-09 Monsanto Company Synthetic plant genes from BT kurstaki and method for preparation
US5188960A (en) 1989-06-27 1993-02-23 Mycogen Corporation Bacillus thuringiensis isolate active against lepidopteran pests, and genes encoding novel lepidopteran-active toxins
US5877012A (en) 1993-03-25 1999-03-02 Novartis Finance Corporation Class of proteins for the control of plant pests
US6137033A (en) 1993-03-25 2000-10-24 Novartis Finance Corporation Class of proteins for the control of plant pests
US6107279A (en) 1993-03-25 2000-08-22 Novartis Finance Corporation Class of proteins for the control of plant pests
US6548291B1 (en) 1996-04-19 2003-04-15 Mycogen Corporation Pesticidal toxins
US6624145B1 (en) 1996-04-19 2003-09-23 Mycogen Corporation Pesticidal toxins
US6083499A (en) 1996-04-19 2000-07-04 Mycogen Corporation Pesticidal toxins
US6127180A (en) 1996-04-19 2000-10-03 Mycogen Corporation Pesticidal toxins
US7504229B2 (en) 1996-09-24 2009-03-17 Monsanto Technology Llc Methods for detecting Bacillus thuringiensis cryET33 and cryET34 polypeptides
US7385107B2 (en) 1996-09-24 2008-06-10 Monsanto Technologies Llc Insect-resistant transgenic plants transformed with CryET33 and CryET34-encoding nucleic acids
US6326351B1 (en) 1996-09-24 2001-12-04 Monsanto Technology Llc Bacillus thuringiensis CryET33 and CryET34 compositions and uses therefor
US6949626B2 (en) 1996-09-24 2005-09-27 Monsanto Technology Llc Bacillus thuringiensis cryET33 and cryET34 compositions and uses therefor
US6399330B1 (en) 1996-09-24 2002-06-04 Monsanto Technology Llc Bacillus thuringiensis cryet33 and cryet34 compositions and uses thereof
US7070982B2 (en) 1996-11-20 2006-07-04 Monsanto Technology Llc Polynucleotide compositions encoding broad spectrum delta-endotoxins
US6962705B2 (en) 1996-11-20 2005-11-08 Monsanto Technolgy Llc Hybrid Bacillus thuringiensis δ-endotoxins with novel broad-spectrum insecticidal activity
US6713063B1 (en) 1996-11-20 2004-03-30 Monsanto Technology, Llc Broad-spectrum δ-endotoxins
US6033874A (en) 1996-11-27 2000-03-07 Ecogen, Inc. CRY1C polypeptides having improved toxicity to lepidopteran insects
US6379946B1 (en) 1997-05-05 2002-04-30 Wisconsin Alumn Research Foundation Insecticidal protein toxins from Xenorhabdus
US6048838A (en) 1997-05-05 2000-04-11 Dow Agrosciences Llc Insecticidal protein toxins from xenorhabdus
US6218188B1 (en) 1997-11-12 2001-04-17 Mycogen Corporation Plant-optimized genes encoding pesticidal toxins
US8513492B2 (en) 1997-12-18 2013-08-20 Monsanto Technology Llc Coleopteran-resistant transgenic plants and methods of their production
WO2000011185A2 (fr) 1998-08-19 2000-03-02 Monsanto Co. Expression amelioree de la proteine insecticide cry3bb dans des plantes
US6340593B1 (en) 1998-10-23 2002-01-22 Mycogen Corporation Plant-optimized polynucleotides encoding approximately 15 kDa and approximately 45 kDa pesticidal proteins
US7064249B2 (en) 1998-11-04 2006-06-20 Monsanto Technology Llc Plants transformed to express Cry2A δ-endotoxins
US8101826B2 (en) 1999-08-19 2012-01-24 Monsanto Technology Llc Expression of Cry3B insecticidal protein in plants
US6248535B1 (en) 1999-12-20 2001-06-19 University Of Southern California Method for isolation of RNA from formalin-fixed paraffin-embedded tissue specimens
US6593273B2 (en) 2000-10-06 2003-07-15 Monsanto Technology Llc Method for reducing pest damage to corn by treating transgenic corn seeds with pesticide
US6586365B2 (en) 2000-10-06 2003-07-01 Monsanto Technology, Llc Method for reducing pest damage to corn by treating transgenic corn seeds with clothianidin pesticide
US6551962B1 (en) 2000-10-06 2003-04-22 Monsanto Technology Llc Method for deploying a transgenic refuge
US8237020B2 (en) 2001-03-30 2012-08-07 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US7615686B2 (en) 2001-03-30 2009-11-10 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US7244820B2 (en) 2001-03-30 2007-07-17 Syngenta Participations Ag Expression and use of novel pesticidal toxins
US7276583B2 (en) 2001-08-31 2007-10-02 Syngenta Participations Ag Modified Cry3A toxins
US8759620B2 (en) 2001-08-31 2014-06-24 Syngenta Participations Ag Transgenic plants expressing modified CRY3A
US7378499B2 (en) 2002-06-26 2008-05-27 Pioneer Hi-Bred International, Inc. Genes encoding proteins with pesticidal activity
US7105332B2 (en) 2002-06-26 2006-09-12 E.I. Du Pont De Nemours And Company Genes encoding proteins with pesticidal activity
US7462760B2 (en) 2002-06-26 2008-12-09 Pioneer Hi-Bred International, Inc. Genes encoding plant protease-resistant pesticidal proteins and method of their use
US7705216B2 (en) 2002-07-29 2010-04-27 Monsanto Technology Llc Corn event PV-ZMIR13 (MON863) plants and compositions and methods for detection thereof
US8084418B2 (en) 2003-01-21 2011-12-27 Dow Agrosciences Llc Methods of inhibiting insects by treatment with a complex comprising a Photorhabdus insecticidal protein and one or two Xenorhabdus enhancer proteins
US7491698B2 (en) 2003-01-21 2009-02-17 Dow Agrosciences Llc Mixing and matching TC proteins for pest control
US20040197916A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-004, a delta-endotoxin gene and methods for its use
US20040197917A1 (en) 2003-02-20 2004-10-07 Athenix Corporation AXMI-014, delta-endotoxin gene and methods for its use
US20040210965A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-007, a delta-endotoxin gene and methods for its use
US20040210964A1 (en) 2003-02-20 2004-10-21 Athenix Corporation AXMI-009, a delta-endotoxin gene and methods for its use
US20040216186A1 (en) 2003-02-20 2004-10-28 Athenix Corporation AXMI-006, a delta-endotoxin gene and methods for its use
WO2004074462A2 (fr) 2003-02-20 2004-09-02 Athenix Corporation Genes de delta-endotoxines et leurs methodes d'utilisation
US20040250311A1 (en) 2003-02-20 2004-12-09 Athenix Corporation AXMI-008, a delta-endotoxin gene and methods for its use
US20060191034A1 (en) 2003-07-07 2006-08-24 Baum James A Insecticidal proteins secreted from bacillus thuringiensis and uses therefor
WO2005021585A2 (fr) 2003-08-28 2005-03-10 Athenix Corporation Axmi-003, un gene de $g(d)-endotoxine, et procede d'utilisation correspondant
US7524810B1 (en) 2003-10-03 2009-04-28 Dow Agrosciences Llc Modified Cry34 proteins
US7939651B2 (en) 2003-10-03 2011-05-10 Dow Agrosciences Llc Modified Cry35 proteins
US7309785B1 (en) 2003-10-03 2007-12-18 Dow Agrosciences Llc Modified chimeric Cry35 proteins
WO2005038032A1 (fr) 2003-10-14 2005-04-28 Athenix Corporation Axmi-010, gene de delta-endotoxine et ses procedes d'utilisation
US8686230B2 (en) 2003-12-15 2014-04-01 Monsanto Technology Llc Corn plant MON88017 and compositions and methods for detection thereof
US7491869B2 (en) 2004-02-20 2009-02-17 Pioneer Hi-Bred International, Inc. Methods of using non-plant insecticidal lipase encoding nucleic acids in transgenic plants
US8884102B2 (en) 2004-03-25 2014-11-11 Syngenta Participations Ag Corn event MIR604
US7875429B2 (en) 2004-09-29 2011-01-25 Pioneer Hi-Bred International, Inc. Corn event DAS-59122-7 and methods for detection thereof
WO2006083891A2 (fr) 2005-01-31 2006-08-10 Athenix Corporation Axmi-018, axmi-020, et axmi-021, famille de genes de delta-endotoxine et leurs methodes d'utilisation
US7803943B2 (en) 2005-03-17 2010-09-28 Biotium, Inc. Methods of using dyes in association with nucleic acid staining or detection and associated technology
US8236757B2 (en) 2005-04-01 2012-08-07 Athenix Corp AXMI-027, AXMI-036 and AXMI-038, a family of delta-endotoxin genes and methods for their use
WO2006119457A1 (fr) 2005-05-02 2006-11-09 Athenix Corporation Axmi-028 et axmi-029, famille de nouveaux genes de delta-endotoxine et procedes d'utilisation associes
US7985893B2 (en) 2005-06-09 2011-07-26 Pioneer Hi Bred International Inc Sclerotinia-resistant Brassica
US20130019349A1 (en) 2006-01-12 2013-01-17 Incima Us Llc Epsps mutants
US7476781B2 (en) 2006-04-14 2009-01-13 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US7329736B2 (en) 2006-04-14 2008-02-12 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US7449552B2 (en) 2006-04-14 2008-11-11 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry gene and protein
US7923602B2 (en) 2006-06-14 2011-04-12 Athenix Corp. AXMI-031, AXMI-039, AXMI-040 and AXMI-049, a family of novel delta endotoxin genes and methods for their use
US20110263488A1 (en) 2006-06-15 2011-10-27 Athenix Corp. Family of pesticidal proteins and methods for their use
US7858849B2 (en) 2006-12-08 2010-12-28 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof
US20100017914A1 (en) 2007-03-28 2010-01-21 Syngenta Participations Ag Insecticidal proteins
US20080295207A1 (en) 2007-04-27 2008-11-27 Monsanto Technology Llc Hemipteran-and Coleopteran Active Toxin Proteins from Bacillus Thuringiensis
WO2008148872A1 (fr) * 2007-06-06 2008-12-11 Cropdesign N.V. Augmentation du rendement de plantes par modulation du facteur de transcription zmm28 de la boîte mads du maïs
US20090144852A1 (en) 2007-10-16 2009-06-04 Athenix Corporation Axmi-066 and axmi-076: delta-endotoxin proteins and methods for their use
US20100005543A1 (en) 2008-06-25 2010-01-07 Athenix Corporation Toxin genes and methods for their use
US8334431B2 (en) 2008-07-02 2012-12-18 Athenix Corporation AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184: insecticidal proteins and methods for their use
US8269069B1 (en) 2008-07-30 2012-09-18 Dow Agrosciences, Llc Modified Bacillus thuringiensis cry proteins that inhibit coleopterans
US9133474B2 (en) 2008-12-16 2015-09-15 Syngenta Participations Ag Corn event 5307
US8084416B2 (en) 2008-12-23 2011-12-27 Athenix Corp. AXMI-150 delta-endotoxin gene and methods for its use
US8319019B2 (en) 2009-01-23 2012-11-27 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
US20100197592A1 (en) 2009-02-05 2010-08-05 Athenix Corporation Variant axmi-r1 delta endotoxin genes and methods for their use
US8318900B2 (en) 2009-02-27 2012-11-27 Athenix Corp. Pesticidal proteins and methods for their use
US20100298211A1 (en) 2009-03-11 2010-11-25 Athenix Corporation Axmi-001, axmi-002, axmi-030, axmi-035, and axmi-045: toxin genes and methods for their use
US8304604B2 (en) 2009-04-17 2012-11-06 Dow Agrosciences, Llc. DIG-3 insecticidal Cry toxins
US8334366B1 (en) 2009-04-29 2012-12-18 The United States Of America, As Represented By The Secretary Of Agriculture Mutant lycotoxin-1 peptide sequences for insecticidal and cell membrane altering properties
US8304605B2 (en) 2009-06-16 2012-11-06 Dow Agrosciences, Llc. DIG-11 insecticidal cry toxins
US20110023184A1 (en) 2009-07-02 2011-01-27 Nalini Manoj Desai Axmi-205 pesticidal gene and methods for its use
US20110064710A1 (en) 2009-09-11 2011-03-17 Benson Terry A Novel bacillus thuringiensis isolate
US20120317682A1 (en) 2009-12-16 2012-12-13 Dow Agrosciences Llc Combined use of vip3ab and cry1fa for management of resistant insects
US20120311746A1 (en) 2009-12-16 2012-12-06 Dow Agroscience Llc Insect resistance management with combinations of cry1be and cry1f proteins
US20120324606A1 (en) 2009-12-16 2012-12-20 Dow Agrosciences Llc Use of cry 1ab in combination with cry1be for management of resistant insects
US20120324605A1 (en) 2009-12-16 2012-12-20 Dow Agrosciences Llc Insectcidal protein combinations for controlling fall armyworm and european corn borer, and methods for insect resistance management
US20120331590A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc Use of cry1da in combination with cry1be for management of resistant insects
US20120331589A1 (en) 2009-12-16 2012-12-27 Dow Agrosciences Llc COMBINED USE OF CRY1Da AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US20120311745A1 (en) 2009-12-16 2012-12-06 Dow Agrosciences Llc Combined use of cry1ca and cry1ab proteins for insect resistance management
US20120317681A1 (en) 2009-12-16 2012-12-13 Thomas Meade COMBINED USE OF CRY1Ca AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT
US8575434B2 (en) 2009-12-17 2013-11-05 Pioneer Hi Bred International Inc Maize event DP-004114-3 and methods for detection thereof
WO2011103248A2 (fr) 2010-02-18 2011-08-25 Athenix Corp. Gènes de delta-endotoxine axmi221z, axmi222z, axmi223z, axmi224z, et axmi225z et leurs procédés d'utilisation
WO2011103247A2 (fr) 2010-02-18 2011-08-25 Athenix Corp. Gènes de delta-endotoxine axmi218, axmi219, axmi220, axmi226, axmi227, axmi228, axmi229, axmi230, et axmi231 et leurs procédés d'utilisation
US20120278954A1 (en) 2011-02-11 2012-11-01 Bowen David J Pesticidal Nucleic Acids and Proteins and Uses Thereof
WO2012139004A2 (fr) 2011-04-07 2012-10-11 Monsanto Technology Llc Famille de toxines inhibitrices d'insectes actives à l'encontre des insectes hemiptères et/ou lépidoptères
US9441240B2 (en) 2012-05-08 2016-09-13 Monsanto Technology Llc Corn event MON 87411
US9688730B2 (en) 2012-07-02 2017-06-27 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US9475847B2 (en) 2012-07-26 2016-10-25 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US20160186204A1 (en) 2013-08-16 2016-06-30 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US20160366891A1 (en) 2013-09-13 2016-12-22 Pioneer Hi Bred Int Insecticidal proteins and methods for their use
US20160347799A1 (en) 2014-02-07 2016-12-01 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
US20170166921A1 (en) 2014-02-07 2017-06-15 Pioneer Hi-Bred International, Inc. Novel insecticidal proteins from plants
US20150257389A1 (en) * 2014-03-14 2015-09-17 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
US20170233440A1 (en) 2014-10-16 2017-08-17 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2016114973A1 (fr) 2015-01-15 2016-07-21 Pioneer Hi Bred International, Inc Protéines insecticides et leurs procédés d'utilisation
WO2017023486A1 (fr) 2015-08-06 2017-02-09 Pioneer Hi-Bred International, Inc. Protéines insecticides d'origine végétale et leurs méthodes d'utilisation
WO2017105987A1 (fr) 2015-12-18 2017-06-22 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2017192560A1 (fr) 2016-05-04 2017-11-09 Pioneer Hi-Bred International, Inc. Protéines insecticides et procédés pour les utiliser
WO2018005411A1 (fr) 2016-07-01 2018-01-04 Pioneer Hi-Bred International, Inc. Protéines insecticides issues de plantes et procédés pour leur utilisation
WO2018075350A1 (fr) 2016-10-21 2018-04-26 Pioneer Hi-Bred International, Inc. Protéines insecticides issues de plantes et procédés pour leur utilisation
WO2018084936A1 (fr) 2016-11-01 2018-05-11 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018111551A1 (fr) 2016-12-14 2018-06-21 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018118811A1 (fr) 2016-12-22 2018-06-28 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018183878A1 (fr) 2017-03-31 2018-10-04 Pioneer Hi-Bred International, Inc. Éléments de modulation d'expression et leur utilisation
WO2018208882A1 (fr) 2017-05-11 2018-11-15 Pioneer Hi-Bred International, Inc. Protéines insecticides et leurs procédés d'utilisation
WO2018232072A1 (fr) 2017-06-16 2018-12-20 Pioneer Hi-Bred International, Inc. Protéines insecticides issues de plantes et procédés pour leur utilisation

Non-Patent Citations (32)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. EU400157
CHEN ET AL., GENOME RES, vol. 9, 1999, pages 492 - 498
CHENG ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 5695 - 5699
CRICKMORE ET AL., BACILLUS THURINGIENSIS TOXIN NOMENCLATURE, 2011
CULLIS, B. R ET AL.: "Biometrics", vol. 54, 1998, VSN INTERNATIONAL LTD, article "Best Linear Unbiased Prediction", pages: 1 - 18
FLEVIN ET AL.: "PCR Protocols: A Guide to Methods and Applications", 1990, KLUWER ACADEMIC PUBLISHERS
GILMOUR, A. R. ET AL., ASREML USER GUIDE 3.0, 2009
GILMOUR, A.R. ET AL., BIOMETRICS, vol. 51, 1995, pages 1440 - 50
HAYMES ET AL.: "Nucleic Acid Hybridization, a Practical Approach", 1985, IRL PRESS
HINCHLIFFE ET AL., THE OPEN TOXICOLOGY JOURNAL, vol. 3, 2010, pages 101 - 118
LI ET AL., PLANT CELL TISS. ORGAN CULT, vol. 89, 2007, pages 159 - 168
LIU ET AL., J. AGRIC. FOOD CHEM., vol. 58, 2010, pages 12343 - 12349
MEINKOTHWAHL: "Anal. Biochem.", vol. 138, 1984, pages: 267 - 284
MONALYSIN, PLOS PATHOGENS, vol. 7, 2011, pages 1 - 13
MONSANTO: "MON810", 24 May 2013 (2013-05-24), XP055753208, Retrieved from the Internet <URL:http://bch.cbd.int/database/record.shtml?documentid=14750> [retrieved on 20201123] *
MORGAN ET AL., APPLIED AND ENVIR. MICRO., vol. 67, 2001, pages 2062 - 2069
NAIMOV ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 74, 2008, pages 7145 - 7151
NIKIFOROV ET AL., NUCLEIC ACID RES., vol. 22, 1994, pages 4167 - 4175
OLESON ET AL., J. ECON. ENTOMOL., vol. 98, no. 1, 2005, pages 1 - 8
PECHY-TARR, ENVIRONMENTAL MICROBIOLOGY, vol. 10, 2008, pages 2368 - 2386
PIONEER HI-BRED: "DP-023211-2", 21 June 2019 (2019-06-21), XP055753188, Retrieved from the Internet <URL:https://www.transgen.de/zulassung/2790.dp-023211-2.html> [retrieved on 20201123] *
PIONEER HI-BRED: "DP202216 Maize", 21 June 2019 (2019-06-21), XP055753194, Retrieved from the Internet <URL:https://www.transgen.de/zulassung/2790.dp-023211-2.html> [retrieved on 20201123] *
PIONEER HI-BRED: "DP-20221-6", 21 June 2019 (2019-06-21), XP055753192, Retrieved from the Internet <URL:https://www.transgen.de/zulassung/2790.dp-023211-2.html> [retrieved on 20201123] *
PIONEER HI-BRED: "DP23211 maize", 21 June 2019 (2019-06-21), XP055753190, Retrieved from the Internet <URL:https://www.transgen.de/zulassung/2790.dp-023211-2.html> [retrieved on 20201123] *
PURCELL ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 15, 1993, pages 1406 - 1413
QIUDENG QUE ET AL: "Trait stacking in transgenic crops: challenges and opportunities", GM CROPS, LANDES BIOSCIENCE, UNITED STATES, vol. 1, no. 4, 1 July 2010 (2010-07-01), pages 220 - 229, XP002677400, ISSN: 1938-1999, DOI: 10.4161/GMCR.1.4.13439 *
SANAHUJA, PLANT BIOTECH JOURNAL, vol. 9, 2011, pages 283 - 300
TYANGI ET AL., NATURE BIOTECH, vol. 14, 1996, pages 303 - 308
VAN FRANNKENHUYZEN, J. INVERT. PATH., vol. 101, 2009, pages 1 - 16
WEISSBACHWEISSBACH: "Molecular Cloning: A Laboratory Manual", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY PRESS
WINGE, INNOV. PHARMA. TECH., vol. 00, 2000, pages 18 - 24
ZHANG ET AL., ANNALS OF MICROBIOLOGY, vol. 59, 2009, pages 45 - 50

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949773A (zh) * 2017-05-18 2018-12-07 萧郁芸 兰花胚珠发育调控基因及其方法
CN108949773B (zh) * 2017-05-18 2023-12-26 萧郁芸 产生转基因植物的方法
WO2023091884A1 (fr) * 2021-11-16 2023-05-25 Pioneer Hi-Bred International, Inc. Événement de maïs das-01131-3 et procédés de détection de celui-ci

Similar Documents

Publication Publication Date Title
US20220002821A1 (en) Maize event dp-004114-3 and methods for detection thereof
JP5529322B2 (ja) トランスジェニック事象ｍｏｎ８９０３４に対応するトウモロコシ植物および種子ならびにそれらを検出および使用するための方法
WO2014116854A1 (fr) Événement de maïs dp-033121-3 et ses procédés de détection
WO2011084632A1 (fr) Evénement de maïs dp-032316-8 et ses procédés de détection
AU2019314261B2 (en) Corn transgenic event MON 95379 and methods for detection and uses thereof
US10407688B2 (en) Maize event DP-004114-3 and methods for detection thereof
EP3370527B1 (fr) Événement transgénique de coton mon88702 et procédés de détection et d&#39;utilisation de celui-ci
WO2021216571A1 (fr) Événement de maïs transgénique mon87403 et procédés pour la détection de celui-ci
WO2021076346A1 (fr) Événement de maïs dp-202216-6 et empilement dp-023211-2
US20230135492A1 (en) Nucleic acid molecule of transgenic maize event me240913 that expresses cry1da protein, cell, plant and transgenic seed, uses thereof, plant product, method, kit and amplicon for detecting the event, and methods to produce a transgenic plant and to control lepidopteran insect pests
WO2019209700A1 (fr) Événement de maïs dp-032218-9 et procédés de détection de celui-ci
US20210381000A1 (en) Maize event dp-915635-4 and methods for detection thereof
CN114174517A (zh) 生物胁迫耐性植物和方法
US20240035100A1 (en) Soybean transgenic event gm_csm63770 and methods for detection and uses thereof
US20230220407A1 (en) Maize event dp-004114-3 and methods for detection thereof
OA18791A (en) Cotton transgenic event MON 88702 and methods for detection and uses thereof.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20793905

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20793905

Country of ref document: EP

Kind code of ref document: A1