阅读说明：本技术 玉米转基因事件mon 95379及其检测方法和用途 (Corn transgenic event MON95379, and detection method and application thereof ) 是由 H·M·安德森 S·L·布朗 R·A·卡瓦尔霍 A·A·卡斯特罗 K·M·邓克曼 A·J· 于 2019-07-26 设计创作，主要内容包括：本发明提供了一种转基因玉米事件MON 95379,包含事件MON 95379的植物、植物细胞、种子、植物部分、后代植物和商品产品。本发明还提供了对事件MON 95379具有特异性的多核苷酸,及使用和检测事件MON 95379的方法以及包含事件MON 95379的植物、植物细胞、种子、植物部分、后代植物和商品产品。(The present invention provides a transgenic corn event MON95379, plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising event MON 95379. The invention also provides polynucleotides specific for event MON95379, and methods of using and detecting event MON95379, as well as plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising event MON 95379.)

1. A recombinant DNA molecule comprising a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and the complete complementary sequences thereof.

2. The recombinant DNA molecule of claim 1, wherein said recombinant DNA molecule is derived from corn event MON95379, a representative sample of seed comprising said event having been deposited under ATCC accession No. PTA-125027.

3. A DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that specifically hybridizes under stringent hybridization conditions to corn event MON95379DNA in a sample, wherein detection of hybridization of the DNA molecule under the stringent hybridization conditions is diagnostic for the presence of corn event MON95379DNA in the sample.

4. The DNA molecule of claim 3, wherein the sample comprises a corn plant, a corn plant cell, a corn seed, a corn plant part, a corn progeny plant, a processed corn seed, an animal feed comprising corn, corn oil, corn flour, corn flakes, corn bran, pasta made from corn, corn biomass, and fuel products produced using corn and corn parts.

5. A pair of DNA molecules comprising a first DNA molecule and a second DNA molecule different from said first DNA molecule that, when used in an amplification reaction with a sample containing corn event MON95379 template DNA, serve as DNA primers to produce an amplicon diagnostic for the presence of said corn event MON95379DNA in said sample, wherein said amplicon comprises a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

6. A method of detecting the presence of a DNA segment diagnostic for corn event MON95379DNA in a sample, the method comprising:

a) contacting the sample with the DNA molecule of claim 3;

b) subjecting the sample and the DNA molecule to stringent hybridization conditions; and

c) detecting hybridization of said DNA molecule to said DNA in said sample,

wherein said detecting is diagnostic for the presence of said corn event MON95379DNA in said sample.

7. A method of detecting the presence of a DNA segment diagnostic for corn event MON95379DNA in a sample, the method comprising:

a) contacting the sample with the pair of DNA molecules of claim 5;

b) performing an amplification reaction sufficient to produce a DNA amplicon; and

c) detecting the presence of said DNA amplicon in said reaction,

wherein the DNA amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

8. A corn plant, corn plant part, corn cell, or part thereof comprising a recombinant polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

9. The corn plant, corn plant part, corn cell, or part thereof of claim 8, wherein the corn plant, corn plant part, corn cell, or part thereof is pesticidal when provided in the diet of a lepidopteran insect pest.

10. The corn plant, corn plant part, corn cell, or part thereof of claim 9, wherein said lepidopteran insect pest is selected from the group consisting of: fall armyworm (spodoptera frugiperda), corn earworm (cotton bollworm), southwest corn borer (southwest corn borer), sugarcane borer (sugarcane borer), and corn stalk borer (corn borer).

11. The maize plant, maize plant part, maize cell, or part thereof of claim 8, wherein said maize plant is further defined as a progeny of any generation of a maize plant comprising said maize event MON 95379.

12. A method of protecting a corn plant from infestation by insects, wherein the method comprises providing a pesticidally effective amount of cells or tissues of the corn plant comprising corn event MON95379 in the diet of a lepidopteran insect pest.

13. The method of claim 12, wherein the lepidopteran insect pest is selected from the group consisting of: fall armyworm (spodoptera frugiperda), corn earworm (cotton bollworm), southwest corn borer (southwest corn borer), sugarcane borer (sugarcane borer), and corn stalk borer (corn borer).

14. A method of producing an insect resistant corn plant, the method comprising:

a) subjecting two different corn plants to sexual cross, wherein at least one of said two different corn plants comprises transgenic corn event MON95379 DNA;

b) sampling seed or tissue from the progeny of the cross;

c) detecting the presence of a DNA segment diagnostic for corn event MON95379DNA in the sample from step b) to identify progeny comprising corn event MON95379 DNA; and

d) selecting said progeny comprising corn event MON95379 DNA.

15. A corn seed comprising a detectable amount of a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 or the complete complement thereof.

16. An inanimate corn plant material comprising a detectable amount of the recombinant DNA molecule of claim 1.

17. A microorganism comprising a detectable amount of the recombinant DNA molecule of claim 1.

18. The microorganism of claim 17, wherein the microorganism is a plant cell.

19. A commodity product comprising the recombinant DNA molecule of claim 1.

20. The commodity product of claim 19, further selected from the group consisting of: whole or processed corn seeds, animal feed comprising corn, corn oil, corn flour, corn flakes, corn bran, corn biomass, and fuel products produced using corn and corn fractions.

21. A corn plant, corn plant part, or corn seed thereof comprising DNA that serves as a template when tested in a DNA amplification method that produces an amplicon diagnostic for the presence of event MON95379 DNA.

22. A method of determining zygosity of a corn plant or corn seed comprising event MON95379, the method comprising:

a) contacting a sample comprising corn DNA with a primer pair capable of producing an amplicon encoding one of the toxin coding sequences of cry1b.868 or Cry1Da _ 7;

b) contacting the sample comprising corn DNA with a primer pair capable of producing an amplicon of an internal standard known to be single copy and homozygous in the corn plant;

c) contacting said DNA sample with a probe set comprising at least a first probe that specifically hybridizes to one of the toxin coding sequences encoding cry1b.868 or Cry1Da _7 and a second probe that specifically hybridizes to internal standard genomic DNA known to be single copy and homozygous in said corn plant;

d) performing a DNA amplification reaction using real-time PCR and determining a cycle threshold (Ct value) of an amplicon corresponding to the toxin-encoding sequence and the single-copy, homozygous internal standard;

e) calculating the difference (Δ Ct) between the Ct value of the single copy, homozygous internal standard amplicon and the Ct value of the toxin-encoding sequence amplicon; and

f) zygosity was determined, where a Δ Ct of about zero (0) indicates homozygosity for the inserted T-DNA of event MON 95739, and a Δ Ct of about one (1) indicates heterozygosity for the inserted T-DNA of event MON 95379.

23. The method of claim 22, wherein the primer pair is selected from the group consisting of: the combination of SEQ ID NO 18 and SEQ ID NO 19 and the combination of SEQ ID NO 21 and SEQ ID NO 22; and wherein the probes are SEQ ID NO:20 and SEQ ID NO: 23.

24. The method of claim 22, wherein the primer pair is selected from the group consisting of: the combination of SEQ ID NO 18 and SEQ ID NO 19 and the combination of SEQ ID NO 24 and SEQ ID NO 25; and wherein the probes are SEQ ID NO 20 and SEQ ID NO 26.

25. The method of claim 22, wherein a Δ Ct of about one (1) indicative of heterozygosity of the inserted T-DNA of event MON95379 is in the range of 0.75 to 1.25.

26. A method of determining zygosity of a corn plant or corn seed comprising event MON95379, the method comprising:

a) contacting a sample comprising corn DNA with a primer pair set comprising at least two different primer pairs capable of producing a first amplicon diagnostic for event MON95379 and a second amplicon diagnostic for native corn genomic DNA not comprising event MON 95379;

b) performing a nucleic acid amplification reaction with the sample and the primer pair set; and

c) detecting a first amplicon diagnostic for event MON95379 or a second amplicon diagnostic for native corn genomic DNA not comprising event MON95379 in the nucleic acid amplification reaction, wherein the presence of only the first amplicon is diagnostic for a corn plant or corn seed homozygous for event MON95379, and the presence of both the first amplicon and the second amplicon is diagnostic for a corn plant or corn seed heterozygous for event MON 95379; or

i) Contacting a sample comprising corn DNA with a set of probes comprising at least a first probe that specifically hybridizes to event MON95379DNA and at least a second probe that specifically hybridizes to corn genomic DNA interrupted by insertion of the heterologous DNA of event MON95379 and does not hybridize to event MON95379 DNA; and

ii) hybridizing said set of probes to said sample under stringent hybridization conditions, wherein detection of hybridization of only said first probe under said hybridization conditions is diagnostic of a corn plant or corn seed that is homozygous for event MON95379, and wherein detection of hybridization of both said first probe and said second probe under said hybridization conditions is diagnostic of a corn plant or corn seed that is heterozygous for event MON 95379.

27. The method of claim 26, wherein the primer pair group comprises a combination of SEQ ID No. 15 and SEQ ID No. 16 and a combination of SEQ ID No. 15 and SEQ ID No. 27.

28. The method of claim 26, wherein the set of probes comprises SEQ ID NOs 17 and 28.

Technical Field

The present invention relates to recombinant DNA molecules present in and/or isolated from corn event MON 95379. The invention also relates to transgenic corn plants, plant parts and seeds, cells, and agricultural products comprising corn event MON95379 and methods of using the same and detecting the presence of corn event MON 95379. Transgenic corn plants, parts, seeds, and cells containing corn event MON95379DNA exhibit resistance to infestation by insects of the lepidopteran family.

Background

Corn (maize) is an important crop and is a major food source in many parts of the world. Biotechnological methods have been applied to corn to improve agronomic traits and product quality. One such agronomic trait is insect resistance, which is achieved through expression of a heterologous insect toxin (also known as a transgene) inserted into the genome of a maize plant.

Expression of such transgenes in transgenic plants, plant parts, seeds or cells may be affected by a number of different factors, including the elements used in the cassette that drive expression of the transgene and the interactions between those elements in the cassette. This is further complicated for transgene insertions comprising two or more expression cassettes, each with a transgene conferring a separate trait, also referred to as polygenic transgenic events. A commercially useful polygenic transgenic event requires that each transgene in the transgene insertion be expressed in the manner necessary for each trait. To this end, a single expression cassette is first designed and tested in plants, and then for each trait an expression cassette is selected that exhibits the best insect activity without causing a negative phenotype due to expression. Next, the selected expression cassettes for one trait are combined with the selected expression cassettes for another trait into a single construct. Multiple constructs were designed using different orientations to provide optimal resistance and prevent negative phenotypes or negative agronomics, such as reduced yield. The constructs were tested to ensure that all expression cassettes functioned well together and that each transgene was correctly expressed. The selected combination and orientation of expression cassettes are then used as a single transgene insert to generate hundreds of transgene events, each event being the result of random insertion of the construct at a different genomic location.

Each transgenic event is unique in its molecular profile and in the point of chromosome insertion. Due to the variability involved in event creation, each unique event must be analyzed through multiple generations of plants-assessing molecular characteristics, protein expression efficacy, and agronomics in each step-in order to select superior events for commercial use. The performance of the event in the transgenic plant, plant part, seed or cell and its effectiveness may be influenced by the genomic location of the transgene insertion. Specifically, cis and/or trans factors influence the effectiveness of an event relative to the site of integration or chromatin structure. Events may have the same transgene insertion, but different transgene expression levels and performance across tissues and developmental stages in various germplasm or under specific growth conditions. There may also be undesirable phenotypic or agronomic differences between some events. Therefore, there is a need to generate and analyze a large number of individual plant transformation events in order to select for events with superior characteristics relative to a desired trait, as well as optimal phenotypic and agronomic characteristics needed to make them suitable for commercial purposes. Furthermore, to create multigenic events for commercial use requires rigorous molecular characterization, greenhouse testing, and field trials over the years at multiple locations and under multiple conditions, so extensive agronomic, phenotypic, and molecular data can be collected. Scientists and agronomic engineers must then analyze the generated data to select events for business purposes. Once selected, such events can be introgressed into new germplasm suitable for specific local growth conditions as a single locus with multiple insect resistance traits using plant breeding methods and stacked/combined by breeding with other different events that confer different traits than those conferred by the events of the invention.

Disclosure of Invention

The present invention provides a novel transgenic corn event-MON 95379 that provides pesticidal control of corn lepidopteran pests. The invention also provides transgenic plants, plant cells, seeds, plant parts, and commodity products comprising event MON 95379. In another embodiment, the invention provides polynucleotides specific for event MON95379 and plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising event MON 95379. In yet another embodiment, a method related to event MON95379 is provided.

Accordingly, in one aspect, the present invention provides a recombinant DNA molecule comprising a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and the complete complementary sequences thereof.

In one embodiment, the recombinant DNA molecule is derived from corn event MON95379 in a seed sample comprising said event that has been deposited under ATCC accession No. PTA-125027.

Another aspect of the invention provides a DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that specifically hybridizes under stringent hybridization conditions to corn event MON95379DNA in a sample, wherein detection of hybridization of said DNA molecule under said stringent hybridization conditions is diagnostic for the presence of corn event MON95379DNA in said sample. In certain embodiments, the sample comprises a corn plant, a corn plant cell, a corn seed, a corn plant part, a corn progeny plant, a processed corn seed, an animal feed comprising corn, corn oil, corn flour, corn flakes, corn bran, pasta made from corn, corn biomass, and fuel products produced using corn and corn parts.

Yet another aspect of the invention provides a pair of DNA molecules comprising a first DNA molecule and a second DNA molecule different from the first DNA molecule, which pair of DNA molecules, when used in an amplification reaction with a sample comprising corn event MON95379 template DNA, serve as DNA primers to produce an amplicon diagnostic for the presence of said corn event MON95379DNA in said sample, wherein said amplicon comprises a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

Another embodiment of the invention is a method of detecting the presence of a DNA segment diagnostic for corn event MON95379DNA in a sample, said method comprising: a) contacting the sample with a DNA molecule that acts as a probe and specifically hybridizes under stringent conditions to corn event MON 95379; b) subjecting the sample and the DNA molecule to stringent hybridization conditions; and c) detecting hybridization of said DNA molecule to said DNA in said sample, wherein said detection is diagnostic for the presence of said corn event MON95379DNA in said sample.

Yet another embodiment of the invention is a method of detecting the presence of a DNA segment diagnostic for corn event MON95379DNA in a sample, said method comprising: a) contacting said sample with a pair of DNA molecules of the invention; b) performing an amplification reaction sufficient to produce a DNA amplicon; and c) detecting the presence of the DNA amplicon in the reaction, wherein the DNA amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

Another embodiment of the invention is a corn plant, corn plant part, corn cell, or part thereof comprising a recombinant polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. The corn plant, corn plant part, corn cell, or part thereof is pesticidal when provided in the diet of a lepidopteran insect pest. Lepidopteran pests may include fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), southwestern corn borer (Diatraea grandiosella), sugarcane borer (Diatraea saccharalis), and corn stalk borer (Elasmopalpus lignosellus). Additionally, a corn plant may be further defined as a progeny of any generation of a corn plant comprising corn event MON 95379.

Yet another embodiment of the present invention is a method of protecting a corn plant from infestation by insects, wherein the method comprises providing a pesticidally effective amount of cells or tissues of the corn plant comprising corn event MON95379 in the diet of a lepidopteran insect pest. Likewise, lepidopteran insect pests that are contemplated include fall armyworm (spodoptera frugiperda), corn earworm (cotton bollworm), southwestern corn borer (southwestern corn borer), sugarcane borer (sugarcane borer), and corn borer (corn borer).

Another embodiment of the invention is a method of producing an insect resistant corn plant comprising: a) subjecting two different corn plants to sexual cross, wherein at least one of said two different corn plants comprises transgenic corn event MON95379 DNA; b) sampling seed or tissue from the progeny of step (a); c) detecting progeny comprising corn event MON95379DNA in the sample from step (b); and d) selecting said progeny comprising corn event MON95379 DNA.

Yet another embodiment of the invention is a corn seed, inanimate corn plant material, or microorganism comprising a detectable amount of a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 or the complete complement thereof.

Yet another embodiment of the invention is a commercial product comprising a nucleotide sequence selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 or the complete complement thereof. Contemplated commodity products include whole or processed corn seeds, animal feed including corn, corn oil, corn flour, corn flakes, corn bran, corn biomass, and fuel products produced using corn and corn fractions.

Another embodiment of the invention is a corn plant, corn plant part, or corn seed thereof comprising DNA that serves as a template when tested in a DNA amplification method that produces an amplicon diagnostic for the presence of event MON95379 DNA.

Yet another embodiment of the invention is a method of determining the zygosity of a corn plant or corn seed comprising event MON95379, comprising: a) contacting a sample comprising corn DNA with a primer pair capable of producing an amplicon encoding one of the toxin coding sequences of cry1b.868 or Cry1Da _ 7; b) contacting said sample comprising corn DNA with a primer pair capable of producing an amplicon of an internal standard known to be single copy and homozygous in corn plants; c) contacting the DNA sample with a probe set comprising at least a first probe that specifically hybridizes to one of the toxin coding sequences encoding cry1b.868 or Cry1Da _7, and a second probe that specifically hybridizes to an internal standard genomic DNA known to be single copy and homozygous in a corn plant; d) performing a DNA amplification reaction using real-time PCR and determining a cycle threshold (Ct value) for amplicons corresponding to the toxin coding sequence and the single copy, homozygous internal standard; e) calculating the difference (delta Ct) between the Ct value of the single copy, homozygous internal standard amplicon and the Ct value of the toxin-encoding sequence amplicon; and f) determining zygosity, wherein a Δ Ct of about zero (0) indicates homozygosity for inserted T-DNA of event MON 95739 and a Δ Ct of about one (1) indicates heterozygosity for inserted T-DNA of event MON 95379. In certain embodiments of this method, the primer pair is selected from the group consisting of: the combination of SEQ ID NO 18 and SEQ ID NO 19 and the combination of SEQ ID NO 21 and SEQ ID NO 22; and the probes are SEQ ID NO 20 and SEQ ID NO 23. In another embodiment, the primer pair is selected from the group consisting of: the combination of SEQ ID NO 18 and SEQ ID NO 19 and the combination of SEQ ID NO 24 and SEQ ID NO 25; and the probes are SEQ ID NO 20 and SEQ ID NO 26. In yet another embodiment of the invention, a Δ Ct of about one (1) indicative of heterozygosity of inserted T-DNA of event MON95379 is in the range of 0.75 to 1.25.

Yet another embodiment of the invention is a method of determining the zygosity of a corn plant or corn seed comprising event MON95379, comprising: a) contacting a sample comprising corn DNA with a primer pair set comprising at least two different primer pairs capable of producing a first amplicon diagnostic for event MON95379 and a second amplicon diagnostic for native corn genomic DNA not comprising event MON 95379; i) carrying out nucleic acid amplification reaction by using the sample and the primer pair group; ii) detecting a first amplicon diagnostic for event MON95379 or a second amplicon diagnostic for native corn genomic DNA not comprising event MON95379 in a nucleic acid amplification reaction, wherein the presence of only the first amplicon is diagnostic for a corn plant or corn seed homozygous for event MON95379 and the presence of both the first amplicon and the second amplicon is diagnostic for a corn plant or corn seed heterozygous for event MON 95379; or b) contacting a sample comprising corn DNA with a probe set comprising at least a first probe that specifically hybridizes to event MON95379DNA and at least a second probe that specifically hybridizes to corn genomic DNA interrupted by insertion of the heterologous DNA of event MON95379 and does not hybridize to event MON95379 DNA; i) hybridizing the probe set to the sample under stringent hybridization conditions, wherein detection of hybridization of only the first probe under the hybridization conditions is diagnostic of a corn plant or corn seed that is homozygous for event MON95379, and wherein detection of hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic of a corn plant or corn seed that is heterozygous for event MON 95379. In one embodiment of this method, the primer pair group comprises the combination of SEQ ID NO. 15 and SEQ ID NO. 16 and the combination of SEQ ID NO. 15 and SEQ ID NO. 27. In another embodiment of this method, the probe set comprises SEQ ID NO 17 and SEQ ID NO 28.

The above and other aspects of the present invention will become more apparent from the following detailed description.

Drawings

Figure 1 shows the sequence of corn event MON 95379. The horizontal lines and boxes correspond to the positions of SEQ ID NO 1([1]), SEQ ID NO 2([2]), SEQ ID NO 3([3]), SEQ ID NO 4 ([4]), SEQ ID NO 5([5]), SEQ ID NO 6([6]), SEQ ID NO 7([7]), SEQ ID NO 8([8]), SEQ ID NO 9([9]), SEQ ID NO 11([11]), and SEQ ID NO 12([12]) relative to SEQ ID NO 10([10 ]). Horizontal arrows labeled SQ51219 (SEQ ID NO:15) ([15]), SQ21524(SEQ ID NO:16) ([16]), SQ50998(SEQ ID NO:21) ([21]), SQ50997(SEQ ID NO:22) ([22]), SQ50485(SEQ ID NO:24) ([24]), and SQ50484(SEQ ID NO:25) ([25]) indicate the approximate positions of the primer sets that can be used to detect corn event MON 95379. The horizontal arrows labeled PB10269(SEQ ID NO:17) ([17]), PB50340(SEQ ID NO:23) ([23]), PB50138(SEQ ID NO:26) ([26]) represent the approximate locations of DNA probes that can be used to detect corn event MON 95379. "E" represents an enhancer element, "P" represents a promoter element, "L" represents a leader (5 'UTR) element, "I" represents an intron element, "T" represents a 3' UTR, "Cry1B.868" represents a Cry1B.868 coding sequence element, "Cry 1Da _ 7" represents a Cry1Da _7 coding sequence element, "LoxP" represents a site at which a Cre-recombinase marker excision occurs, leaving one of two LoxP sites after the marker excision, and "LB" represents a left T-DNA border.

FIG. 2 is a schematic of the T-DNA cassette before integration to form event MON95379, after integration, and after Cre-excision. The top horizontal box represents the T-DNA cassette in the plasmid vector used for transformation of event MON95379, presented as SEQ ID NO 13([13]) ("Pre-integrated T-DNA"). The horizontal arrows [13] below represent the individual genetic elements contained in the two transgene cassettes. "LB" represents the left T-DNA border element, "E" represents the enhancer element, "P" represents the promoter element, "L" represents the leader (5 'UTR) element, "I" represents the intron element, "T" represents the 3' UTR, "Cry 1 B.868" represents the Cry1B.868 coding sequence element, "Cry 1Da _ 7" represents the Cry1Da _7 coding sequence element, "CP 4" represents the CP4 selectable marker, "TS" represents the targeting sequence, "LoxP" represents the site where the Cre recombinase marker excision occurs, and "RB" represents the right T-DNA border element. The middle horizontal box "inserted T-DNA after integration" denotes the T-DNA cassette integrated into the maize genome after transformation, wherein the right T-DNA border (RB) is lost during integration. The bottom horizontal box "inserted T-DNA after Cre excision" indicates the integrated T-DNA cassette after excision of the CP4 selectable marker cassette, leaving one of the two LoxP sites and the LB region.

Fig. 3 is a schematic of a breeding process that produces marker-free corn event MON 95379. R₀A generative event ("transformant") is the event obtained from the initial transformation using the binary transformation vector used to generate corn event MON 95379. Subsequent generation of "R" (R)₁And R₂) Representing successive generations produced by self-pollination of plants resulting from the initial R producing maize event MON95379₀The transformant was obtained. Inserting R homozygous for T-DNA₂Transformants were cross-pollinated with elite transgenic maize lines containing the transgenic cassette for expression of Cre recombinase, resulting in generation F1, many of which were deprived of the CP4 selectable marker cassette by Cre recombinase excision. Selection of hemizygous T-DNA positive, CP4 negative plants and self-pollination to yield F₂And (4) generation. Selection of F homozygous for the inserted T-DNA allele and lacking the CP4 marker and lacking the Cre recombinase transgene cassette₂Plants and self-pollinated to produce F₃And (4) generation. F₃The progeny are self-pollinated to produce F₄Pure lines of Gold Standard seeds (Gold Standard Seed).

Brief description of the sequences

SEQ ID NO 1 is a 50 nucleotide sequence representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO 1 is found at nucleotide positions 838-887 within SEQ ID NO 10.

SEQ ID NO 2 is a 50 nucleotide sequence representing the 3' junction region of the integrated transgene expression cassette and the maize genomic DNA. SEQ ID NO 2 is found at nucleotide position 14,156-14,205 within SEQ ID NO 10.

3 is a 100 nucleotide sequence representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO 3 is found at nucleotide position 813-912 within SEQ ID NO 10.

SEQ ID NO 4 is a 100 nucleotide sequence representing the 3' junction region of the integrated transgene expression cassette and the maize genomic DNA. SEQ ID NO. 4 is found at nucleotide position 14,131 and 14,230 within SEQ ID NO. 10.

SEQ ID NO 5 is a 200 nucleotide sequence representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO 5 is found at nucleotide position 763-962 within SEQ ID NO 10.

SEQ ID NO 6 is a 200 nucleotide sequence representing the 3' junction region of the integrated transgene expression cassette and the maize genomic DNA. SEQ ID NO 6 is found at nucleotide position 14,081-14,280 within SEQ ID NO 10.

SEQ ID NO 7 is a 1,160 nucleotide sequence representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO. 7 is found at nucleotide positions 1-1,160 within SEQ ID NO. 10.

SEQ ID NO 8 is a sequence of 1,178 nucleotides representing the 3' junction region of the integrated transgene expression cassette and the maize genomic DNA. SEQ ID NO 8 is found at nucleotide position 14,039-15,216 within SEQ ID NO 10.

SEQ ID NO 9 is a 13,318 nucleotide sequence corresponding to the T-DNA of the transgene insertion of maize event MON 95379.

10 is a sequence of 15,216 nucleotides corresponding to the contig nucleotide sequence of the 5 'genomic flanking DNA nucleotide sequence, the T-DNA nucleotide sequence inserted in event MON95379 and the 3' genomic flanking DNA nucleotide sequence; and includes SEQ ID NO 11 (nucleotides 1-862), SEQ ID NO 9 (nucleotides 863-14,180) and SEQ ID NO 12 (nucleotides 14,181-15,216).

SEQ ID NO 11 is a sequence of 862 nucleotides representing the 5' flanking maize genomic DNA up to the inserted T-DNA. SEQ ID NO. 11 is found at nucleotide positions 1-862 within SEQ ID NO. 10.

SEQ ID NO 12 is a sequence of 1,036 nucleotides representing the 3' flanking maize genomic DNA after the inserted T-DNA. SEQ ID NO. 12 is found at nucleotide positions 14,181-15,216 within SEQ ID NO. 10.

SEQ ID No. 13 is a sequence of 18,376 nucleotides representing a transgene cassette contained within a binary plasmid transformation vector used to transform maize to produce maize event MON 95379.

SEQ ID NO 14 is a 35 nucleotide sequence representing a LoxP site for Cre-mediated excision and recombination. The remaining LoxP site after marker excision can be found at nucleotide position 1,080-1,114 within SEQ ID NO. 10.

SEQ ID NO. 15 is a 20 nucleotide sequence corresponding to the thermal amplification primer designated SQ51219 that is used to identify maize event MON95379DNA in a sample and is identical to the nucleotide sequence corresponding to position 833-852 of SEQ ID NO. 10.

16 is a 30 nucleotide sequence corresponding to the thermal amplification primer designated SQ21524 which is used to identify the maize event MON95379DNA in a sample and is identical to the reverse complement of the nucleotide sequence corresponding to position 905-934 of SEQ ID NO 10.

SEQ ID NO 17 is a 16 nucleotide sequence corresponding to the probe designated PB10269, which is used to identify maize event MON95379DNA in a sample and is identical to the reverse complement of the nucleotide sequence corresponding to position 886-901 of SEQ ID NO 10.

18 is a 24 nucleotide sequence corresponding to the thermal amplification primer named SQ20222, which serves as an internal control for the event and zygosity assays of MON95379 and hybridizes to a region of the maize genome.

SEQ ID NO 19 is a 28 nucleotide sequence corresponding to a thermal amplification primer designated SQ20221, which serves as an internal control for event and zygosity assays of MON95379 and hybridizes to a region of the maize genome.

SEQ ID No. 20 is a 29 nucleotide sequence corresponding to a probe designated PB50237, which serves as an internal control for event and zygosity assays of MON95379 and hybridizes to a region of the maize genome.

21 is a 20 nucleotide sequence corresponding to the thermal amplification primer named SQ50998, which was used in the zygosity assay of event MON95379 and hybridizes to the coding sequence of cry1b.868 within SEQ ID No. 10; and is identical to the nucleotide sequence corresponding to position 2,809-2,828 of SEQ ID NO 10.

22 is a 20 nucleotide sequence corresponding to the thermal amplification primer named SQ50997 used in the zygosity assay of event MON95379 and hybridizes to the coding sequence of cry1b.868 within SEQ ID No. 10; and is identical with the reverse complement of the nucleotide sequence corresponding to position 2,852-one 2,871 of SEQ ID NO. 10.

SEQ ID No. 23 is an 18 nucleotide sequence corresponding to probe designated PB50340, which was used in the zygosity assay of event MON95379 and hybridizes to the coding sequence of cry1b.868 within SEQ ID No. 10; and is identical with the reverse complement of the nucleotide sequence corresponding to position 2,833-and 2,850 of SEQ ID NO. 10.

24 is a 19 nucleotide sequence corresponding to the thermal amplification primer named SQ50485, used in the zygosity assay of event MON95379 and hybridized to the coding sequence of Cry1Da — 7 within SEQ ID NO 10; and is identical to the nucleotide sequence corresponding to position 12,820-12,838 of SEQ ID NO. 10.

SEQ ID NO. 25 is an 18 nucleotide sequence corresponding to a thermal amplification primer named SQ50484, which was used in the zygosity assay of event MON95379 and hybridizes to the coding sequence of Cry1Da _7 within SEQ ID NO. 10; and is identical with the reverse complement of the nucleotide sequence corresponding to position 12,855-12,872 of SEQ ID NO. 10.

26 is a 14 nucleotide sequence corresponding to the probe designated PB50138, used in the zygosity assay of event MON95379 and hybridized to Cry1Da — 7 coding sequence within SEQ ID NO 10; and is identical with the reverse complement of the nucleotide sequence corresponding to position 12,840-12,853 of SEQ ID NO 10.

SEQ ID No. 27 is a 21 nucleotide sequence corresponding to a thermal amplification primer called PNEGDNA that was used in the zygosity assay of event MON95379 and hybridizes to a region of corn genomic DNA that was deleted when the T-DNA used to produce event MON95379 was inserted into the corn genome. The amplicon derived from primer SQ51219 in combination with PNEGDNA was diagnostic for the wild type allele of T-DNA lacking the event MON95379 insertion.

28 is a 14 nucleotide sequence corresponding to a probe called PRBNEGDNA used in the zygosity assay of event MON95379 and which hybridizes to a region of corn genomic DNA that is deleted when the T-DNA used to produce event MON95379 is inserted into the corn genome.

Detailed Description

The present invention provides a transgenic corn event-MON 95379 that achieves pesticidal control of corn lepidopteran pests by expressing cry1b.868 and Cry1Da _ 7. In particular, expression of cry1b.868 and Cry1Da _7 insect arrestin in corn event MON95379 provides resistance to the following lepidopteran insect pests: fall armyworm (spodoptera frugiperda), corn earworm (cotton bollworm), southwest corn borer (southwest corn borer), sugarcane borer (sugarcane borer), and corn stalk borer (corn borer). Event MON95379 would satisfy the great demand in the corn market for control of these insects because chemical insecticides generally do not provide adequate control of these insects, or require multiple applications during the growing season, thereby increasing the environmental input of chemical insecticides and increasing corn production costs.

It should be understood that reference to event MON95379 is equivalent to reference to event MON 95379; they are interchangeable and represent the same transgenic maize event.

Plant transformation techniques are used to randomly insert foreign DNA (also known as transgenic DNA) into the chromosome of the genome of a cell to produce genetically engineered cells, also known as "transgenic" or "recombinant" cells. Many individual cells are transformed using this technique, each cell producing a unique "transgenic event" or "event" due to random insertion of foreign DNA into the genome. Transgenic plants are then regenerated from each individual transgenic cell. The transgenic plants thus produced each contain a unique inserted transgenic event as a stable part of their genome. This transgenic plant can then be used to produce progeny plants, each containing a unique transgenic event.

Corn event MON95379 was produced by an agrobacterium-mediated transformation process of a corn immature embryo with a single T-DNA binary system. In this system, Agrobacterium strains are used which employ a binary plasmid vector with a single T-DNA. The T-DNA construct comprises two transgenic cassettes for expression of insect toxin coding sequences encoding cry1b.868 and Cry1Da _7, and a transgenic cassette for selection of transformed maize cells by glyphosate selection (CP 4). The T-DNA construct is SEQ ID NO 13 and is shown in FIG. 2 ("T-DNA before integration"). During integration, the right T-DNA border is lost, as shown in FIG. 2 ("inserted T-DNA after integration"). The glyphosate selection cassette is flanked by LoxP recognition sites that are recognized by the Cre-recombinase derived from Enterobacter phage P1(Larry Gilbertson (2003) Cre-lox recombination: Cre-active tools for plant Biotechnology TRENDS in Biotechnology,21:12, 550-555).

As specifically described herein, corn event MON95379 was produced by a complex development process in which: (1) hundreds of plasmid vector constructs were developed-which differed in the coding sequence for the pesticidal protein, the coding sequence for the transcriptional regulatory elements, and the number and orientation of cassettes within the constructs-and these constructs were transformed into maize cells to create thousands of events that were tested and analyzed, thereby selecting the construct for generating event MON 95379; (2) transforming hundreds of corn cells with a construct for producing event MON95379, generating a population of transgenic plants, wherein each plant comprises a unique transgenic event that is regenerated and tested; (3) the final event MON95379 was selected after a rigorous multi-year event selection process involving testing and analysis of molecular characteristics, efficacy, protein expression, and agronomic characteristics in a variety of genetic backgrounds; and (4) removal of the glyphosate selection cassette from corn event MON95379 by in vivo Cre excision to create a "marker-free" final event MON 95379. Corn event MON95379 is thus produced and selected as a unique elite event suitable for large-scale agronomic purposes.

Plasmid DNA inserted into the genome of corn event MON95379 was characterized by detailed molecular analysis. This analysis includes: the number of insertions (the number of integration sites within the maize genome), the location of the genome insertion (the specific site in the maize genome at which the insertion occurs), the number of copies (the number of T-DNA copies within a locus), and the integrity of the DNA into which the transgene is inserted. Detailed molecular analysis showed that the integrated T-DNA comprising the cry1b.868 and Cry1Da _7 expression cassettes remained intact following integration of the glyphosate (CP4) selection cassette and Cre excision. As used herein, an "expression cassette" or "cassette" is a recombinant DNA molecule comprising a combination of different elements to be expressed by a transformed cell. Table 1 provides a list of the elements contained in SEQ ID No. 10, the DNA sequence corresponding to corn event MON 95379.

TABLE 1 description of corn event MON95379

Maize event MON95379 is characterized by insertion into a single locus of the maize genome, thereby creating two new loci or linked sequences (e.g., the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8) that are not known to occur naturally in the maize genome or other transgenic maize event-they are unique to event MON 95379-spanning a portion of the inserted DNA and the maize genomic DNA. These linker sequences are useful for detecting the presence of event MON95379 in corn cells, corn tissue, corn seeds, and corn plants or corn plant products (such as corn commodity products). Described herein are DNA molecular probes and primer pairs that have been developed to identify the presence of these different ligated segments in a biological sample containing or suspected of containing corn cells, corn seeds, corn plant parts, or corn plant tissue having event MON 95379.

A sample is intended to refer to a composition of substantially pure corn DNA or a composition comprising corn DNA. In either case, the sample is a biological sample, i.e., it comprises biological material, including but not limited to DNA obtained or obtained directly or indirectly from the genome of corn event MON 95379. "directly" refers to the ability of one skilled in the art to obtain DNA directly from the maize genome by disrupting maize cells (or by obtaining a maize sample containing disrupted maize cells) and exposing the genomic DNA for detection purposes. By "indirectly" is meant the ability of one of skill in the art to obtain target DNA or specific reference DNA (i.e., the novel and unique linking segments described herein) that is diagnostic for the presence of event MON95379 in a particular sample by means other than directly obtaining or obtaining a corn sample comprising disrupted corn cells via corn cell disruption. Such indirect means include, but are not limited to, amplification of DNA segments comprising DNA sequences targeted by specific probes designed to specifically bind to the target sequence; or amplifying a segment of DNA that can be measured and characterized, i.e., by separation from other segments of DNA via some effective matrix such as agarose or acrylamide gels; or by direct sequence analysis of the amplicons, or cloning the amplicons into vectors and direct sequencing of the inserted amplicons present within such vectors.

Detailed molecular analysis showed that event MON95379 contained a single T-DNA insertion with one copy of each of cry1b.868 and Cry1Da _7 expression cassettes. In event MON95379, no additional elements were identified from the transformation construct except for the agrobacterium tumefaciens left border region used to transfer the transgenic DNA from the plant transformation plasmid to the corn genome. Finally, thermal amplification and DNA sequence analysis of the specific amplicon producing the presence of the diagnosable event MON95379 were performed to determine the genomic ligation of the randomly assigned 5 'and 3' inserts into the plant (insert-to-plant), confirm the organization of the elements within the insert, and determine the complete DNA sequence of the inserted transgenic DNA (SEQ ID NO: 9). 11 is a sequence representing: the 5' LH244 maize genomic DNA sequence of eight hundred sixty-two (862) base pairs (bp) flanked by the inserted T-DNA sequence shown as SEQ ID NO 9. 12 is a sequence representing: thirty-six (1,036) bp of the 3' LH244 maize genomic DNA sequence was flanked by the inserted T-DNA sequence shown as SEQ ID NO 9. SEQ ID NO 7 is a sequence representing: the 5' LH244 maize genomic DNA sequence of eight hundred sixty-two (862) base pairs (bp) flanking the inserted T-DNA sequence, combined with two hundred ninety-eight (298) bp of the inserted T-DNA sequence represented as SEQ ID NO 9. SEQ ID NO 8 is a sequence representing: one hundred forty-two (142) bp of inserted T-DNA sequence, and one thousand thirty-six (1,036) bp of 3' LH244 maize genomic DNA sequence flanked by the inserted T-DNA sequence shown as SEQ ID NO: 9. 10 corresponds to maize event MON95379 and contains a contiguous sequence (contig) comprising the 5 'LH 244 flanking sequence, the transgene insert of event MON95379 and the 3' LH244 flanking sequence, and thus contains the genomic junction sequence inserted into the plant.

Unless otherwise indicated herein, terms should be understood by those of ordinary skill in the relevant art based on conventional usage. Definitions of terms commonly used in Molecular biology can be found in Rieger et al, Glossary of Genetics: Classical and Molecular, 5 th edition, Springer-Verlag: New York,1991 and Lewis, Genes V, Oxford University Press: New York, 1994, as well as other sources known to those of ordinary skill in the art. As used herein, the term "corn" refers to a species belonging to the genus zea, preferably corn, and includes all plant varieties that can be bred with corn plants containing event MON95379, including wild zea mays species as well as those belonging to the genus zea that allow breeding between species.

The present invention provides transgenic plants transformed with a DNA construct containing an expression cassette that expresses toxic amounts of the insecticidal proteins cry1b.868 and Cry1Da _ 7. A toxic amount refers to an effective amount, an insecticidal amount, an insecticidally effective amount, a target insect inhibitory amount, an effective insecticidal amount, an insecticidal amount in a lepidopteran insect diet, and other similar terms as would be understood by one of ordinary skill in the relevant art in light of routine usage. Corn plants transformed according to the methods disclosed herein and with the DNA constructs disclosed herein are resistant to lepidopteran insect pests.

Transgenic "plants" were generated by: transforming a plant cell with heterologous DNA (i.e., a polynucleic acid construct comprising a number of useful target features); inserting the transgene into the genome of the plant cell to realize plant regeneration; and selecting a specific plant characterized by the number of effective features of the transgenic plant inserted into the specific genomic position and regenerated. The term "event" refers to DNA from the original transformant that contains the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA. Such DNA is unique and is expected to be transferred into progeny that receive inserted DNA comprising the transgene of interest as a result of sexual crossing of the parental line comprising the inserted DNA (e.g., progeny produced by original transformants and selfing) with the parental line not comprising the inserted DNA. The invention also provides original transformant plants comprising the heterologous DNA and progeny of the transformants. Such progeny may be produced by a sexual outcross between a plant comprising the event and another plant, wherein the progeny comprises the heterologous DNA. This event is present in the progeny of the cross at the same chromosomal location even after repeated backcrossing with the recurrent parent.

As used herein, the term "recombinant" refers to a non-native DNA, protein, or organism that is not normally found in nature and that results from human intervention. A "recombinant DNA molecule" is a DNA molecule that comprises a combination of DNA molecules that do not occur together in nature and are the result of human intervention. For example, a DNA molecule consisting of a combination of at least two DNA molecules heterologous to each other, such as a DNA molecule comprising a transgene and plant genomic DNA adjacent to the transgene, is a recombinant DNA molecule.

The terms "DNA" and "DNA molecule" as referred to herein refer to deoxyribonucleic acid (DNA) molecules. The DNA molecule may be of genomic or synthetic origin and is conventionally from the 5 '(upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. Conventionally, the DNA sequences of the invention and fragments thereof are disclosed with reference to only one of the two complementary DNA sequence strands. By implication and intent, the complement of the sequences provided herein (the sequence of the complementary strand) is also referred to in the art as the reverse complement, which is within the scope of the present invention and is expressly intended to be within the scope of the claimed subject matter.

As used herein, the term "fragment" refers to a smaller portion of the whole. For example, SEQ ID NO:10 will include the following sequences, which are SEQ ID NOs: 10, at least about 12 consecutive nucleotides, at least about 13 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 15 consecutive nucleotides, a sequence of at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, at least about 50 consecutive nucleotides, at least about 60 consecutive nucleotides, at least about 70 consecutive nucleotides, at least about 80 consecutive nucleotides, at least about 90 consecutive nucleotides, or at least about 100 consecutive nucleotides of the complete sequence.

Reference in this application to an "isolated DNA molecule" or equivalent term or phrase is intended to mean that the DNA molecule is one that exists alone or in combination with other compositions, but is not in its natural environment. For example, a nucleic acid element that is naturally found within the genomic DNA of an organism, such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcription termination sequence, and the like, is not considered "isolated" as long as the element is at a location within the genome of the organism and within its naturally occurring genome. However, as long as each of these elements, and sub-portions of these elements, are not at a location within the genome of an organism and not within its naturally occurring genome, the elements will be "isolated" within the scope of the present disclosure. Similarly, a nucleotide sequence encoding a pesticidal protein or any naturally occurring pesticidal variant of that protein will be an isolated nucleotide sequence as long as the nucleotide sequence is not within the DNA of the bacterium in which the sequence encoding the protein is naturally found. For the purposes of this disclosure, synthetic nucleotide sequences encoding the amino acid sequences of naturally occurring pesticidal proteins are considered isolated. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a cell of a plant or bacterium or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it be present in a plasmid or similar structure used to transform cells within the genome of a plant or bacterium, or in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacterium. In any case, an isolated DNA molecule is a chemical molecule, whether referred to as a nucleic acid, a nucleic acid sequence, a polynucleotide sequence, or the like. It is a novel inventive molecule that exhibits industrial utility both when present in a plant cell or plant genome and when present outside of a plant cell, and therefore exhibits and is intended to exhibit such utility wherever the molecule is located.

DNA sequences spanning the region of linkage to the phosphodiester bond of the flanking maize genomic DNA through one end of the transgene insert are referred to as "links". Ligation is the point of attachment of the transgene insert to the flanking DNA as one continuous molecule. One linkage was found at the 5 'end of the transgenic insert and the other linkage was found at the 3' end of the transgenic insert, referred to herein as the 5 'linkage and the 3' linkage, respectively. "linker sequence" refers to a DNA sequence of any length spanning either the 5 'or 3' linkage of an event. The linker sequence for maize event MON95379 using SEQ ID NO 10 will be apparent to those skilled in the art. Examples of linker sequences for event MON95379 are provided as SEQ ID NOs 1-8. FIG. 1 shows the physical arrangement of the linker sequences arranged from 5 'to 3' relative to SEQ ID NO 10. The linker sequence of event MON95379 can be present as part of the genome of a plant, seed, or cell containing event MON 95379. The identification of any one or more of the connecting sequences in a sample from a plant, plant part, seed, or cell indicates that the DNA was obtained from corn containing event MON95379 and is diagnostic for the presence of event MON 95379.

The linker sequence of event MON95379 may be represented by a sequence selected from the group consisting of seq id no:1, 2, 3, 4, 5, 6, 7, 8 and 10. For example, the linker sequence can be arbitrarily represented by the nucleotide sequences provided by SEQ ID NO. 1 and SEQ ID NO. 2. Alternatively, the linker sequence may be arbitrarily represented by the nucleotide sequences provided by SEQ ID NO. 3 and SEQ ID NO. 4. Alternatively, the linker sequence may be arbitrarily represented by the nucleotide sequences provided by SEQ ID NO. 5 and SEQ ID NO. 6. Alternatively, the linker sequence may be arbitrarily represented by the nucleotide sequences provided in SEQ ID NO. 7 and SEQ ID NO. 8. These nucleotides are linked by phosphodiester bonds and in maize, event MON95379 exists as part of the recombinant plant cell genome.

Identification of one or more of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 10 in a sample derived from a maize plant, maize seed or maize plant part can diagnose: DNA was obtained from corn event MON 95379. Accordingly, the present invention provides a DNA molecule comprising at least one of the nucleotide sequences provided as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10. Any segment of DNA derived from transgenic corn event MON95379 sufficient to comprise at least one of the sequences provided as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10 is within the scope of the present invention. In addition, any polynucleotide comprising a sequence complementary to any of the sequences described in this paragraph is within the scope of the invention.

The present invention provides exemplary DNA molecules that can be used as primers or probes for detecting the presence of corn plant-derived DNA comprising event MON95379DNA in a sample. Such primers or probes are specific for a target nucleic acid sequence and are therefore suitable for use in identifying a corn event MON95379 nucleic acid sequence by the methods of the invention described herein.

A "probe" is a nucleic acid molecule that is complementary to a target nucleic acid strand and is suitable for use in hybridization methods. The probe may be linked to a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent or enzyme. Such probes are complementary to target nucleic acid strands, and in the context of the present invention, to DNA strands from event MON95379, whether from a plant containing event MON95379 or from a sample comprising event MON95379 DNA. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence. An exemplary DNA sequence suitable for use as a probe for detecting corn event MON95379 is provided as: SEQ ID NO 17(PB10269), SEQ ID NO 23 (PB50340), SEQ ID NO 26(PB 50138).

"primers" are typically DNA molecules designed for use in a particular annealing or hybridization method involving thermal amplification. A pair of primers can be used in a thermal amplification, such as a Polymerase Chain Reaction (PCR), with a template DNA, such as a corn genomic DNA sample, to produce an amplicon, wherein the amplicon produced by the reaction will have a DNA sequence corresponding to the template DNA sequence located between the two sites of hybridization of the primers to the template.

A primer is typically designed to hybridize to a complementary target DNA strand to form a hybrid between the primer and the target DNA strand, and the presence of the primer is a recognition point for a polymerase, thereby initiating extension of the primer (i.e., polymerizing additional nucleotides into an extended nucleotide molecule) using the target DNA strand as a template. A primer pair refers to a polynucleotide segment between the locations to which individual members of the primer pair are targeted for binding, typically in a thermal amplification reaction or other conventional nucleic acid amplification method, using two primers that bind to opposite strands of a double-stranded nucleotide segment. Exemplary DNA molecules suitable for use as primers are provided as SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 25 and SEQ ID NO 27.

Primer pairs SEQ ID NOs 15 and 16 are useful as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both have sufficient length of contiguous nucleotides of SEQ ID No. 10 to serve as DNA primers that, when used in a thermal amplification reaction with template DNA derived from corn event MON95379, produce an amplicon that is diagnostic for corn event MON95379DNA in a sample. The primer pairs SEQ ID NOs 21 and 22 are useful as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both have sufficient length of contiguous nucleotides of SEQ ID No. 10 to serve as DNA primers that, when used in a thermal amplification reaction with a template DNA derived from corn event MON95379, can produce an amplicon that can diagnose the zygosity of corn event MON95379DNA in a sample. Primer pairs SEQ ID NOs 24 and 25 are suitable for use as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both have sufficient long contiguous nucleotides of SEQ ID No. 10 to serve as DNA primers that, when used in a thermal amplification reaction with template DNA derived from corn event MON95379, produce an amplicon that can diagnose the zygosity of corn event MON95379DNA in a sample. The primer pairs SEQ ID No. 18 and SEQ ID No. 19 are useful as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both have contiguous nucleotides of a locus within the corn genome long enough to serve as DNA primers that, when used in a thermal amplification reaction with template DNA derived from corn event MON95379, can produce an amplicon that serves as an internal control for diagnosing both corn event MON95379 and the zygosity of corn event MON95379DNA in a sample.

The length of the DNA probes and DNA primers is typically eleven (11) or more polynucleotides, often eighteen (18) or more polynucleotides, twenty-four (24) or more polynucleotides, or thirty (30) or more polynucleotides. Such probes and primers are selected to be of sufficient length to specifically hybridize to the target sequence under high stringency hybridization conditions. Preferably, the probes and primers according to the present invention have complete sequence similarity with the target sequence, although probes that differ from the target sequence and retain the ability to hybridize to the target sequence can be designed by conventional methods.

The nucleic acid probes and primers of the invention hybridize to a target DNA molecule under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from the transgenic plant in the sample. Polynucleic acid molecules, also referred to as nucleic acid segments or fragments thereof, are capable of specifically hybridizing to other nucleic acid molecules in certain instances.

As used herein, two polynucleic acid molecules are said to be capable of specifically hybridizing to each other if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. One nucleic acid molecule is said to be "complementary" to another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit "perfect complementarity" when each nucleotide of the molecule is complementary to a nucleotide of another molecule. Two molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability to allow them to remain annealed to each other under at least conventional "low stringency" conditions. Similarly, molecules are said to be "complementary" if they are capable of hybridizing to each other with sufficient stability to allow them to remain annealed to each other under conventional "high stringency" conditions. Conventional stringent conditions are described by Sambrook et al, 1989 and by Haymes et al in Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Thus, deviations from complete complementarity are permitted as long as such deviations do not completely preclude the ability of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to be used as a primer or probe, it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure at the particular solvent and salt concentrations used.

As used herein, a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize under high stringency conditions to the complement of the nucleic acid sequence to which it is compared. Suitable stringency conditions to facilitate DNA hybridization, for example, 6.0 XSSC at about 45 ℃ followed by a 2.0 XSSC wash at 50 ℃, are known to those skilled in the art or are found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 XSSC at 50 ℃ to a high stringency of about 0.2 XSSC at 50 ℃. In addition, the temperature in the washing step can be raised from low stringency conditions at about 22 ℃ at room temperature to high stringency conditions at about 65 ℃. Both temperature and salt may be varied, or either temperature or salt concentration may be held constant while the other variable may be varied. In a preferred embodiment, the polynucleic acid of the invention will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10 or a complementary sequence thereof or a fragment thereof under moderately stringent conditions, such as at about 2.0x SSC and about 65 ℃. In a particularly preferred embodiment, the nucleic acid of the invention will specifically hybridize under high stringency conditions to one or more of the nucleic acid molecules set forth in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10, or to complementary sequences or fragments thereof. In one aspect of the invention, preferred marker nucleic acid molecules of the invention have the nucleic acid sequences as set forth in SEQ ID NO 1, or SEQ ID NO 2, or SEQ ID NO 3, or SEQ ID NO 4, or SEQ ID NO 5, or SEQ ID NO 6, or SEQ ID NO 7, or SEQ ID NO 8, or SEQ ID NO 9, or SEQ ID NO 10, or the complementary sequences thereof, or fragments thereof. Hybridization of a probe to a target DNA molecule can be detected by a number of methods known to those skilled in the art, which may include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels.

With respect to amplification of a target nucleic acid sequence using a particular amplification primer pair (e.g., by PCR), a "stringent condition" is a condition that allows 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) is bound and preferably produces a unique amplification product (i.e., amplicon) in a DNA thermal amplification reaction.

The term "specific for (a target sequence)" indicates that the probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.

As used herein, "amplified DNA" or "amplicon" refers to the product of a polynucleic acid amplification process directed against a target polynucleic acid molecule that is part of a polynucleic acid template. For example, to determine whether a corn plant resulting from a sexual cross contains transgenic plant genomic DNA from a corn plant comprising event MON95379 of the present invention, DNA extracted from a corn plant tissue sample can be subjected to a polynucleic acid amplification method using a primer pair comprising a first primer in the region of the heterologous insert DNA flanking event MON95379, the first primer derived from a genomic DNA sequence and extended 5 'to 3' in the direction of the insert DNA by a polymerase. The second primer is derived from a heterologously inserted DNA molecule and is extended 5 'to 3' by a polymerase in the direction of the flanking genomic DNA from which the first primer is derived. The length of the amplicon may be in the following range: the total length of the primer pair is plus one nucleotide base pair, or plus about fifty nucleotide base pairs, or plus about two hundred fifty nucleotide base pairs, or plus about four hundred fifty nucleotide base pairs or more. Alternatively, the primer pair may be derived from genomic sequences flanking the inserted heterologous DNA to produce an amplicon comprising the entire inserted polynucleotide sequence (e.g., a forward primer isolated from the genomic portion 5 'of SEQ ID NO:10 and a reverse primer isolated from the genomic portion 3' of SEQ ID NO:10 that amplify a DNA molecule comprising the inserted DNA sequence identified herein in the genome of event MON95379 (SEQ ID NO: 9)). Members of the primer pair derived from the plant genomic sequence adjacent to the inserted transgenic DNA are at a distance from the inserted DNA sequence that can range from one nucleotide base pair to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers that may form in a DNA thermal amplification reaction.

For practical purposes, primers should be designedTo produce amplicons of a limited size range, e.g., 100 to 1000 bases. In general, amplicons of smaller size (shorter polynucleotide length) can be more reliably produced in a thermal amplification reaction, allow shorter cycle times, and can be easily separated and viewed on agarose gels, or are suitable for end-point useAnd (4) sample determination. Smaller amplicons can be generated and detected by methods known in the art of DNA amplicon detection. Alternatively, amplicons generated using the primer pairs can be cloned into vectors, propagated, isolated and sequenced, or can be directly sequenced using well-established methods in the art. One aspect of the invention is any primer pair derived from the combination of SEQ ID NO. 11 and SEQ ID NO. 9 or the combination of SEQ ID NO. 12 and SEQ ID NO. 9 suitable for use in a DNA amplification method to produce an amplicon diagnostic for event MON95379 or its progeny. One aspect of the invention is any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID No. 11 or the complement thereof, suitable for use in a DNA amplification method to produce an amplicon diagnostic for event MON95379 or progeny thereof. One aspect of the invention is any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID NO 12 or the complement thereof, suitable for use in a DNA amplification method to produce an amplicon diagnostic for a plant comprising event MON95379 or its progeny. One aspect of the invention is any single isolated DNA polynucleotide primer molecule comprising at least 15 contiguous nucleotides of SEQ ID No. 9 or the complement thereof, suitable for use in a DNA amplification method to produce an amplicon diagnostic for event MON95379 or progeny thereof.

Polynucleic acid amplification can be accomplished by any of a variety of polynucleic acid amplification methods known in the art, including Polymerase Chain Reaction (PCR). 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 PCR Protocols A Guide to Methods and Applications, editor Innis et al Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify genomic DNA up to 22kb (kilobases) and phage DNA up to 42kb (Cheng et al, Proc. Natl. Acad. Sci. USA 91: 5695-. These methods, as well as other methods known in the art of DNA amplification, can be used to practice the present invention. The heterologous DNA insert or flanking genomic DNA sequence from corn event MON95379 can be verified (and corrected if necessary) by the following method: amplifying such DNA molecules in corn seed containing event MON95379DNA or corn plants grown from corn seed containing event MON95379DNA using primers derived from the sequences provided herein, the event MON95379DNA deposited at the ATCC under accession No. PTA-125027; standard DNA sequencing of the PCR amplicons or cloned DNA fragments thereof is then performed.

Diagnostic amplicons produced by these methods can be detected by a variety of techniques. One such method is Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res.22:4167-4175,1994) in which DNA oligonucleotides are designed which overlap both the adjacent flanking genomic DNA sequence and the inserted DNA sequence. The oligonucleotides are immobilized in wells of a microtiter plate. After PCR of the target region (using one primer in the insert and one primer in the adjacent flanking genomic sequence), the single stranded PCR product can be hybridized to the immobilized oligonucleotide and used as a template for a single base extension reaction using a DNA polymerase and a labeled dideoxynucleotide triphosphate (ddNTP) specific for the expected next base. The readings may be fluorescence-based or ELISA-based. The signal indicates that the transgene/genomic sequence is present due to successful amplification, hybridization, and single base extension.

Another method is the pyrosequencing technique described by Winge (Innov. Pharma. Tech.00:18-24, 2000). In this method, an oligonucleotide is designed that overlaps adjacent genomic DNA and inserted DNA junctions. The oligonucleotides are hybridized to single-stranded PCR products from the target region (one primer in the insert and one primer in the flanking genomic sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate, and fluorescein. DNTP was added separately and incorporation produced the optical signal that was measured. The light signal indicates the presence of the transgene/genomic sequence due to successful amplification, hybridization, and single or multiple base extension.

Fluorescence polarization, described by Chen et al (Genome Res.9: 492-498, 1999), is one method that can be used to detect the amplicons of the present invention. Using this method, an oligonucleotide is designed that overlaps the genomic flanking and inserted DNA junction. The oligonucleotides hybridize to single-stranded PCR products from the target region (one primer in the inserted DNA and one primer in the flanking genomic DNA sequence) and are incubated in the presence of a DNA polymerase and a fluorescently labeled ddNTP. Single base extension leads to the incorporation of ddntps. Incorporation can be measured as a change in polarization using a fluorometer. The change in polarization indicates the presence of the transgene/genomic sequence due to successful amplification, hybridization, and single base extension.

Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of PCR as it occurs (i.e., in real-time). Data was collected throughout the PCR rather than at the end of the PCR. In real-time PCR, the reaction is characterized by the point in time during the cycle when target amplification is first detected, rather than the amount of target accumulated over a fixed number of cycles. In real-time PCR assays, positive reactions are detected by the accumulation of fluorescent signals. The higher the initial copy number of the nucleic acid target, the earlier a significant increase in fluorescence is observed. The cycle threshold (Ct value) is defined as the number of cycles required for the fluorescence signal to cross the threshold (i.e. exceed the background level). The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct value, the greater the amount of target nucleic acid in the sample).

(PE Applied Biosystems, Foster City, Calif.) is described as a method for detecting and quantifying the presence of DNA sequences using real-time PCR, and is well understood in the manufacturer's instructions. Briefly, a FRET oligonucleotide probe is designed that overlaps the genomic flanking and inserted DNA junction. FRET probes and PCR primers (one of the inserted DNA sequences)Primers and one primer in the flanking genomic sequence) 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. The fluorescent signal indicates the presence of the transgene/genomic sequence due to successful amplification and hybridization.

Molecular beacons have been described for use in sequence detection, such as Tyangi, et al. (Nature Biotech.14:303-308, 1996). Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and inserted DNA junction. The unique structure of the FRET probe results in it comprising a secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the inserted DNA sequence and one in the flanking genomic 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 moiety from the quencher moiety. A fluorescent signal is generated. The fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization.

DNA detection kits based on DNA amplification methods comprise DNA primer molecules that specifically hybridize to the target DNA and amplify the diagnostic amplicon under appropriate reaction conditions. The kit may provide an agarose gel based detection method or a number of methods known in the art for detecting diagnostic amplicons. DNA detection kits may be developed using the compositions disclosed herein and are suitable for use in methods of identifying corn event MON95379DNA in a sample, and may be applied to methods of breeding corn plants containing event MON95379 DNA. One object of the present invention is a kit comprising DNA primers homologous or complementary to any portion of the maize genomic region set forth in SEQ ID NO. 10 and any portion of the inserted transgenic DNA set forth in SEQ ID NO. 10. The DNA molecules can be used in DNA amplification methods (PCR) or as probes in polynucleic acid hybridization methods (i.e.southern analysis, northern analysis). The kits of the invention may also optionally comprise reagents or instructions for performing the detection or diagnostic reactions described herein.

Probes and primers according to the invention can have complete sequence identity to the target sequence, although primers and probes that differ from the target sequence in that they retain the ability to preferentially hybridize to the target sequence can be designed by conventional methods. In order for a nucleic acid molecule to be used as a primer or probe, it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure at the particular solvent and salt concentrations used. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA from corn event MON95379 in a sample. Probes and primers are typically at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides in length or longer. Such probes and primers specifically hybridize to the target DNA sequence under stringent hybridization conditions. Conventional stringent conditions are described by Sambrook et al, 1989 and by Haymes et al, in Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

Many methods well known to those skilled in the art can be used to isolate and manipulate the DNA molecules disclosed in the present invention or fragments thereof, including thermal amplification methods. The DNA molecule or fragment thereof may also be obtained by other techniques, such as direct synthesis of the fragment by chemical methods, as is commonly practiced by using an automated oligonucleotide synthesizer.

Thus, the DNA molecules and corresponding nucleotide sequences provided herein are particularly useful for identifying corn event MON95379, detecting the presence of DNA derived from transgenic corn event MON95379 in a sample, and monitoring the presence and/or absence of corn event MON95379 or a plant part derived from a corn plant comprising event MON95379 in a sample.

The present invention provides corn plants, corn plant cells, corn seeds, corn plant parts (such as pollen, ovules, silks, ears, anthers, cobs, root tissue, stalk tissue, leaf tissue), corn progeny plants, and corn commodity products. These corn plants, corn plant cells, corn seeds, corn plant parts, corn progeny plants, and corn commodity products contain detectable amounts of polynucleotides of the invention, such as, for example, polynucleotides having a sequence provided as at least one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10. The maize plants, plant cells, seeds, plant parts, and progeny plants of the invention may also contain one or more additional transgenes. Such additional transgenes may be any nucleotide sequence encoding a protein or RNA molecule conferring a desired trait, including but not limited to increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, and/or increased herbicide tolerance.

The present invention provides maize plants, maize plant cells, maize seeds, maize plant parts (such as pollen, ovules, silks, ears, anthers, cobs, root tissue, stalk tissue, leaf tissue), maize progeny plants derived from a transgenic maize plant comprising event MON95379 DNA. A representative sample of corn seed containing event MON95379DNA has been deposited under the Budapest treaty at the American type culture CollectionThe ATCC information repository has assigned the patent deposit designation PTA-125027 to seeds containing event MON95379 DNA.

The invention provides a microorganism comprising a DNA molecule having at least one sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10 present in its genome. An example of such a microorganism is a transgenic plant cell. Microorganisms (such as the plant cells of the present invention) are useful in many industrial applications, including but not limited to: (i) as a research tool for scientific research or industrial research; (ii) in culture for the production of endogenous or recombinant carbohydrate, lipid, nucleic acid or protein products or small molecules that can be used for subsequent scientific research or as industrial products; and (iii) use in conjunction with modern plant tissue culture techniques to produce transgenic plants or plant tissue cultures that can be used in agricultural research or production. The production and use of microorganisms, such as transgenic plant cells, utilizes modern microbial technology and human intervention to produce man-made unique microorganisms. In this process, recombinant DNA is inserted into the genome of a plant cell to create a transgenic plant cell that is isolated and unique from naturally occurring plant cells. Such transgenic plant cells can then be cultured like bacterial and yeast cells using modern microbial technology and can exist in an undifferentiated single-cell state. The new genetic composition and phenotype of transgenic plant cells is a technical effect of the integration of heterologous DNA into the cell genome. Another aspect of the invention is a method of using a microorganism of the invention. Methods of using the microorganisms of the invention, such as transgenic plant cells, include: (i) methods for generating transgenic cells by integrating recombinant DNA into the genome of a cell, and then using the cell to derive additional cells having the same heterologous DNA; (ii) methods of culturing cells containing recombinant DNA using modern microbial technology; (iii) methods of producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid, or protein products from cultured cells; and (iv) methods of using modern plant tissue culture techniques with transgenic plant cells to produce transgenic plants or transgenic plant tissue cultures.

Plants of the invention can transmit event MON95379DNA (including the transgene inserted in corn event MON 95379) to progeny. As used herein, "progeny" includes any plant, plant cell, seed, and/or regenerable plant part comprising event MON95379DNA derived from an ancestral plant and/or a DNA molecule comprising at least one sequence selected from SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10. Plants, progeny, and seeds may be homozygous or heterozygous for the transgene for event MON 95379. Progeny may be grown from seeds produced from a plant containing corn event MON95379 and/or from a plant fertilized with pollen of a plant containing corn event MON 95379.

Progeny plants may be self-pollinated (also referred to as "selfed") to produce a true plant breeding line, i.e., a plant that is homozygous for the transgene. Selfing of suitable progeny may produce plants that are homozygous for both added exogenous genes.

Alternatively, the progeny plant may be outcrossed, e.g., bred, with another unrelated plant to produce a variety or hybrid seed or plant. Other unrelated plants may be transgenic or non-transgenic. Thus, the variety or hybrid seed or plant of the invention may be obtained by: sexually hybridizing a first parent lacking the specific and unique DNA of corn event MON95379 with a second parent comprising corn event MON95379 to produce a hybrid comprising the specific and unique DNA of corn event MON 95379. Each parent may be a hybrid or an inbred line/variety, as long as the cross or breeding produces a plant or seed of the invention, i.e., a seed having at least one allele that contains the DNA of corn event MON95379 and/or a DNA molecule having at least one sequence selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10. Thus, two different transgenic plants can be crossed to produce a progeny hybrid comprising two independently isolated, added exogenous genes. For example, event MON95379, comprising cry1b.868 and Cry1Da _7 conferring resistance to corn insects, can be crossed with other transgenic corn plants to produce plants characterized by two transgenic parents. An example of this is crossing event MON95379, which contains cry1b.868 and Cry1Da _7 conferring lepidopteran resistance in corn, with a plant having one or more additional traits, such as herbicide tolerance, insect resistance, or drought tolerance, to produce a progeny plant or seed that is resistant to lepidopteran insect pests and has at least one or more additional traits. As with vegetative propagation, backcrossing of parent plants and outcrossing with non-transgenic plants is also contemplated. Descriptions of other Breeding Methods commonly used for different traits and crops can be found in one of several references, for example, Fehr, Breeding Methods for Cultivar Development, Wilcox J. eds., American Society of agriculture, Madison Wis (1987).

The plants, progeny, seeds, cells, and plant parts of the invention may further comprise one or more additional corn traits or transgenic events, particularly those introduced by crossing a corn plant comprising corn event MON95379 with another corn plant comprising an additional trait or transgenic event. Such traits or transgenic events include, but are not limited to, increased insect resistance, herbicide tolerance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, increased nutritional quality, hybrid seed production, or disease or fungal resistance. Maize transgenic events are known to those of skill in the art. For example, a list of such traits is provided by the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) and can be found on its website: www.aphis.usda.gov are provided. Thus, two or more transgenic events can be combined in progeny seed or plants by crossing two parent plants each comprising one or more transgenic events, collecting the progeny seed and selecting a progeny seed or plant comprising two or more transgenic events. These steps can be repeated until the desired combination of transgenic events is obtained in the offspring. As with vegetative propagation, backcrossing of parent plants and outcrossing with non-transgenic plants is also contemplated.

The present invention provides a plant part derived from a corn plant comprising event MON 95379. As used herein, "plant part" refers to any part of a plant that is comprised of material derived from a corn plant comprising event MON 95379. Plant parts include, but are not limited to, pollen, ovules, silks, ears, anthers, cobs, root tissue, stalk tissue, and leaf tissue. The plant part may be viable, non-viable, regenerable and/or non-regenerable.

The present invention provides a commodity product derived from a corn plant comprising event MON95379 and comprising a detectable amount of a nucleic acid specific for event MON 95379. As used herein, "commodity product" refers to any composition or product comprised of material derived from corn event MON95379DNA comprising: a corn plant, whole or processed corn seed, or one or more plant cells and/or plant parts. Non-viable commodity products include, but are not limited to, non-viable seeds, whole or processed seeds, seed parts, and plant parts; animal feed comprising corn, corn oil, corn flour, corn flakes, corn bran, pasta made from corn, corn biomass, and fuel products produced using corn and corn fractions. Viable commodity products include, but are not limited to, seeds, plants, and plant cells. Thus, a corn plant comprising event MON95379 can be used to make any commodity product typically obtained from corn. Any such commercial product derived from a corn plant comprising event MON95379 may contain at least a detectable amount of specific and unique DNA corresponding to corn event MON95379, and in particular may contain a detectable amount of a polynucleotide comprising a DNA molecule having at least one sequence selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10. Any standard method of detection of nucleotide molecules may be used, including the detection methods disclosed herein. Commercial products are within the scope of the present invention if any detectable amount of a DNA molecule having at least one sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10 is present in the commercial product.

The corn plants, corn plant cells, corn seeds, corn plant parts (such as pollen, ovules, silks, ears, anthers, cobs, root tissue, stalk tissue, leaf tissue), corn progeny plants, and commodity products of the invention are therefore particularly useful for growing plants to produce seeds and/or plant parts comprising corn event MON95379 for agricultural purposes, to produce progeny comprising corn event MON95379 for plant breeding and research purposes, for industrial and research applications and for sale to consumers along with microbial technology.

Methods are provided for producing insect-resistant corn plants comprising a DNA sequence specific and unique to event MON95379 of the present invention. The transgenic plants used in these methods may be homozygous or heterozygous for the transgene. The progeny plants produced by these methods may be varieties or hybrid plants; can be grown from a seed produced by a plant comprising corn event MON95379 and/or a seed produced by a plant fertilized with pollen of a plant comprising corn event MON 95379; and may be homozygous or heterozygous for the transgene. The progeny plant may then be self-pollinated to produce a true plant breeding line, i.e., a plant that is homozygous for the transgene, or may be outcrossed, e.g., bred, with another unrelated plant to produce a variety or hybrid seed or plant.

Methods of detecting the presence of DNA derived from a corn cell, corn tissue, corn seed, or corn plant comprising corn event MON95379 in a sample are provided. One method comprises (i) extracting a DNA sample from at least one corn cell, corn tissue, corn seed, or corn plant; (ii) contacting the DNA sample with at least one primer capable of producing a DNA sequence specific for event MON95379DNA under conditions suitable for DNA sequencing; (iii) carrying out DNA sequencing reaction; then (iv) confirming that the nucleotide sequence comprises a nucleotide sequence of the construct comprised therein having specificity for event MON95379, such as a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10. Another method comprises (i) extracting a DNA sample from at least one corn cell, corn tissue, corn seed, or corn plant; (ii) contacting the DNA sample with a primer pair capable of producing an amplicon from event MON95379DNA under conditions suitable for DNA amplification; (iii) carrying out DNA amplification reaction; then (iv) detecting the amplicon molecule and/or confirming that the nucleotide sequence of the amplicon comprises a nucleotide sequence specific for event MON95379, such as a nucleotide sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10. The amplicon should be an amplicon having specificity for event MON95379, such as an amplicon comprising SEQ ID NO 1, or SEQ ID NO 2, or SEQ ID NO 3, or SEQ ID NO 4, or SEQ ID NO 5, or SEQ ID NO 6, or SEQ ID NO 7, or SEQ ID NO 8, or SEQ ID NO 9, or SEQ ID NO 10. The detection of a nucleotide sequence specific for event MON95379 in the amplicon can determine and/or diagnose the presence of corn event MON95379 specific DNA in the sample. An example of a primer pair capable of producing an amplicon from event MON95379DNA under conditions suitable for DNA amplification is provided as SEQ ID No. 15 and SEQ ID No. 16. Other primer pairs can be readily designed by one skilled in the art and will produce amplicons comprising SEQ ID NO 1, or SEQ ID NO 2, or SEQ ID NO 3, or SEQ ID NO 4, or SEQ ID NO 5, or SEQ ID NO 6, or SEQ ID NO 7, or SEQ ID NO 8, wherein such primer pairs comprise at least one primer within the genomic region flanking the insert and a second primer within the insert. Another method of detecting the presence of DNA derived from a corn cell, corn tissue, corn seed, or corn plant comprising corn event MON95379 in a sample comprises: (i) extracting a DNA sample from at least one corn cell, corn tissue, corn seed, or corn plant; (ii) contacting the DNA sample with a DNA probe specific for event MON95379 DNA; (iii) hybridizing the probe and the DNA sample under stringent hybridization conditions; and then (iv) detecting hybridization between the probe and the target DNA sample. An example of a sequence of a DNA probe specific for event MON95379 is provided as SEQ ID No. 17. Other probes can be readily designed by one skilled in the art and will comprise at least one fragment of the genomic DNA flanking the insert and at least one fragment of the insert DNA, such as the sequences provided in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 10. Detection of hybridization of the probe to the DNA sample can diagnose the presence of corn event MON95379 specific DNA in the sample. The absence of hybridization can alternatively diagnose the absence of corn event MON 95379-specific DNA in the sample.

DNA detection kits are provided that are suitable for use in identifying corn event MON95379DNA in a sample, and also for use in methods of breeding corn plants containing appropriate event DNA. Such kits comprise DNA primers and/or probes comprising fragments of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10. One example of such a kit includes at least one DNA molecule having contiguous nucleotides of SEQ ID NO:10 of sufficient length to serve as a DNA probe suitable for detecting the presence and/or absence in a sample of DNA derived from a transgenic corn plant comprising event MON 95379. DNA derived from a transgenic corn plant comprising event MON95379 will comprise a DNA molecule having at least one sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10. DNA molecules sufficient for use as DNA probes are provided that are suitable for determining, detecting, or diagnosing the presence and/or absence of corn event MON95379DNA in a sample, as provided in SEQ ID No. 17. Other probes can be readily designed by those of skill in the art and will comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 consecutive nucleotides of SEQ ID No. 10 and are sufficiently unique to corn event MON95379DNA to identify DNA derived from that event.

Another class of kits comprises a primer pair for producing an amplicon suitable for detecting the presence and/or absence of DNA derived from transgenic corn event MON95379 in a sample. Such kits will employ a method comprising contacting a target DNA sample with a primer pair as described herein, followed by a nucleic acid amplification reaction sufficient to produce an amplicon comprising a DNA molecule having at least one sequence selected from SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10, and then detecting the presence and/or absence of the amplicon. Such a method can further comprise sequencing the amplicon or fragment thereof, which will determine, i.e., diagnose, the presence of corn event MON95379 specific DNA in the target DNA sample. Other primer pairs can be readily designed by those skilled in the art and should comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotide sequences of the sequences provided in, but not limited to, SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10, and are sufficiently unique to the corn event MON95379DNA to identify the DNA derived from the event.

The kits and detection methods of the invention are particularly useful for identifying corn event MON95379, selecting plant varieties or hybrids comprising corn event MON95379, detecting the presence of DNA derived from a transgenic corn plant comprising event MON95379 in a sample, and monitoring the sample for the presence and/or absence of a corn plant comprising event MON95379 or a plant part derived from a corn plant comprising event MON 95379.

The heterologous DNA insert, linker sequence, or flanking sequence from corn event MON95379 may be verified (and corrected if necessary) by: such sequences are amplified from the event using primers derived from the sequences provided herein, and standard DNA sequencing is then performed on the amplicons or cloned DNA.

Methods of detecting the zygosity of a transgenic allele of DNA derived from a corn cell, corn tissue, corn seed, or corn plant comprising corn event MON95379 in a sample are provided. One method comprises (i) extracting a DNA sample from at least one corn cell, corn tissue, corn seed, or corn plant; (ii) contacting the DNA sample with a primer pair capable of producing a first amplicon diagnostic for event MON 95379; (iii) contacting the DNA sample with a primer pair capable of producing a second amplicon that is diagnostic for native corn genomic DNA that does not comprise event MON 95379; (iv) carrying out DNA amplification reaction; then (v) detecting the amplicons, wherein the presence of only the first amplicon diagnoses homozygous event MON95379DNA in the sample, and the presence of both the first amplicon and the second amplicon diagnoses a corn plant that is heterozygous for the event MON95379 allele. An exemplary set of primer pairs is presented as: 15 and 16, which produce an amplicon diagnostic for event MON 95379; 15 and 27, which produce an amplicon diagnostic for non-inserted wild-type corn genomic DNA that does not comprise event MON 95379. Probe sets may also be incorporated into such an amplification method to be used in a real-time PCR format using the primer set sets described above. An exemplary set of probes is presented as SEQ ID NO:17 (an amplicon diagnostic for event MON 95379) and SEQ ID NO:28 (an amplicon diagnostic for wild-type corn genomic DNA that does not include event MON 95379).

Another method for determining engageability includes: (i) extracting a DNA sample from at least one corn cell, corn tissue, corn seed, or corn plant; (ii) contacting the DNA sample with a probe set comprising at least a first probe that specifically hybridizes to event MON95379DNA and at least a second probe that specifically hybridizes to corn genomic DNA interrupted by insertion of the heterologous DNA of event MON95379 but does not hybridize to event MON95379 DNA; (iii) hybridizing the set of probes to the sample under stringent hybridization conditions, wherein detection of hybridization of only the first probe under the hybridization conditions is diagnostic of a homozygous allele of event MON95379DNA in the sample; and wherein detecting hybridization of both the first probe and the second probe under hybridization conditions is diagnostic of a heterozygous allele of event MON95379 in the DNA sample.

Yet another method for determining engageability includes: (i) extracting a DNA sample from at least one corn cell, corn tissue, corn seed, or corn plant; (ii) contacting the DNA sample with a primer pair capable of producing an amplicon encoding one of the toxin coding sequences of cry1b.868 or Cry1Da _ 7; (iii) contacting the DNA sample with a primer pair capable of producing an amplicon of an internal standard known to be single copy and homozygous in the maize plant; (iv) contacting the DNA sample with a probe set comprising at least a first probe that specifically hybridizes to one of the toxin coding sequences encoding cry1b.868 or Cry1Da _7 and at least a second probe that specifically hybridizes to an internal standard genomic DNA known to be single copy and homozygous in a corn plant; (v) performing a DNA amplification reaction using real-time PCR and determining a cycle threshold (Ct value) for amplicons corresponding to the toxin coding sequence and the single copy, homozygous internal standard; (vi) calculating the difference (delta Ct) between the Ct value of the single copy, homozygous internal standard amplicon and the Ct value of the toxin-encoding sequence amplicon; and (vii) determining zygosity, wherein a Δ Ct of about zero (0) indicates homozygosity of the inserted T-DNA and a Δ Ct of about one (1) indicates heterozygosity of the inserted T-DNA. Heterozygous and homozygous events are distinguished by Δ Ct value units of about one (1). Given that the normal variability observed in real-time PCR is due to a number of factors such as amplification efficiency and ideal annealing temperature, a range of "about one (1)" is defined as a Δ Ct of 0.75 to 1.25. Primer pairs and probes for use in the above method of determining zygosity may amplify and detect amplicons from the cry1b.868 coding sequence and internal standards, or from Cry1Da _7 coding sequence and internal standards. Exemplary primer pairs for detecting amplicons corresponding to the Cry1B.868 coding sequence and internal standard are presented as SEQ ID NO:18 in combination with SEQ ID NO:19 (internal standard) and SEQ ID NO:21 in combination with SEQ ID NO:22 (Cry1B.868). The accompanying exemplary probes are presented as SEQ ID NO:20 (internal standard) and SEQ ID NO:23 (Cry1B.868). Exemplary primer pairs for detecting amplicons corresponding to Cry1Da _7 coding sequence and internal standard are presented as SEQ ID NO:18 in combination with SEQ ID NO:19 (internal standard) and SEQ ID NO:24 in combination with SEQ ID NO:25 (Cry1Da _ 7). The accompanying exemplary probes are presented as SEQ ID NO:20 (internal standard) and SEQ ID NO:26(Cry1Da _ 7).

Registering information

According to the budapest treaty, a representative corn seed sample comprising event MON95379 was deposited at 20 days 4 months 2018 at the American Type Culture Collection (ATCC) address 10801 University Boulevard, Manassas, Virginia USA, Zip Code 20110 and assigned ATCC accession No. PTA-125027. The deposit is made available to the patent and trademark office personnel and to the personnel identified by the office personnel as being entitled to the required authorization during the pendency of the application. All restrictions on public availability will be irrevocably removed after the release of the patent. The deposit will be stored in the depository for thirty (30) years or five (5) years after the last request or the validity period of the patent (whichever is longer), and will be replaced as required during this period.

Examples

The following examples are included to more fully describe the invention. Summarizing the construction and testing of one hundred twenty five (125) constructs, the generation of about one million-seven hundred eighty five (10,785) events (proof of concept and commercial proof) and the analysis of hundreds of thousands of individual plants through rigorous molecular, agronomic and field testing required to create and select the maize event MON95379 over six (6) years.

The examples illustrate certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Expression cassette testing, construct design, plant testing and construct selection

Transgene expression in plants is affected by many different factors. The correct combination of insecticidal proteins and different expression elements driving expression in plants must be found without causing phenotypic deviation. Furthermore, in addition to the expression elements themselves and their combination and orientation in the cassette, it is known that expression of a transgene in plants is affected by chromosomal insertion sites, which may be due to chromatin structure (e.g., heterochromatin) or transcriptional regulatory elements (e.g., enhancers) close to the integration site (Kurt Weising et al, (1988) Forein genes in plants: transfer, structure, expression and applications, Annu. Rev. Gene.22: 421-77). For example, it has been observed that in plants and other organisms, there may be large differences in the expression levels of introduced genes from the same construct in events with different chromosomal insertion positions. Different chromosomal insertion positions may also produce differences in spatial or temporal patterns of expression that may not correspond to the pattern expected from the transcriptional regulatory elements present in the introduced genetic construct.

For these reasons, it is often necessary to create and screen a large number of constructs and transformation events to identify a construct, followed by an event that demonstrates optimal expression of the introduced gene of interest, while not producing an agronomic or phenotypic allotype.

For these reasons, the development of transgenic corn plants comprising insecticidal proteins active against lepidopterans without any negative impact on agronomic, yield or stacking feasibility requires extensive research, development and analysis. Specifically, over a period of six (6) years, approximately zero million seven hundred eighty five (10,785) proof-of-concept and commercial transgenic events were developed, tested, and analyzed, which were derived from one hundred twenty five (125) different plasmid vector constructs.

This example describes the design and testing of one hundred twenty five (125) different constructs in maize plants to identify preferred constructs for event creation. Each construct differs in the coding sequence for the pesticidal protein and the transcriptional regulatory elements. Tests have been performed to select the optimal construct for expressing a pesticidal protein in a plant. Each construct has a unique configuration, which varies with the expression cassette composition (both pesticidal protein and expression element), orientation, and whether the protein is targeted to the chloroplast.

In a preliminary conceptual validation and development phase, one hundred seventeen (117) constructs contained twenty-six (26) different promoters, twenty-six (26) different introns, and ten (10) different insect toxin coding sequences in different combinations, and these constructs were used to generate approximately six thousand (6,000) transformation events. After preliminary molecular characterization for the presence of transgenes, fifty-zero fifty-two (5,052) single-copy and double-copy transformed maize events were selected for further characterization and efficacy testing. These events were evaluated with respect to: phenotype or agronomic phenotype, expression level of insect toxin protein, and efficacy of a selected lepidopteran insect species. The resulting efficacy and protein expression data, as well as any information about phenotype and agronomic phenotype, were used to eliminate null proteins, expression elements and combinations, and to design smaller quantities of binary commercial transformation plasmid constructs for the next stage of development.

In the next development phase, eight (8) new constructs are created. These constructs comprise a combination of two (2) to four (4) insect toxin transgene expression cassettes in different orientations (converging or diverging). These eight (8) constructs were used to generate a total of five thousand seven hundred thirty-three (5,733) transformation events (also referred to as "transformants"). After shoots have formed in the culture, a subset of transformation events are selected based on visual characteristics and early molecular analysis. A total of eight hundred twenty-three (823) transformation events were selected and transplanted into pots and planted for further study.

With respect to the following aspects for the resulting R₀Analysis was performed on transformation events: efficacy against selected lepidopteran species, toxin protein expression, plant health, seed return, and phenotypic and agronomic traits. R is also characterized molecularly₀Events were generated to ensure cassette integrity and correct insertion in the maize genome. Many events were deleted from the test due to failure to pass agronomic analysis and molecular characterization tests. In addition, R here₀Stage (1) of the eight (8) constructs were deleted from further studies as it produced events of phenotypic deviation. In addition to these agronomic problems, subsequent mode of action ("MOA") studies performed showed that the insect toxin protein contained in this construct demonstrated MOA overlap with commercially available proteins.

Mode of action studies were conducted on an insect protein common to four (4) of eight (8) constructs. These studies indicate that this insect protein has an overlapping MOA with the commercially available protein. A protein exhibiting a similar or overlapping MOA as the commercial insecticidal proteins currently used is not desirable because it will developResistance, which may render proteins with similar MOAs ineffective against the insect population. Thus, the four (4) constructs and the events resulting therefrom were deleted. As previously described, one (1) of the four (4) deleted constructs was in R₀The phase also produces events of phenotypic deviation.

In the next development phase, at F₁(hybrid seed)/R₁(homozygous inbred line) and R₂The generation was further evaluated for the efficacy, seed return and segregation, phenotype and agronomic allotypes and further molecular characterization of one hundred fifty (150) events derived from the remaining four (4) constructs. Two (2) of the remaining four (4) constructs were deleted from further studies at this stage due to failure to meet one or more progression criteria, leaving events derived from (2) constructs for further evaluation.

Seventeen (77) events (forty-one (41) events derived from the construct used to produce event MON95379 ("construct MON 95"), and thirty-six (36) events derived from another construct ("construct 1")) were treated as R with respect to the following aspects₂Inbred lines and F₁Evaluation of hybrid species: efficacy, seed return and segregation, phenotype and agronomic allotypes and further molecular characterization. Based on these evaluations, the events associated with construct 1 were prioritized down, set aside and stored.

Thus, multiple rounds of testing and comparison of the various constructs revealed that the transgene cassette provided as SEQ ID NO:13 (i.e., construct MON95) is the best choice for efficacy against the lepidopteran pest species fall armyworm (FAW, spodoptera frugiperda), corn earworm (CEW, cotton bollworm), southwestern corn borer (SWCB, southwestern corn borer), sugarcane borer (SCB, sugarcane borer), and corn borer (LSCB, corn borer), with optimal molecular characterization and agronomic performance.

Table 2 shows the number of derived ("inserted") transformation events, the number of transformation events selected for growth as the R0 event ("transplanted"), and the point at which each construct was deleted in the evaluation, study, and development process (which led to the selection of construct MON 95).

TABLE 2 event construct selection

Example 2

Field trials, molecular testing and event selection

This example describes molecular characterization, analysis, and testing in field trials of events created with construct MON95 at multiple locations over several years, thereby resulting in the selection of the final event MON 95379.

Table 3 shows a process for selecting the final event MON 95379. In the commercial transformation of R₀In the screening, two hundred and ten (210) R's were derived from construct MON95₀Events were transformed and selected for growth. At the first two hundred and ten (210) selected Rs₀Of the transformation events, one hundred forty-seven events (147) were deleted due to problems with efficacy, protein expression, seed return and plant health, or molecular characterization. The remaining sixty-three (63) events are required in the next development phase (F)₁Screening and R₁Seedling stage) were analyzed and tested. At this stage, eleven (11) events were deleted due to efficacy issues in the greenhouse test. Three (3) additional events were deleted due to insufficient seed returned from the nursery and/or segregation analysis of the resulting seeds. Finally, five (5) more events were removed due to problems found in molecular characterization, and three (3) events were removed due to problems found in molecular southern analysis, leaving forty-one (41) events for the next generation of assays. R under test₂/F₁Stage, two (2) of the remaining forty-one (41) events were deleted, leaving thirty-nine (39) events, due to problems found in further molecular southern characterization.

The remaining thirty-nine (39) events progress in two different simultaneous parallel test phases: 1) Further field tests; 2) cre excision of the selection cassette and generation of gold standard seeds. Events are deleted in each of these simultaneous parallel testing phases.

During Cre excision, eleven (11) events were deleted due to problems found in molecular characterization after Cre excision of the glyphosate selection cassette. In addition, six (6) additional events were deleted due to problems found in molecular characterization during gold standard seed generation.

During the concurrent Field testing, based on the data collected from the 2016US Field rails, four (4) additional events were deleted for efficacy issues and twelve (12) additional events were deleted for agronomic considerations. Then, based on the data collected from the Brazil field trial, another event was deleted due to efficacy issues. Next, bioinformatic analysis performed during the 2017u.s. field trial resulted in three (3) additional events being removed from further testing, leaving two events: event 1 and MON 95379. After further agronomic analysis of the events from multiple field trials in the united states, brazil, argentina, and puerto rico, event MON95379 was selected as a commercialized event because the event was ranked higher than event 1 when all features of molecular characterization, protein expression, efficacy, and agronomic of each event were compared.

Table 3 MON95379 event selection.

Example 3

Cre excision of glyphosate selection cassette from maize event MON95379

This example describes the removal of the glyphosate selection cassette from corn event MON95379 by in vivo Cre excision. The glyphosate selection cassette was used to select for transformed events. By removing the selection cassette, a "marker-free" event is created, wherein only the pesticidal protein expression cassette remains in the final event.

Fig. 3 shows a breeding process for generating the marker-free event MON95379 corn event. Maize variety LH244 immature embryos were transformed with construct MON95 (presented as SEQ ID NO:13, shown in FIG. 2) using an Agrobacterium-mediated transformation process. Construct MON95 contained three (3) expression cassettes: two (2) expression cassettes were used to express the insecticidal proteins cry1b.868 and Cry1Da _7, while a single cassette was used to select for transformed plant cells using glyphosate selection. Both sides of the selection cassette flank a LoxP Cre recombinase recognition site.

After transformation, R is₀Transformants were self-pollinated for two (2) generations during which many events were removed according to various assays such as efficacy, protein expression, seed return and plant health and molecular characterization. To R₂Instead, thirty-nine (39) events are retained from the first two hundred and ten (210) events. Thirty-nine (39) homozygous R₂The generation event breeds with a superior line of transformed maize plants expressing Cre recombinase derived from enterobacter bacteriophage P1.

Make R₂The generation event is identified as a "Cre cross" with this stage of plant breeding where the Cre recombinase is expressed. In particular, in this stage, the de-tasseled (female) R that is homozygous for SEQ ID NO:13₂The generative plants are cross-pollinated with a transgenic maize plant (male) homozygous for the transgenic cassette for expressing the Cre recombinase. Male donor pollen expressing the Cre recombinase germinates after landing on the tassel tissue of the female plant comprising SEQ ID NO 13. After the pollen tube enters the embryo sac, the pollen tube ruptures, releasing the two sperm of the male donor expressing Cre recombinase. The nucleus of a sperm fuses with the egg nucleus to form a zygote. The other sperm nucleus fuses with one of the two polar nuclei, which in turn fuses with the other polar nucleus, thereby establishing a primary endosperm nucleus.

Thus, when using a plant expressing Cre recombinase as a male pollen donor, as the cell divides and develops into kernels (i.e., seeds), both the embryo and endosperm of the resulting cross will express Cre recombinase. Cre recombinase binds to the inverted repeat in the LoxP site and catalyzes an exchange in the eight base pair spacer flanking both LoxP sites of the expression cassette, resulting in excision of the marker cassette, one of which LoxP sites remains in the integrated T-DNA due to recombination (see fig. 2, "T-DNA inserted after Cre excision").

F resulting from Cre hybridization was selected due to the absence of the CP4 selection cassette₁Progeny, and self-pollinating them. By this process, two alleles (Cre recombinase allele and T-DNA allele for generating event MON 95379) are present in the resulting F₂Segregating the population to produce offspring that are homozygous or heterozygous for one or both alleles.

Selection of F showing absence of Cre recombinase allele and homozygosity for SEQ ID NO 9 (transgene inserted T-DNA after Cre excision)₂The progeny. These selected F₂Progeny are self-pollinated, producing F homozygous for SEQ ID NO 9₃And (4) generation.

Further self-pollination to produce F₃Progeny seed (F)₄Seeds), the purity thereof was determined, and designated as "gold standard seeds". F4 is the first generation gold standard seed.

Excision of the glyphosate selectable marker cassette did not affect expression of cry1b.868 and Cry1Da _ 7. Removal of the glyphosate selection cassette from corn event MON95379 by Cre excision provides a transgenic corn event that is resistant to lepidopteran pests without increasing tolerance to glyphosate in the final event. This "marker-free" event ensures that flexibility is provided in constructing a maize breeding complex with other maize transgenic events to provide multiple products incorporating event MON95379 and to allow multiple options for providing more traits in the final breeding complex.

Example 4

Corn event MON95379 exhibits resistance to the lepidopteran insect pests fall armyworm, corn earworm, southwestern corn borer, sugarcane borer

This example describes the activity of the MON95379 event on lepidopteran insect pests. Insect toxin proteins Cry1B.868 and Cry1Da _7, when expressed together in corn event MON95379, are resistant to fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), southwestern corn borer (Diatraea zea) and sugarcane borer (Cnaphalocrocis medinalis).

Transformation and insertion constructsForty-one (41) Rs were selected after construction of MON95₀Events were used for bioassays using leaf discs. Bioassay using plant leaf disks was performed similarly to the method described in U.S. patent No. 8,344,207. Tissues to be used as negative controls were obtained using untransformed LH244 maize plants. Plates containing wells with one insect per leaf disc per well were incubated for three (3) days. After three (3) days, the plates were examined. If at least fifty percent (50%) of the leaf discs in the negative control were consumed, measurements were taken on transgenic event leaf discs. If less than fifty percent (50%) of the leaf discs in the negative control have not been consumed, the insects are allowed to continue feeding until a fifty percent (50%) target is reached. Leaf damage ("leaf damage rating" or "LDR") and mortality were measured for each well. The average of each metric is determined. Leaf damage ratings ranged from one (1) to eleven (11), reflecting the percentage of leaf discs that had been consumed. Table 4 shows the results for R₀Leaf damage scale measure determined for leaf discs. On this scale LDR of the negative control was always at least 10.

TABLE 4.R₀Leaf disc determined Leaf Damage Rating (LDR) measure.

Table 5 shows the mean leaf damage rating for forty-one (41) events transformed with construct MON95, including MON95379 event. As can be seen from table 5, expression of two insecticidal proteins cry1b.868 and Cry1Da _7 provided resistance to Fall Armyworm (FAW), Corn Earworm (CEW) and southwestern corn borer (SWCB). The LDR of the negative control was between 10 and 11. FAW and SWCB consumed only about five percent (5%) of the event MON95379 leaf disc compared to the negative control, which consumed at least fifty percent (50%) of the leaf disc. With respect to CEW, only about 6.25% of leaf discs were consumed, in contrast to negative controls consuming at least fifty percent (50%) of leaf discs. In addition, one hundred percent (100%) of FAW and CEW were killed after consumption of the leaf discs containing event MON 95379.

TABLE 5 expression of Cry1B.868 and R of Cry1Da _7₀Mean Leaf Damage Rating (LDR) score and mean mortality of plants.

Forty-one (41) events were crossed with a non-transgenic 93IDI3 variety plant. F comprising construct MON95 was selected₁Heterozygous progeny plants. In the greenhouse, for each insect pest species, about five (5) plants F were treated for each event, respectively₁The plants are artificially infected. With respect to FAW, approximately forty (40) newborns were used to infest each F at the V6 to V8 recurrent stage₁A plant. With respect to SWCB, approximately thirty (30) newborns are used to infect F at the recurrent stage of V6 to V10₁A plant. Leaf damage was measured about fourteen (14) days after infestation for FAW and SWCB. Table 6 and table 7 show the lesion grade metrics used to evaluate leaf lesions.

Table 6 leaf damage rating measures for corn plants infected with FAW.

TABLE 7 leaf damage rating measurements for SWCB infected maize plants.

SWCB-infected F was also evaluated₁Length of stalk perforations made by SWCB by the plants. To determine the length of the stalk perforations, corn plant jades were usedThe rice straw was broken at approximately eye level and the top was used to check for perforation damage. The culms were cut with a double-handled knife, and the length of the tract drilled by the SWCB was measured in centimeters (cm). In these experiments, the upper limit of the length of the channels was ten centimeters (10 cm).

In addition, five (5) strains F were also infected with CEW for each event₁Plants to measure the extent of damage caused by CEW to ears of corn. Approximately forty (40) CEW nymphs were used to infest each plant and placed at R₁Stage plants on green silks. Twenty-one (21) days after infestation, developing ears were examined and lesions were recorded as cm²Ear damage.

Table 8 shows F infected with FAW and SWCB₁Mean leaf damage rating of events, stalk perforation length by SWCB, and ear damage by CEW, where "NT" indicates not tested.

TABLE 8 FAW and SWCB infected F₁Average leaf damage rating, stalk perforation length by SWCB, and ear damage by CEW of transgenic corn plants.

As can be seen in table 8, both FAW and SWCB had minimal leaf damage to corn event MON95379 compared to the negative control. Essentially, once the insect begins with event MON 95379F₁Leaf feeding, expression of cry1b.868 and Cry1Da _7 insecticidal proteins in corn leaves containing event MON95379 resulted in the insects ceasing to feed the leaves. No SWCB drilling was observed in event MON95379, while the negative control showed extensive drilling. With respect to CEW ear damage, there was much less damage to the ear compared to the negative control, and comparable to ear damage observed in several commercial transgenic maize events. F₁The magnitude of infestation used in the assay is much higher than is common in nature. F₁The measurement shows that the content of the active carbon,corn event MON95379 provides excellent control over FAW, SWCB, and CEW.

R as described in example 2/Table 3 was determined in field experiments using artificial infestation in 2016 summer₂/F₁F of the last thirty-nine (39) events₁Resistance of progeny to FAW, CEW and SWCB. Multiple sites were used to determine resistance.

FAW resistance was determined at three (3) sites: jerseyville, IL; thomsboro, IL; and Union City, TN. At each site, each event was measured in three (3) field plots, one (1) row per plot and thirty (30) seeds per row. Forty (40) FAW newworms were used to infect each plant twice during the early and middle recurrent stages (V4 and V7 vegetative stages). Leaf feeding damage ratings were assessed using the measures provided in table 6.

SWCB resistance was determined at three (3) sites: one (1) in Jonesboro, AR and two (2) in Union City, TN. At each site, each event was measured in three (3) field plots, one (1) row per plot and thirty (30) seeds per row. During the mid-rotation stage (V7-V8), thirty (30) SWCB neoworms were used to infect each plant. At fifty (50%) percent pollen shed, each plant is again infested with thirty (30) SWCB neoworms. Culm drilling damage was assessed as previously described.

CEW resistance was determined at five (5) sites: jerseyville, IL; jonesboro, AR; montouth, IL; thomasboro, IL and Union City, TN. At each site, each event was measured in three (3) field plots, one (1) row per plot and thirty (30) seeds per row. When the silks are fresh and green, the plants are infested and some ears have begun to form (stages R1-R3). Infection was performed using CEW egg strips. Each strip contained approximately forty (40) eggs. One (1) strip was placed between the ear and stalk of each plant with the eggs facing the ear and near the silks. Assessment of ear damage was determined twenty-one (21) to twenty-eight (28) days after infestation. By this time, the insect has progressed from larval to pupal stages. Damage to the ear was measured as described previously.

For FAW and SWCB, data from all three (3) sites is used. For CEW, only data from Jonesboro, AR can be used due to various field conditions. Table 9 shows the average FAW leaf damage rating, SWCB pore channel length, and CEW ear damage measurements for each test event and negative control.

Table 9.2016 mean FAW leaf damage rating, SWCB tunnel length, and CEW ear damage for field efficacy testing.

As shown in table 9, corn event MON95379 provided superior control over FAW, SWCB, and CEW compared to the negative control. The level of infestation in these assays is much higher than is often encountered in the field under natural conditions, demonstrating the superior performance of event MON95379 under high insect pressure.

The events were further characterized during the concurrent field trials and Cre excision of the selection cassette and generation of gold standard seeds. As a result of extensive molecular characterization, efficacy, expression, and agronomic studies, events were deleted from the test, leaving two (2) events: event 1 and MON 95379. Event 1 was reduced in priority and event MON95379 was upgraded according to the yield resistance observed in agronomic studies.

During the growing season of argentina from 2016 to 2017, the resistance of event MON95379 to FAW, CEW and SCB was determined under natural infestation conditions in temperate and subtropical regions. Using the metrics provided in table 6, FAW leaf damage ratings were determined for event MON95379 grown in the subtropical regions of argentina. SCB pore channel data for event MON95379 was obtained from two (2) sites in the argentine temperate zone. CEW ear damage data for event MON95379 was obtained from two (2) sites in the argentine temperate zone and three (3) sites in the argentine subtropical zone. Table 11 shows the average FAW leaf lesion rating, SCB cell channel length, and CEW ear lesion under naturally infested conditions for event MON95379 and the negative control during the growth season of argentina in 2016-.

Table 10.2016-2017 average FAW leaf damage rating, SCB cell length, and CEW ear damage for field efficacy trials in argentina.

As can be seen from table 10, event MON95379 provided resistance to FAW, SCB, and CEW under natural infestation conditions of argentina compared to negative controls.

Also during three (3) growing seasons of puerto rico (2016 month 1, 2016 month 7, and 2017 month 1), the resistance of event MON95379 to FAW compared to a commercial corn event (MON89034, which expresses cry1a.105 and Cry2Ab2) was evaluated. Table 11 shows the mean leaf damage levels for each season of the three (3) growing seasons based on the measures shown in table 6, compared to event MON89034 and the negative control.

Table 11 mean leaf damage ratings for event MON95379 and event MON89034 naturally infected with FAW resistant to event MON 89034.

As can be seen in table 11, corn event MON95379 exhibited resistance to FAW resistant to event MON89034 at high natural pressure relative to the negative control.

In the summer of 2017, the resistance of event MON95379 to FAW, SWCB, and CEW was evaluated in the united states using a method similar to that described in the summer of 2016. FAW resistance was determined at three (3) sites: jerseyville, IL; thomsboro, IL; and Monmouth, IL. At each site, each event was measured in three (3) field plots, one (1) row per plot and thirty (30) seeds per row. Forty (40) FAW newworms were used to infect each plant twice. The first infestation occurred around stage V5. In Monmouth, IL and Jerseyville, the second plant infestation of IL occurs around stage V8. Due to low hatchability and bad weather, no second infection of IL is possible in thomas boro. FAW leaf feeding damage ratings were evaluated using the metrics provided in table 6.

SWCB resistance was determined at three (3) sites: one (1) in Jonesboro, AR and two (2) in Union City, IL. At each site, each event was measured in three (3) field plots, one (1) row per plot and thirty (30) seeds per row. Each plant was infested twice with thirty (30) SWCB neoworms. Under normal conditions, the first infestation is carried out in the middle of the recurrent stage of half the rows (V7-V8), but the infestation is delayed by about one week. In any event, strong insect pressure is established. At fifty percent (50%) of pollen shed, thirty (30) SWCB neoworms per plant infest the second half of the row of plants. Culm drilling damage was assessed as previously described.

CEW resistance was determined at six (6) sites as follows: jerseyville, IL; jonesboro, AR; paraguld, AR; montouth, IL; and two sites in Union City, TN. At each site, each event was measured in three (3) field plots, one (1) row per plot and thirty (30) seeds per row. Due to insect deficits, in Monmouth, IL and Jerseyville, IL infestations two (2) to three (3) weeks later than when silks are fresh and green. At Monmouth, each plant was infested with approximately twenty-two (22) newborns. In Jerseyville, IL, partially opened ears of corn were infested with twenty-three (23) to twenty-four (24) neoworms. Each plant in one (1) of the Jonesboro, AR, three (3) rows receives approximately thirty (30) neoworms, while each plant in the other two (2) rows receives sixteen (16) to (18) neoworms. In Paraguld, AR, approximately thirty (30) neoworms were received per plant in all three (3) rows. Infestation of both sites in Union City, TN is delayed due to insect availability. Each plant from both sites received eighteen (18) newworms. Assessment of ear damage was determined twenty-one (21) to twenty-eight (28) days after infestation. Damage to the ear was indicated as described previously. Both the tagged and non-tagged event MON95379 plants were artificially infected. In addition, natural insect pressure was also used to perform the assay at the site of the plant containing the marker event MON 95379. Table 12 and table 13 show FAW leaf damage grade, SWCB pore canal length, and CEW ear damage for the labeled and unlabeled event MON95379 plants.

Table 12 mean FAW leaf damage grade, SWCB pore length and CEW ear damage of event MON95379 plants prior to excision of the selectable marker Cre under artificial and natural infestation conditions.

Table 13 mean FAW leaf damage rating, SWCB pore canal length and CEW ear damage of marker-free event MON95379 plants under artificial infestation.

As can be seen in tables 12 and 13, event MON95379 provides resistance to FAW, SWCB, and CEW under both artificial (labeled and unlabeled) and natural (unlabeled) infection conditions.

In 2018, the resistance of hybrid hybrids of event MON95379 and event MON89034 to FAW was determined under natural infestation conditions in a brazilian field trial. The field tests were carried out on Santa Helena de Goi-s, State of Goi-s. At this site, there was a FAW population that was resistant to transgenic corn event MON 89034. Transgenic corn plants and conventional corn plants (negative control) corresponding to crosses of event MON95379 x MON89034, event MON89034 were grown. At stage V6, the measures shown in table 6 were used to determine leaf damage score for sixty (60) plants corresponding to the hybrid of event MON95379 x MON89034, thirty (30) plants corresponding to event MON89034, and thirty (30) negative controls. In addition, the number of FAW newworms, larvae greater than two millimeters (2mm) and less than or equal to 1.5 centimeters, and larvae greater than 1.5 centimeters was recorded for each plant. Table 14 shows the average leaf damage rating for hybrids of event MON95379 x MON89034, event MON89034, and the negative control, as well as the number of newborns and larvae observed on corn plants.

Table 14 average FAW leaf damage rating and number of newborns and larvae for hybrids of event MON95379 x MON89034, event MON89034 and negative control in brazil 2018 field trials.

As can be seen in table 14, hybrids of event MON95379 x MON89034 provided resistance to FAW under natural infestation conditions relative to the negative control. Hybrids of event MON95379 x MON89034 also performed better than event MON89034 under conditions where event MON89034 resistant FAW was within the FAW population. No newborns and larvae were observed on the plants corresponding to the hybrid of event MON95379 x MON 89034. On event MON89034 plants, newworms and larvae ranging from two (2) millimeters to one and a half (1.5) centimeters were observed. More larvae were observed on the negative control plants than on the event MON89034 plant, and had larvae that were greater than 1.5 cm in length.

Example 5

Determination of Activity of corn event MON95379 on corn stalk borer

This example describes the determination of the activity of transgenic corn event MON95379 on the lepidopteran insect pest borer (LSCB, corn borer).

Event MON95379 was grown in the greenhouse with negative control plants and infested with LSCB neoworms. Ten (10) event MON95379 plants and nine (9) negative control plants were grown in a single pot. Nine (9) days after planting, each plant was infested with ten (10) LSCB neoworms. Twenty-two (22) days after infestation, plants were examined and the lesions were rated using a 0-4 lesion rating scale as shown in table 15.

Table 15 LSCB plant damage scale measure.

Table 16 lists the resulting LSCB damage rating for each plant.

Table 16. LSCB plant damage per infected plant.

As can be seen in table 16, LSCB produced extensive damage to negative control plants, of which four (4) were scored as "dead", four (4) were scored as "severe damage", and only one (1) was scored as "average damage". In contrast, the plants infected with event MON95379 LSCB showed no damage.

Transgenic corn event MON95379 provides resistance to corn borer (LSCB, corn borer).

Example 6

Corn event MON95379 provides consistent yield and similar agronomic performance to untransformed LH244 maize plants

This example demonstrates that transgenic corn event MON95379 provides consistent yield and similar agronomic in the field as untransformed LH244 corn plants.

Prior to Cre excision of the glyphosate selection cassette, plants corresponding to event MON95379 were field tested to determine yield and agronomic aspects compared to control plants. Yield measurements were calculated and expressed as bushels/acre (bu/acre). Plant height and ear height are measured in inches (in). Fifty percent (50%) of pollen shed and fifty percent (50%) of silks were expressed as days post-planting (DAP). The test weight is a measure of the bulk density or weight per unit volume of the grain, expressed in pounds per bushel (lb/bu). The USDA rated a standard test weight of corn bushels at fifty-six pounds per bushel (56lb/bu) based on 15.5% moisture content. The moisture percentage of the corn kernels is expressed on a wet weight basis. Moisture content is the amount of water in a seed and is usually expressed in percent. It can be expressed on a wet weight (expressed as a percentage of the fresh weight of the seed) or dry weight (expressed as a percentage of the dry weight of the seed). The determination of the moisture percentage is destructive to the seed. The percent moisture (wet basis) can be calculated using the following simple formula:

M_wb＝(W_w/W_w+W_d)x 100

wherein W_wEqual to the weight of water, and W_dEqual to the weight of dry matter.

Yield and agronomic measures of the pre-Cre excision event MON95379 inbred lines and hybrids of the glyphosate-tagged cassette were determined during the 2016 U.S. growing season. Tables 17 and 18 show the yield and agronomic characteristics measured for the event MON95379 inbred and hybrid lines, respectively. The negative control plant used for inbred line comparison was untransformed variety LH 244. Hybrids containing event MON95379 can be created by cross-pollinating the inbred event MON95379 with maize variety 93IDI3, while the untransformed control is LH244 x 93IDI3 hybrid.

Table 17. yield and agronomic of event MON95379 inbred lines relative to non-transgenic controls.

Table 18. yield and agronomic of event MON95379 hybrids relative to non-transgenic controls.

As can be seen in tables 17 and 18, the yield and other agronomic measurements of event MON95379 were relatively identical for both inbred and hybrid lines in the 2016 united states field trial relative to the control. The variation between inbred lines and hybrids and their respective controls was within acceptable limits and showed no negative impact on yield and other agronomic characteristics obtained by T-DNA insertion event MON95379 in the maize genome.

The yield and agronomic of the Cre pre-excision event MON95379 inbred lines and hybrids of the glyphosate-tagged cassette were also studied during the growing season of argentina 2016 to 2017. Tables 19 and 20 show the yield and agronomic characteristics measured for the event MON95379 inbred line. The negative control plant used for inbred line comparison was untransformed variety LH 244. Hybrids containing event MON95379 were created by cross-pollination event MON89034 x MON 895379. The transgenic control was event MON88017 x event MON 89034. The non-transgenic control was the LH244 × 93IDI3 hybrid. Table 21 shows the yields and agronomic characteristics measured for the event MON95379 hybrid, where "NC" means not calculated.

Table 19. yield and agronomic of event MON95379 inbred lines relative to non-transgenic controls.

Table 20. yield and agronomic of event MON95379 inbred lines relative to non-transgenic controls.

Table 21. yield and agronomic of event MON95379 hybrids relative to transgenic and non-transgenic controls.

As can be seen in tables 19-21, the yield and other agronomic characteristic measurements for the event MON95379 inbred and hybrid relative to the control were relatively the same.

In 2017, the yield and agronomic of event MON95379 inbred lines and hybrids was again measured in a field trial in the united states prior to Cre excision of the glyphosate-tagged cassette. Inbred and hybrid controls were similar to those used in the 2016 U.S. field trial. Table 22 shows the yield and agronomic characteristics of the event MON95379 inbred relative to the non-transgenic control, and tables 23 and 24 show the yield and agronomic characteristics of the event MON95379 hybrid relative to the non-transgenic control as measured in the us field trial of 2017.

Table 22. yield and agronomic of event MON95379 inbred lines relative to non-transgenic controls.

Table 23. yield and agronomic of event MON95379 hybrids relative to non-transgenic controls.

Table 24. yield and agronomic of event MON95379 hybrids relative to non-transgenic controls.

As can be seen in tables 22-24, the yield and other agronomic characteristics of event MON95379 demonstrated in 2017 in the us field trial were similar to the untransformed controls of the inbred and hybrid lines.

Agronomic and yield measurements were made in field trials of event MON95379 inbred lines and hybrids after Cre excision of the glyphosate-tagged cassette during the growing season of argentina from 2018 to 2019. Inbred controls were similar to those used in the us field trial of 2017. The hybrid was produced by crossing with elite variety 80IDM 2. Tables 25 and 26 show yield and agronomic characteristics of the event MON95379 inbred lines relative to the non-transgenic control, and table 27 shows yield and agronomic characteristics of the event MON95379 hybrid relative to the non-transgenic control measured in the argentina field trial between 2018 and 2019.

Table 25. yield and agronomic of event MON95379 inbred lines relative to non-transgenic controls.

Table 26. yield and agronomic of event MON95379 inbred lines relative to non-transgenic controls.

Table 27. yield and agronomic of event MON95379 hybrids relative to non-transgenic controls.

As can be seen in tables 25-27, the yield and other agronomic characteristics of event MON95379 demonstrated in the argentina field trials from 2017 to 2018 were similar to the untransformed controls of the inbred and hybrid lines.

Thus, in summary, in the united states and argentina, corn event MON95379 exhibited similar yield and other agronomic characteristics in four (4) separate growing seasons. Event MON95379 did not negatively impact yield nor cause changes in other agronomic characteristics compared to the non-transgenic and transgenic controls.

Example 7

Corn event MON95379 event-specific endpointMeasurement of

The following example describes methods suitable for identifying the presence of event MON95379 in a corn sample. Design of a pair of PCR primers and probes for use at event-specific endpointsThe unique linkage formed between the corn genomic DNA and the inserted DNA of event MON95379 was identified in the PCR. The methods used for event-specific endpoints are described in tables 28 and 29An example of conditions in PCR to identify the presence of event MON95379 in a corn sample.

The sequence of oligonucleotide forward primer SQ21529(SEQ ID NO:15) is identical to the nucleotide sequence corresponding to position 833-852 of SEQ ID NO: 10. The sequence of the oligonucleotide reverse primer SQ21524(SEQ ID NO:16) is identical to the reverse complement of the nucleotide sequence corresponding to position 905-934 of SEQ ID NO: 10. The sequence of oligonucleotide probe PB10269(SEQ ID NO:17) is identical to the reverse complement of the nucleotide sequence corresponding to position 886-901 of SEQ ID NO: 10. Can be at the end pointPrimers SQ21529(SEQ ID NO:15) and SQ21524(SEQ ID NO:16) with probes PB10269(SEQ ID NO:17) that can be fluorescently labeled (e.g., 6-FAM) are used in the PCR assay^TMFluorescent label) to identify the presence of DNA derived from event MON95379 in the sample.

In addition to SQ21529(SEQ ID NO:15), SQ21524(SEQ ID NO:16), and PB10269(SEQ ID NO:17), it will be apparent to those skilled in the art that other primers and/or probes may also be designed to amplify or hybridize to sequences within SEQ ID NO:10 that are unique to and useful for detecting the presence of DNA derived from event MON95379 in a sample.

Following standard molecular biology laboratory practice, PCR assays for event identification were developed for the detection of event MON95379 in samples. With primer pairs and probes (e.g., labeled with a fluorescent label such as 6-FAM)^TM) Probes of (d) for a standard PCR assay orThe parameters of the PCR assay were optimized for detecting the presence of DNA derived from event MON95379 in the sample. Controls for the PCR reaction included internal control primers and internal control probes specific for regions within the corn genome used as internal controls (e.g.,labeled), and are the primers SQ20222(SEQ ID NO:18), SQ20221(SEQ ID NO:19) andlabeled probe PB50237(SEQ ID NO: 20).

Typically, parameters optimized for detection of event MON95379 in a sample include primer and probe concentrations, amount of templated DNA, and PCR amplification cycle parameters. Controls for this analysis included a positive control from corn containing event MON95379, a negative control from non-transgenic corn, and a negative control without template DNA.

TABLE 28 MON95379 event-specific endpointsAnd (4) PCR reaction components.

TABLE 29 end pointsThermocycler conditions.

Example 8

Use ofAssays for determining zygosity of event MON95379 and detecting insect toxin transgene

The following example describes methods suitable for identifying the zygosity of event MON95379 in a corn sample and detecting the insect toxin transgene in event MON 95379. PCR primer pairs and probes were designed to identify specific properties of the alleles positive and negative for T-DNA insertion that produced event MON 95379.

The zygosity assay is useful for determining whether a plant comprising an event is homozygous for the event DNA (i.e., comprises exogenous DNA located at the same position on each chromosome of the chromosome pair), heterozygous for the event DNA (i.e., comprises exogenous DNA on only one chromosome of the chromosome pair), or wild-type (i.e., the event DNA is null).

Will end upThe thermal amplification method was used to develop a zygosity assay for event MON 95379. This assay uses a primer pair and a probe to detect an amplicon corresponding to one of the two insect toxin coding sequences encoding cry1b.868 and Cry1Da _7 contained in the T-DNA used to produce corn event MON 95379. In addition, primer pairs and probes were used to detect single copy internal controls located within the maize genome and known to be present as homozygous alleles.

For this assay, two (2) primer pairs and two (2) probes were mixed together with the sample. The DNA primers used in the zygosity assay to detect the presence of the Cry1B.868 toxin coding sequence were primers SQ50998(SEQ ID NO:21) and SQ50997(SEQ ID NO: 22). Use in a zygosity assay to detect the presence of the Cry1B.868 toxin coding sequenceThe labeled DNA probe was PB54340(SEQ ID NO: 23). The DNA primers used in the zygosity assay to detect the presence of the Cry1Da _7 toxin coding sequence were primers SQ50485(SEQ ID NO:24) and SQ50484(SEQ ID NO: 25). Use in a zygosity assay to detect the presence of Cry1Da _7 toxin coding sequenceThe labeled DNA probe was PB50138(SEQ ID NO: 26). The same internal control was used for both zygosity testing assays. The primers of the internal control were SQ20222(SEQ ID NO:18) and SQ20221(SEQ ID NO:19), while the internal control was 6FAM^TMThe labeled probe was PB50237(SEQ ID NO: 20). As shown in tables 30 and 31, DNA primers and probes for cry1b.868 or Cry1Da _7 were mixed with primers and probes for internal controls.

TABLE 30 corn event MON95379 zygosity for detection of Cry1B.868PCR。

TABLE 31 corn event MON95379 zygosity for detection of Cry1Da _7PCR。

Depending on which toxin coding sequence was used for the assay, separate reactions were mixed using DNA from leaf samples with unknown zygosity, negative controls for DNA from untransformed maize plants, negative controls lacking DNA, and positive controls using DNA from transgenic plants homozygous for cry1b.868 or Cry1Da _ 7. The reaction was then subjected to thermal cycling as shown in table 32.

TABLE 32 bonding PropertiesThermocycler conditions.

After amplification, the cycle threshold (Ct value) of the amplicon corresponding to the toxin coding sequence and the single copy homozygous internal standard was determined. The difference (Δ Ct) between the Ct value of the single copy homozygous internal standard amplicon and the Ct value of the toxin coding sequence amplicon was determined. With respect to zygosity, a Δ Ct of about zero (0) indicates homozygosity for the inserted event MON 95379T-DNA, while a Δ Ct of about one (1) indicates heterozygosity for the inserted event MON 95379T-DNA. The absence of the amplicon corresponding to the insect toxin-encoding sequence indicates that the sample is empty for the inserted event MON 95379T-DNA. Due to multiple factors such as amplification efficiency and ideal annealing temperature,there is some variation in Ct values in the thermal amplification method. Thus, a range of "about one (1)" is defined as a Δ Ct of 0.75 to 1.25.

For each progeny derived from a hybrid with event MON95379, both toxin coding sequences were assayed to ensure accuracy of the progeny zygosity assay.

Example 9

Determining zygosity of corn event MON95379 using TAQMAN

The following example describes a method suitable for identifying the zygosity of event MON95379 in a corn sample.

PCR primer pairs and probes were designed to identify specific properties of the alleles positive and negative for T-DNA insertion that produced event MON 95379. Tables 33 and 34 provide event-specific engagementExamples of conditions that can be used in PCR. For this assay, three primers and two probes were mixed together with the sample. The DNA primers used in the zygosity assay were primers SQ50219(SEQ ID NO:15), SQ21524(SEQ ID NO:16) and PWTDNA (SEQ ID NO: 27). The probe used for measuring the zygosity was 6FAM^TMLabeled probes PB10269(SEQ ID NO:17) andthe labeled probe PRWcDNA (SEQ ID NO: 28). Primers SQ50219(SEQ ID NO:15) and SQ21524(SEQ ID NO:16) and 6FAM^TMLabeled probe PB10269(SEQ ID NO:17) was diagnostic for event MON95379 DNA. SQ50219(SEQ ID NO:15) and PWTDNA (SEQ ID NO:27) andthe labeled probe PRWTDNA (SEQ ID NO:28) has diagnostic significance; that is, they can diagnose wild type alleles.

When three primers and two probes were mixed together in a PCR reaction with DNA extracted from plants heterozygous for event MON95379, existing was from 6FAM^TMThe labeled probe PB10269(SEQ ID NO:17) also hasFromThe fluorescent signal of the labeled probe PRWTWDNA (SEQ ID NO:28), which is indicative of and diagnosable of a plant heterozygous for event MON 95379. When three primers and two probes were mixed together in a PCR reaction with DNA extracted from plants homozygous for event MON95379, only from 6FAM^TMFluorescent signal of labeled probe PB10269(SEQ ID NO:17) without fromFluorescent signal of the labeled probe PRWTDNA (SEQ ID NO: 28). When three primers and two probes were mixed together in a PCR reaction with DNA extracted from plants empty for event MON95379 (i.e., wild type), only from those that were emptyFluorescent signal of the labeled probe PRWTDNA (SEQ ID NO: 28). The template DNA samples and controls used for this analysis were a positive control from corn containing event MON95379DNA (from known homozygous and known heterozygous samples), a negative control from non-transgenic corn, and a negative control containing no template DNA.

TABLE 33 event MON95379 zygosityPCR

TABLE 34 bondabilityThermal cycler Condition

Example 10

Identification of maize event MON95379 in any MON95379 breeding event

The following example describes how the corn event MON95379 can be used to identify the MON95379 event in progeny of any breeding event.

The DNA primer pair was used to produce an amplicon diagnostic for corn event MON 95379. The amplicon of diagnosable event MON95379 comprises at least one junction sequence. The linkage sequence of event MON95379 is SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 (in FIG. 1, [1], [2], [3], [4], [5], [6], [7], and [8], respectively). SEQ ID NO 1 is a fifty (50) nucleotide sequence representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO 1 is located at nucleotide positions 838-887 of SEQ ID NO 10. SEQ ID NO 2 is a fifty (50) nucleotide sequence representing the 3' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO 2 is located at nucleotide position 14156-14205 of SEQ ID NO 10. SEQ ID NO 3 is a one hundred (100) nucleotide sequence representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. The SEQ ID NO 3 is located at nucleotide positions 813-912 of SEQ ID NO 10. SEQ ID NO 4 is a one hundred (100) nucleotide sequence representing the 3' junction region of the maize genomic DNA and the integrated transgene expression cassette. The SEQ ID NO. 4 is located at nucleotide positions 14,131 and 14,230 of SEQ ID NO. 10. SEQ ID NO 5 is a two hundred (200) nucleotide sequence representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO 5 is located at nucleotide position 763-962 of SEQ ID NO 10. SEQ ID NO 6 is a two hundred (200) nucleotide sequence representing the 3' junction region of the maize genomic DNA and the integrated transgene expression cassette. The SEQ ID NO 6 is located at nucleotide position 14,081-14,280 of SEQ ID NO 10. SEQ ID NO 7 is a sequence of one thousand one hundred sixty (1,160) nucleotides representing the 5' junction region of the maize genomic DNA and the integrated transgene expression cassette. SEQ ID NO. 7 is located at nucleotide positions 1-1,160 of SEQ ID NO. 10. SEQ ID NO 8 is a sequence of one thousand one hundred seventy eight (1,178) nucleotides representing the 3' junction region of the integrated transgene expression cassette and the maize genomic DNA. SEQ ID NO 8 is located at nucleotide position 14,039-15,216 of SEQ ID NO 10.

The primer pair that will produce the amplicon diagnostic for event MON95379 includes a primer pair based on the flanking sequences (SEQ ID NO:11 and SEQ ID NO:12) and the inserted T-DNA (SEQ ID NO: 9). To obtain a diagnostic amplicon in which SEQ ID NO 1, or SEQ ID NO 3, or SEQ ID NO 5 or SEQ ID NO 7 is visible, a forward primer molecule may be designed based on 5' flanking maize genomic DNA (SEQ ID NO 11; bases 1 to 862 from SEQ ID NO 10) and a reverse primer molecule may be designed based on the inserted T-DNA (SEQ ID NO 9; positions 863 to 14,180 from SEQ ID NO 10), wherein the primer molecules have a sufficient contiguous nucleotide length to specifically hybridize with SEQ ID NO 11 and SEQ ID NO 9. To obtain a diagnostic amplicon in which SEQ ID NO 2, or SEQ ID NO 4, or SEQ ID NO 6 or SEQ ID NO 8 is visible, a forward primer molecule may be designed based on the inserted T-DNA (SEQ ID NO 9; positions 863 to 14,180 from SEQ ID NO 10) and a reverse primer molecule may be designed based on the 3' flanking maize genomic DNA (SEQ ID NO 12; positions 14,181 to 15,216 from SEQ ID NO 10), wherein the primer molecules have a sufficient contiguous nucleotide length to specifically hybridize with SEQ ID NO 9 and SEQ ID NO 12.

For practical purposes, primers should be designed that produce amplicons of a limited size range, preferably between 200 and 1000 bases. In general, amplicons of smaller size are more reliably generated in a PCR reaction, allowing shorter cycle times, and they can be easily separated and viewed on agarose gels, or are suitable for end-point useIn the sample assay. Alternatively, amplicons generated using the primer pairs can be cloned into vectors, propagated, isolated and sequenced, or can be directly sequenced using well established methods in the art. One aspect of the inventionIs any primer pair derived from the combination of SEQ ID No. 11 and SEQ ID No. 9 or the combination of SEQ ID No. 12 and SEQ ID No. 9 suitable for use in a DNA amplification method to produce an amplicon diagnostic for event MON95379 or progeny thereof. One aspect of the invention is any single isolated DNA polynucleotide primer molecule comprising at least eleven (11) contiguous nucleotides of SEQ ID No. 11, SEQ ID No. 9 or SEQ ID No. 12 or the complement thereof, suitable for use in a DNA amplification method to produce an amplicon diagnostic for event MON95379 or its progeny.

Tables 28 and 29 show examples of amplification conditions for this assay. Any modification of these methods or the use of DNA primers homologous or complementary to SEQ ID No. 11 or SEQ ID No. 12 or the DNA sequence of the genetic element contained in the transgenic insert of event MON95379 (SEQ ID No. 9) that produces an amplicon diagnostic for event MON95379 is within the art. The diagnostic amplicon comprises a DNA molecule that is homologous or complementary to at least one transgene/genomic junction DNA or substantial portion thereof.

Analysis of plant tissue samples comprising event MON95379 should include a positive tissue control from a plant comprising event MON95379, a negative control (e.g., LH244) from a corn plant without event MON95379, and a negative control without corn genomic DNA. The primer pair will amplify an endogenous maize DNA molecule and will serve as an internal control for the DNA amplification conditions. Those skilled in the art of DNA amplification methods can select additional primer sequences from SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 9. The conditions selected for amplicon production by the methods shown in table 28 and table 29 may vary, but will produce an amplicon diagnostic for event MON95379 DNA. The use of DNA primer sequences within or modified from the methods of tables 28 and 29 is within the scope of the present invention. One aspect of the invention is an amplicon generated by at least one DNA primer sequence derived from SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 9 that is diagnostic for event MON 95379.

One aspect of the present invention is a DNA detection kit comprising at least one DNA primer having a sufficient length of contiguous nucleotides derived from SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO. 9 when said DNA primerWhen used in a DNA amplification method, produces a diagnostic amplicon of event MON95379 or progeny thereof. One aspect of the invention is a corn plant or seed, wherein the genome thereof will produce an amplicon diagnostic for event MON95379 when tested in a DNA amplification method. Can be obtained by using Applied Biosystems GeneAmp^TM PCR System 9700、Stratagene The determination of event MON95379 amplicon was performed by a Gradient thermocycler or any other amplification system that can be used to produce an amplicon diagnostic for event MON95379 as shown in table 29.

All publications and published patent documents cited in this specification, which are material to the present invention, are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Having illustrated and described the principles of the present invention, it will be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. Such modifications are also within the scope of the appended claims.