Nucleic acid molecule from the ME240913 maize transgenic event.

Abstract

The present invention relates to a novel transgenic maize event expressing the truncated or modified cry1da insecticidal protein, designated event me240913. The invention describes the nucleic acids that are unique to event me240913. Also defined are primers, amplicons, methods and kits for detecting the presence of event me240913. The invention also relates to maize plants containing said event, uses thereof, methods and compositions for controlling lepidopteran insect pests. The invention describes a maize event that has demonstrated a high level of plant protection against feeding damage produced by lepidoptera, including S.frugiperda, as well as insects resistant to cry1f. The event of the invention proved to be highly toxic to S. frugiperda.

Description

Descriptive Report of the Invention Patent for NUCLEIC ACID MOLECULE FROM THE TRANSGENIC EVENT OF CORN ME240913. FIELD OF THE INVENTION

[001] The present invention relates to the field of plant molecular biology, plant transformation, and plant reproduction and pest control. More specifically, the invention relates to transgenic maize plants (Zea mays) resistant to lepidopteran insect pests comprising a novel transgenic genotype, uses thereof, methods of controlling lepidopteran insect pests and detection of the presence of nucleic acids unique to transgenic maize plants in a plant product and compositions thereof. BACKGROUND

[002] Consistent advances in genetic engineering techniques have enabled the development of commercially important transgenic plants containing heterologous genes of interest, which confer desirable characteristics to such plants. Among the genes of interest are genes that confer resistance to herbicides, environmental stresses, diseases, and invertebrate pests.

[003] In the context of genes encoding proteins that are useful for controlling invertebrate pests, the cry gene, originating from the Gram-positive bacterium Bacillus thuringiensis (Bt), can be cited. This bacterium, which occurs naturally in various habitats, including soil, phylloplane, grain residues, dust, water, plant matter, and insects, has the innate characteristic of forming protein crystals during the stationary and / or sporulation phase. The protein crystals or delta-endotoxins, representing 20 to 30% of the total cell protein (Boucias & Pendland, 1998), can exhibit properties Petition 870240068507, dated 12 / 08 / 2024, page 17 / 96 2 / 58 Specific insecticides have various shapes, such as: bipyramidal, spherical, rectangular, cuboid, and irregular. Bipyramidal crystals exhibit a higher frequency of toxicity than crystals with other shapes, particularly against lepidopterans.

[004] The mechanism of action of Cry proteins with insecticidal effect generally involves the solubilization of crystals in the midgut of target insects, the digestive action of proteases present in the intestine of these insects on the pro-toxins, the adhesion of the active toxins to the receptors of the midgut and the insertion of these active Cry toxins into the apical cell membrane, creating ion channels or pores (cytolysis). These channels, when formed, can alter the insect's feeding and disrupt the integrity of the intestine, leading to decreased growth and even death.

[005] One advantage of using Cry proteins in agriculture is the selective toxicity of each of these proteins against various species of insect pests, and their non-toxic nature to other vertebrate organisms, such as fish, amphibians, reptiles, birds, and mammals. Another advantage of Cry proteins is their relative specificity of toxicity in relation to insect pests of different crops. Currently, several cry genes are recognized. The cry1, cry2, and cry9 genes are generally active against Lepidoptera; the cry2, cry4A, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40 genes are generally active against Diptera; the cry3, cry7, and cry8 genes are generally active against Coleoptera; and the cry5, cry12, cry13, and cry14 genes are generally active against Nematodes. Commercial products containing Bt Cry proteins with insecticidal activity are produced today and used as biopesticides.These products have a high sales percentage in the market and have been used for over 60 years, primarily to control pests of the orders Lepidoptera and Diptera. Another use of these proteins... Petition 870240068507, dated 12 / 08 / 2024, page 18 / 96 3 / 58 Cry refers to its expression in transgenic plants.

[006] Despite the use of specific Bt proteins in transgenic corn plants for the control of insect pests, their use has produced populations of Lepidoptera and Coleoptera resistant to some of these Bt proteins (Tabashnik, BE; Brévault, T.; Carrière, Y. Insect resistance to genetically engineered crops: successes and failures. ISB News Report: Agricultural and Environmental (Biotechnology, Jan. 2014). A specific case is the emergence of Spodoptera frugiperda populations resistant specifically to the Cry1F and Cry1A genes, which are more commonly found in tropical conditions. Spodoptera is the main pest in several regions of the world that cultivate corn, and the development of resistance to Cry1F and Cry1A proteins represents a significant challenge for corn production in these locations worldwide. Spodoptera populations found in the field identified as resistant to Cry1F proteins were recorded (1) in 2010, Puerto Rico (Storer, NP; Babcock, JM; Schlenz, M.; Meade, T.; Thompson, GD; Bing, JW; Huckaba, RM Discovery and characterization of field resistance to Bt corn: S. frugiperda (Lepidoptera: Noctuidae) in Puerto Rico. Journal of Economic Entomologia, v. 103, p. 1031-1038, 2010.), (2) in the United States (Huang, F.; Qureshi, JA; Meagher JR, RL; Reisig, DD; Head, GP; Andow, DA; NI, X.; Kerns, D.; Buntin, GD; Niu, Y.; Yang, F.; Dangal, V. Cry1F resistance in fall armyworm S. frugiperda: single gene versus pyramided Bt maize.) and (3) in Brazil (Farias, JR; Andow, DA; Horiksoshi, RJ; Sorgatto, RJ; Fresia, P.; Santos, AC; Omoto, C. Field-evolved resistance to Cry1F maize by S. frugiperda (Lepidoptera: Noctuidae) in Brazil. Crop Protection, v.64, p.150-158, 2014). In Brazil, populations of S. frugiperda resistant to Cry1Ab have also been identified (Omoto, C.; Berbardi, O. Salmeron, E.; Sorgatto, RJ; Dourado, PM; Crivellari, A.; Carvalho, RA; Willse, A.; Martinelli, S.; Head, G. Petition 870240068507, dated 12 / 08 / 2024, page 19 / 96 4 / 58 P. Field-evolved resistance to CrylAb maize by S. frugiperda in Brazil. Pest Management Science, Chichester, 2016). Some of these Cry1F-resistant S. frugiperda populations develop rapidly due to selective pressure from products based on transgenic plants containing only one cry1 gene active against S. frugiperda.

[007] Due to this large-scale problem of the development of resistance to single genes expressing Cry1F in maize plants, commercial cultivars are made available containing the combination of two or more insecticidal genes such as crylAb, crylF, vip3A, cry1A.105, cry2Ab, cry3Bb, cry34Ab, cry35Ab, mcry3A, ecry3.1Ab, and dvsnf7.

[008] It is therefore critical to use proteins with different modes of action in the so-called protein pyramiding that slows the rate of resistance development. Consequently, there has been great interest in the continued discovery of new proteins active against S. frugiperda that do not have cross-resistance with those that already exhibit resistance to, for example, Cry1F.

[009] The dvsnf7 gene is another insect pest control technology that uses double-stranded RNA to inhibit genes important for insect survival. However, it has little effect on the order Lepidoptera, which is extremely voracious, causing defoliation in very short times.

[0010] There is therefore a great need for the development of new alternative proteins that are toxic to both wild-type insect populations and populations resistant to Cry1F / Cry1A. These new proteins must have high value for use in transgenic strategies, be applicable in transgenic technology, and be able to control lepidopteran insect pests in transgenic crops. Petition 870240068507, dated 12 / 08 / 2024, page 20 / 96 5 / 58

[0011] One of these proteins is CrylDa, produced by some Bt strains and known to be toxic to S. frugiperda (Costa, ML, Lana, UG, Barros, EC, Paiva, LV and Valicente, FH, J of Agricultural Science, 2014, vol 6, pp128-136). However, the CrylDa protein is described in previous articles as having a limited spectrum of toxicity against lepidopteran pests (von Frankenhuyzen, 2009) and variable toxicity for different populations within a lepidopteran species, such as S. frugiperda. For example, Monnerat et al (2006). They found that the CrylDa protein was toxic to S. frugiperda collected in Colombia and Mexico, but that it was non-toxic to a population collected in Brazil (Applied and Environmental Microbiology, 2006, vol 72, p. 7029-7035).

[0012] The relatively narrow spectrum of activity against lepidopteran pests and its variability in toxicity levels has been a major limitation in the use of CrylDa. Specifically, von Frankenhuyzen (Frankenhuyzen, K. 2009. Minireview: Insecticidal activity of Bacillus thuringiensis crystal proteins. Journal of Invertebrate Pathology. 101: 1-16) reported that CrylDa had the narrowest spectrum of activity of the Cry1 proteins against a variety of lepidopteran species (toxic against only 44% of the species tested) compared to the leading Cry1A and 2Aa proteins tested (active against > 80% of the species tested).Due to the limitations of the native CrylDa protein in its activity spectrum and its variability in toxicity level, researchers have been working to improve the structure of the native Cry1 Da protein to make it more efficient against a wider spectrum of insect pests of the order Lepidoptera, more specifically the corn earworm (Helicoverpa zea).

[0013] Document W02007107302 describes the use of Cry1C, Cry1D, or CrylDa sequences to produce a novel chimeric protein potentially active against the corn earworm. Da Petition 870240068507, dated 12 / 08 / 2024, page 21 / 96 6 / 58 Similarly, documents WO2015143311 and WO2016061377 describe structural modifications of the Cry1Da protein in an attempt to broaden its spectrum against other lepidopteran pests, specifically corn earworm (Helicoverpa zea), including modifications to the native amino acid sequence of Cry1Da or fusion with other insecticidal proteins to increase insecticidal activity against a wider spectrum of lepidopteran pests. Because the focus of these experiments and findings was on increasing the spectrum of Cry1Da activity against other lepidopterans, there is essentially no data available regarding the toxicity of Cry1Da or its naturally occurring variants against populations of S. frugiperda, including those resistant to Cry1F. The only study involving the evaluation of Cry1Da expressed in transgenic maize plants showed that this protein has a small to moderate protective effect against foliar damage caused by S.frugiperda (population not specified in the study). Furthermore, no specific data on the toxicity of this protein against this pest are shown. Given the level of damage produced in the study cited above, it is reasonable to conclude that the transgenic plants had only a small toxic effect on the insects.

[0014] With regard to genetically modified plants, DNA constructs that encode desired proteins are individually inserted into the plant genome by genetic transformation. Current plant transformation methods primarily utilize Agrobacterium tumefaciens, which generally leads to a low number of gene construct copies within the host plant genome. Gene constructs are composed of a promoter, coding region, and terminator. The promoter is the regulatory and determinant element of gene expression, both temporally and spatially. Generally, for quantity expression Petition 870240068507, dated 12 / 08 / 2024, page 22 / 96 7 / 58 sufficiently large, as is the case in the production of insecticidal protein for the control of lepidopteran insect pests, the constructs utilize constitutive promoters such as the one that controls the expression of the ubiquitin gene in maize.

[0015] The integration of the DNA construct into the host genome is random, and this random insertion into the plant genome's DNA can impact a gene whose product is critical for plant survival, rendering the resulting plant unviable. Furthermore, random insertion can target a region of the host genome that may negatively influence the expression of the gene of interest, regardless of the use of constitutive promoters. In other examples, overproduction of the construct's genetic product has detrimental effects on the cell, primarily leading to decreased productivity. Due to these potential problems, it is common to produce dozens (in some cases hundreds) of different events and track these events to a single event that possesses the desired patterns and levels of transgenic expression for commercial purposes.

[0016] An event that possesses desired levels or patterns of transgenic expression is useful for transfer to other genetic backgrounds of the same species through traditional sexual crossing. There are reports of genes that, even when expressed at high and constitutive levels in the parent genotype, did not necessarily show the same response in other genetic backgrounds. Therefore, it is desirable that, in addition to adequate spatial and temporal expression, transformation events exhibit low gene expression variation in crosses with other genotypes (different backgrounds).

[0017] In this case, the offspring of such crosses retain the transgenic expression characteristics of the host plant. Petition 870240068507, dated 12 / 08 / 2024, page 23 / 96 8 / 58 original transformant. This strategy is used to ensure reliable gene expression in various varieties that are well adapted to local growing conditions. This is impacted by having inserted the integrated DNA at ideal locations within the genome, thus providing the best levels of temporal and spatial expression, stability across multiple generations of breeding and across various genetic origins. As such, the physical genomic location of the inserted DNA becomes a fundamental characteristic of the resulting product's effectiveness and is therefore novel.

[0018] It would also be of great interest to establish a method capable of detecting the presence of a specific transformation event, in order to determine the presence of said event for seed quality testing, field release, and in processed plants or samples. Such methods could also be used to determine and monitor gene segregation in offspring from sexual crosses, traceability of the event in crosses, and detection in foods derived from recombinant plants. A well-known, but not limited to, nucleic acid detection method is in vitro DNA amplification by PCR (Polymerase Chain Reaction) using polynucleotide primers. Another method is DNA hybridization using nucleic acid probes. Detection methods may use primers or probes based on common elements between different gene constructs or based on specific regions of the construct.

[0019] For the reasons mentioned above, there is a need to identify detection methods based on novel nucleic acid sequences that are unique to the transgenic maize event, useful for identifying the transgenic maize event and for detecting nucleic acids from the transgenic maize event in a plant product, as well as kits comprising the reagents necessary for use in. Petition 870240068507, dated 12 / 08 / 2024, p. 24 / 96 9 / 58 detection of these nucleic acids in a plant product. SUMMARY

[0020] The present invention relates to a transgenic maize event, designated ME240913, comprising a novel transgenic genotype containing a crylDa nucleic acid sequence optimized for expression in maize. The crylDa coding sequence encodes a truncated variant of the native Cry1 Da protein with SEQ ID NO: 3, which surprisingly confers a high level of plant protection against leaf damage caused by various populations of S. frugiperda that occur naturally in Brazil (wild type and Cry1F resistant). Importantly, the leaf tissue of the ME240913 event produces an unexpectedly high toxicity to lepidopteran insect pests, such as Noctuidae insects, particularly S. frugiperda.In addition to the crylDa coding sequence, the present invention also provides other nucleic acids that are unique to the ME240913 event, namely the sequence of an amplicon resulting from a PCR reaction using specific primers to confirm the 5' junction sequence, the 3' junction sequence, 5' and 3' flanking sequences, and / or complements thereof. The invention also provides amplicons comprising the unique nucleic acids of the ME240913 event, transgenic maize plants comprising the unique nucleic acids of the ME240913 event, and seeds of the transgenic maize plants.

[0021] The present invention also relates to methods for producing a transgenic maize plant comprising the unique nucleic acids of the invention by sexual crossing of a first parent maize plant with a second parent maize plant to produce a plurality of first-generation offspring plants, wherein at least one of said parent plants comprises a unique nucleic acid for the event ME240913, selection of a first-generation offspring plant. Petition 870240068507, dated 12 / 08 / 2024, page 25 / 96 10 / 58 that is resistant to infestation by lepidopteran insect pests, self-pollination of the first-generation offspring plant to produce a plurality of second-generation offspring plants, and selection, among the second-generation offspring plants, of a plant that is resistant to lepidopteran insect pests.

[0022] The present invention further describes methods for controlling lepidopteran insect pests, such as those of the Noctuidae family, particularly S. frugiperda. Corn plants comprising the ME240913 event and methods of the present invention are effective for controlling specific lepidopteran insect pest populations, such as S. frugiperda, that have become resistant to plants expressing Cry1F protein.

[0023] Methods for producing hybrid maize seeds are also disclosed. Such methods comprise the steps of planting seeds of a first congenital maize line comprising at least one unique nucleotide sequence for the ME240913 event and seeds of a second congenital line having a different genotype, cultivating maize plants resulting from said planted seeds until flowering time, emasculating the flowers of plants of one of the congenital maize lines, sexual crossing of the two different congenital lines with each other, and harvesting the hybrid seed thus produced.

[0024] A sample of the seed, and consequently maize plants grown from the seed, comprising unique nucleic acids for the ME240913 event, was deposited in the American Type Culture Collection (ATCC) with accession number PTA-126224. The transgenic maize plants of the invention exhibit essentially all the corresponding morphological and physiological characteristics of the non-transgenic isogenic maize plants in addition to those conferred on the maize plants by the novel genotype of the invention. Products of Petition 870240068507, dated 12 / 08 / 2024, page 26 / 96 11 / 58 Plant and plant extracts from corn ME240913, tissues and seeds are also provided by the present invention.

[0025] The present invention further provides methods for introgressing the ME240913 event into maize lines by means of sexual crossing of plants containing the ME240913 event, such as, for example, but not limited to, plants obtained from seeds deposited in the American Type Culture Collection (ATCC) with accession number PTA-126224.

[0026] According to the present invention, the ME240913 event can be combined with other transgenic maize events by means of methods known in the art, such as gene pyramiding. The teaching that ME240913 produces a high level of control against plant damage produced by various Brazilian S. frugiperda populations (including Cry1F-resistant insects) establishes that the new event is toxic to Cry1F-resistant S. frugiperda populations in Brazil. These results also indicate that the ME240913 event acts through a mechanism of action distinct from that of Cry1F and, therefore, the maize event will be very effective when combined with other insecticidal genes incorporated into the plant using gene pyramiding. The ME240913 event also expresses the pat(bar) herbicide resistance gene. Therefore, the ME240913 event can also be used to reduce the rate of herbicide resistance using gene pyramiding.

[0027] Examples of gene pyramiding include, but are not limited to, herbicide and insect pest resistance events. Through such methods, the ME240913 event can be combined, for example, with events containing one or more genes selected from a pat gene, a cp4 epsps gene (5-enolpyruvylshikimate-3-phosphate synthase), a crylAb gene, a crylF gene, a vip3A gene, a cry1A.105 gene, a cry2Ab gene, a cry3Bb gene, a cry34Ab gene, a Petition 870240068507, dated 12 / 08 / 2024, page 27 / 96 12 / 58 cry35Ab gene, a mcry3A gene, ecry3.1Ab, a dvsnf7 gene, and an amy797E gene.

[0028] The present invention also provides a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer, which function together in the presence of a template DNA of the ME240913 event in a sample to produce a diagnostic amplicon of the ME240913 event, wherein the first polynucleotide primer comprises a portion of the 5' flanking sequence, the 3' flanking sequence, and / or their complements, wherein the second polynucleotide primer comprises a portion of a specific inserted sequence, or its complements, and wherein said maize event ME240913 presents a specific sequence containing the event insertion and flanking sequences in the maize genome.

[0029] The present invention relates to transgenic maize plants as well as to cells and tissues thereof, which comprise a nucleic acid molecule of the invention.

[0030] In one embodiment, the transgenic maize plant is resistant to lepidopteran insect pests. In a more preferred embodiment, the transgenic maize plant is highly resistant to leaf damage caused by naturally occurring S. frugiperda populations found in maize-producing regions of Brazil.

[0031] In another embodiment, the transgenic corn plant is highly toxic to S. frugiperda, producing 100% mortality when fed fresh leaf tissue. In another embodiment, the transgenic corn plant is highly toxic to S. frugiperda, as evidenced by high mortality and morbidity in S. frugiperda after dilution of lyophilized leaves from event ME240913 at a ratio of 1:25 (weight / weight) with artificial diet. The high toxicity of Petition 870240068507, dated 12 / 08 / 2024, page 28 / 96 13 / 58 freeze-dried plant leaf tissue from event ME240913 shows that said event is very efficient in controlling S. frugiperda and useful for pyramiding several insect control genes.

[0032] Corn seeds comprising a nucleic acid molecule of the invention are also disclosed. In one embodiment, the corn seeds are deposited in the American Type Culture Collection under accession number PTA-126224 and are used to produce transgenic corn plants.

[0033] Furthermore, the present invention relates to a maize plant product event ME240913, tissue or seed, which comprises a nucleotide sequence of the present invention or its complements, and in which the sequence is detectable in the plant product using a nucleic acid amplification or nucleic acid hybridization method.

[0034] The invention also covers plant products, such as, but not limited to, corn kernels, fodder, corn flour, corn starch, corn syrup, corn oil, corn starch, and cereals manufactured wholly or partly with corn-derived products.

[0035] The present invention also provides kits for detecting nucleic acids that are unique to the ME240913 event, which comprise at least one nucleic acid molecule that is a primer or probe comprising a nucleic acid sequence comprising a specific sequence, and complements thereof, wherein said primer or probe, by amplifying or hybridizing a target nucleic acid sequence in a sample followed by amplicon detection or hybridization with the target sequence, is diagnostic for the presence of nucleic acid sequences unique to the ME240913 event in the sample.

[0036] Methods are also revealed for detecting the presence of at least one nucleic acid molecule that is unique to the event. Petition 870240068507, dated 12 / 08 / 2024, p. 29 / 96 14 / 58 ME240913 in a sample comprising maize nucleic acids, wherein said methods comprise the following steps: contacting the sample with a pair of specific primers of the present invention, performing a nucleic acid amplification reaction so as to produce an amplicon, and detecting said amplicon, wherein the amplicon comprises nucleic acid sequences unique to the ME240913 event, or its complements, and wherein said maize event ME240913 exhibits a specific sequence containing the event insertion and flanking sequences in the maize genome.

[0037] Another method for detecting the presence of a nucleic acid molecule that is unique to the ME240913 event in a sample comprising maize nucleic acids comprises the steps of: contacting the sample with a probe that hybridizes under high stringency conditions with the genomic DNA of the ME240913 event and does not hybridize under high stringency conditions with the DNA of a control maize plant, wherein the probe comprises a nucleotide sequence unique to the ME240913 event and its complements, subjecting the sample and the probe to high stringency hybridization conditions, and detecting the hybridization of the probe with the nucleic acid molecule, wherein the said maize event ME240913 presents a specific sequence containing the event insertion and flanking sequences in the maize genome.

[0038] Furthermore, methods are disclosed for controlling lepidopteran insect pests of maize plants, wherein the maize plants comprise a nucleic acid molecule comprising unique nucleic acid sequences for the event ME240913, or its complements, wherein said methods comprise the steps of planting seeds obtained from a plant comprising said unique nucleic acid sequences. Petition 870240068507, dated 12 / 08 / 2024, page 30 / 96 15 / 58 for event ME240913 in a corn cultivation area susceptible to lepidopteran insect pests.

[0039] In addition, methods are described for the control of lepidopteran insect pests of corn plants, in which corn plants contain truncated and modified Cry1Da protein, produced from the expression of unique nucleic acid sequences for the ME240913 event, leading to the accumulation of expression levels in leaves of the truncated and modified Cry1Da protein which are highly protective against foliar damage caused by populations that occur naturally in Brazil and are also highly toxic against S. frugiperda as can be seen in the high mortality rates in the studies carried out.

[0040] Further disclosed are the use of a plant, plant cell, plant part or seed comprising the event ME240913 to cross with a second plant, regenerate a plant, plant or cultivate a field of plants or produce a plant product.

[0041] These and other aspects of the invention will become more apparent from the detailed description that follows. DESCRIPTION OF SEQUENCES IN SEQUENCE LISTING

[0042] SEQ ID NO: 1 is the nucleotide sequence encoding the truncated Cry1Da protein, optimized for expression in maize, present in event ME240913.

[0043] SEQ ID NO: 2 is the nucleic acid sequence of the complete transgene construct of the ME240913 event comprising in the following order: the 3' UTR terminator region of the Tvsp gene; coding region of the phosphinothricin acetyltransferase gene (bar), translational enhancer region (tev); promoter region of the duplicated CaMV 35S gene, promoter region of the ubiquitin gene (ubi); codon-optimized nucleic acid sequence of the cry1Da gene (SEQ ID NO: 1); and 3' UTR terminator region of the nopalin synthase gene. Petition 870240068507, dated 12 / 08 / 2024, page 31 / 96 16 / 58

[0044] SEQ ID NO: 3 is the amino acid sequence of the protein CrylDa truncated and expressed by event ME240913.

[0045] SEQ ID NO: 4 is the join sequence 5'.

[0046] SEQ ID NO: 5 is the join sequence 3'.

[0047] SEQ ID NO: 6 is the flanking sequence 5'.

[0048] SEQ ID NO: 7 is the flanking sequence 3'.

[0049] SEQ ID NO: 8 is the nucleotide sequence comprising the 5' flanking sequence (nucleotides 1-116), the complete insert sequence (nucleotides 117-6306) and the 3' flanking sequence (nucleotides 6307-6424) of event ME240913.

[0050] SEQ ID NO: 9 is a useful forward initiator sequence in the amplification of the 5' join sequence.

[0051] SEQ ID NO: 10 is a reverse initiator sequence useful in 5' junction sequence amplification.

[0052] SEQ ID NO: 11 is an illustrative amplicon sequence resulting from PCR using specific primers to confirm the 5' junction sequence.

[0053] SEQ ID NO: 12 is a useful probe sequence in detecting the 5' junction sequence.

[0054] SEQ ID NO: 13 is a forward initiator sequence useful in 3' join sequence amplification.

[0055] SEQ ID NO: 14 is a reverse initiator sequence useful in 3' junction sequence amplification.

[0056] SEQ ID NO: 15 is an illustrative amplicon sequence resulting from PCR using specific primers to confirm the 3' junction sequence.

[0057] SEQ ID NO: 16 is a useful probe sequence in detecting the 3' junction sequence. BRIEF DESCRIPTION OF THE FIGURES

[0058] Figure 1 is a representative diagram of the insert of Petition 870240068507, dated 12 / 08 / 2024, p. 32 / 96 17 / 58 event ME240913 and the respective nucleotide sequences that define said event, including 5' and 3' joining and flanking sequences of the maize genome.

[0059] Figure 2 is a representative diagram of the Ubi::cry1Da::NOS and 2x35S::bar::Tvsp gene constructs of the ME240913 event insert inserted into the HindIII and EcoRI enzyme sites of the pTF101.1 binary vector, between the right and left edges of the TDNA.

[0060] Figure 3 shows the result of the event evaluation. ME240913 regarding the control of S. frugiperda using fresh corn leaves for a period of 5 days. Samples of fresh non-GMO corn leaves (a) and genetically modified corn with Cry1Da (b) are shown.

[0061] Figure 4 shows the result of S exposure tests. Survival of S. frugiperda (%) was evaluated up to 3 days after exposure of newly hatched larvae to fresh leaves of control corn and corn leaves expressing the ME240913 event in two different genetic backgrounds (hybrid and RC1F1).

[0062] Figure 5 shows the survival rate of newly hatched larvae after 14 days of exposure to lyophilized leaves at a 1:25 dilution in an artificial diet of the control and the ME240913 event.

[0063] Figure 6 shows the sizes of live caterpillars after 7 (A) and 10 (B) days of exposure to 1:25 diluted lyophilized leaf tissue of Event ME240913 on the artificial diet compared to the control diet.

[0064] Figure 7 shows the result of damage level on the leaves of control (conv) and Event ME240913 (GMO) corn plants in plots infested by six different populations of S. frugiperda.

[0065] Figure 8 shows photographs of damage to control corn plants (left) and of Event ME240913 (right) in the field. Petition 870240068507, dated 12 / 08 / 2024, page 33 / 96 18 / 58 infested by populations of S. frugiperda from Palotina / PR (8A), Rondonópolis / MT (8B), Rondonópolis + Campo Verde / MT (8C), Paracatu / MG (8D), Sete Lagoas / MG (8E), and Ivatuba / PR (8F).

[0066] Figure 9 refers to the graph of the injury score (±IC, P = 0.05) caused by S. frugiperda infestation on a scale of Carvalho, 1970. Treatment 1 = transgenic maize comprising the cry1Da codon-optimized nucleic acid molecule of the present invention (SEQ ID NO: 1) + Cry1F resistant caterpillar population; Treatment 2 = non-transgenic L3 maize line + Cry1F resistant caterpillar population; Treatment 3 = transgenic maize comprising the cry1Da codon-optimized nucleic acid molecule of the present invention (SEQ ID NO: 1) + susceptible caterpillar population; Treatment 4 = non-transgenic L3 maize line + susceptible caterpillar population. DETAILED DESCRIPTION OF THE INVENTION

[0067] Unless otherwise defined, all technical terms, annotations and other scientific terminology used herein are intended to have the meanings normally understood by those skilled in the art in the field of the present invention. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is generally understood in the prior art.

[0068] The techniques and procedures described or referred to in this document are generally well understood and employed using conventional methodology by those skilled in the art. As appropriate, processes involving the use of commercially available kits and reagents are generally carried out Petition 870240068507, dated 12 / 08 / 2024, page 34 / 96 19 / 58 in accordance with protocols and / or parameters defined by the manufacturer, unless otherwise indicated.

[0069] It is worth noting that the present invention, where appropriate, is not limited to the methodology, protocols, cell lines, genera or species of animals, constructs and specific reagents described, which, obviously, may vary. Furthermore, the terminology used in this document is only for the purpose of describing examples of specific embodiments, and is not intended to limit the scope of the present invention.

[0070] Throughout this document, the singular forms “a”, “an”, and “the”, or singular forms of any term or expression, include references to the plural, unless the context clearly dictates otherwise.

[0071] Throughout this document, the word “comprises”, and any variations such as “comprises” or “comprises”, should be interpreted as “open terms”, which may imply the inclusion of additional elements or groups of elements, which have not been explicitly described, and are not limiting in nature.

[0072] Throughout this document, the word “consists”, and any variations such as “consist” or “consisting”, should be interpreted as “closed terms”, and cannot imply the inclusion of additional elements or groups of elements that have not been explicitly described, and are of a limiting nature.

[0073] Throughout this document, exact values ​​or ranges of exact values ​​given with respect to a particular factor, quantity, concentration or preference should be interpreted as also giving corresponding approximate values ​​or ranges of values, such as by means of the expression “about”.

[0074] Throughout this document, words and expressions such as “preferably”, “particularly”, “for example”, “such as”, “such as” Petition 870240068507, dated 12 / 08 / 2024, page 35 / 96 Terms like “20 / 58 as”, “more particularly” and similar expressions, and their variations, should be interpreted as entirely optional features, preferred realizations, or possible non-exhaustive examples, without conferring a limiting scope.

[0075] Throughout this document, words and expressions such as “nucleic acids,” “nucleotides,” and the like are to be interpreted as naturally occurring, synthetic, or artificial nucleic acids or nucleotides. They include deoxyribonucleotides (DNA) or ribonucleotides (RNA) or any nucleotide analog and polymers or hybrids thereof in sense or antisense configuration, single-stranded or double-stranded. Unless otherwise stated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. The term “nucleic acid” is used interchangeably in this document with the terms “gene,” “cDNA,” “mRNA,” “oligonucleotide,” “nucleic acid molecule,” or “primer.”

[0076] The expressions “nucleic acid molecule”, “nucleic acid sequence” and the like refer to a polymer of single-stranded or double-stranded DNA or RNA bases, read from the 5' end to the 3' end. This includes chromosomal DNA, self-replicating plasmids, infectious DNA or RNA polymers that play a primarily structural role, among others. They also refer to a consecutive list of abbreviations, letters, characters or words, representing nucleotides or genes, as commonly used in the technical field of the present invention.

[0077] As used in this document, the term “amplified” Petition 870240068507, dated 12 / 08 / 2024, p. 36 / 96 21 / 58 refers to the construction of multiple copies of a nucleic acid molecule or multiple complementary copies of a nucleic acid molecule using at least one of the nucleic acid molecules as a template. Amplification systems include, but are not limited to, Polymerase Chain Reaction (PCR) systems, Ligase Chain Reaction (LCR) systems, Nucleic Acid Sequence-Based Amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, Transcription-Based Amplification (TAS) systems, and Strand Displacement Amplification (SDA). See, for example: Diagnostic Molecular Microbiology: Principles and Applications, DH Persing, et al., Ed., American Society for Microbiology, Washington, DC (1993). The product of amplification is described as an amplicon.

[0078] A “coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. Preferably, the RNA is then translated in an organism to produce a protein.

[0079] Throughout this document, words and expressions such as “sequence similarity,” “identity,” and the like, with respect to another sequence, shall be interpreted as the percentage of nucleotides in the sequence that is identical to the nucleotides of another sequence, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. According to the present invention, the expression “at least 70% similarity,” for example, is defined as similarity or identity of 70 to 100%. Preferably, the percentage of similarity is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0080] A “gene” is a defined region located within a genome and which, despite the coding sequence mentioned Petition 870240068507, dated 12 / 08 / 2024, page 37 / 96 22 / 58 above, may include other, primarily regulatory nucleic acid sequences, responsible for controlling expression, i.e., transcription and translation of the coding region. A gene may also contain other untranslated 5' and 3' sequences and termination sequences. Other elements that may be present are, for example, introns.

[0081] “Gene of interest” refers to any gene that, when transferred to a plant, confers on the plant a desirable characteristic, such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process, or altered reproductive capacity.

[0082] “Genotype” as used in this document is the genetic material inherited from the parent maize plants. The genotype ME240913 refers to the transformed heterologous genetic material within a plant's genome as well as the genetic material flanking the inserted sequence.

[0083] A “heterologous” nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including the unnatural occurrence of multiple copies of a nucleic acid sequence.

[0084] A “homologous” nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0085] “High toxicity” of corn leaf tissue refers to the ability of leaf tissue samples to produce 100% mortality for lepidopteran species, such as S. frugiperda, within 7 days of exposure to the leaf tissue. Another part of the definition of high toxicity is the ability of dried leaf tissue, diluted 1:25 with conventional corn leaf tissue, to produce > 95% mortality and Petition 870240068507, dated 12 / 08 / 2024, page 38 / 96 23 / 58 morbidity within 14 days after dietary exposure of leaf tissue.

[0086] “Operationally linked” refers to the association of nucleic acid sequences in a single nucleic acid fragment such that the function of one affects the function of the other. For example, a promoter is operationally linked to a coding sequence or functional RNA when it is able to affect the expression of the coding sequence or functional RNA (i.e., when the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences in sense or antisense orientation can be operationally linked to regulatory sequences.

[0087] “Plant protection,” as used in this document, refers to the ability of an intact maize plant to resist leaf damage caused by susceptible lepidopteran pests, including but not limited to protection against leaf damage caused by S. frugiperda. Protection can be observed by examining plants or photographs of plants in order to compare the damage observed in control maize plants with that observed in maize plants expressing Cry1Da. Plant protection, as used in this document, also refers to the ability of an intact plant to resist damage caused by susceptible lepidopteran pests, including but not limited to the use of standard and accepted methods of plant damage grading.

[0088] “Primers,” as used in this document, are isolated nucleic acids that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a polymerase, such as DNA polymerase. Pairs or sets of primers can be used for amplification of a nucleic acid molecule, for example, by Petition 870240068507, dated 12 / 08 / 2024, p. 39 / 96 24 / 58 Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.

[0089] A “probe” is an isolated nucleic acid to which a conventional detectable marker or reporter molecule, such as a radioactive isotope, ligand, chemiluminescent agent, or enzyme, is attached. Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from the maize event ME240913. The DNA from the ME240913 event may be from a maize plant or from a sample that includes DNA from the ME240913 event. Probes according to the present invention include not only ribonucleic or deoxyribonucleic 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 that target DNA sequence.

[0090] Primers and probes are generally between 10 and 15 nucleotides or more in length. Primers and probes may also be at least 20 nucleotides or more in length, or at least 25 nucleotides or more, or at least 30 nucleotides or more in length. Such primers and probes specifically hybridize to a target sequence under high-stringency hybridization conditions. Primers and probes according to the present invention may have a complete sequence complementary to the target sequence, although probes that differ from the target sequence and retain the ability to hybridize to the target sequences may be designed by conventional methods.

[0091] “Stringence conditions” or “stringing hybridization conditions” include references to conditions under which a probe will hybridize to its target sequence to a greater detectable degree than to other sequences. Stringence conditions are dependent on the target sequence and will differ depending on the structure of the Petition 870240068507, dated 12 / 08 / 2024, p. 40 / 96 25 / 58 polynucleotide. By controlling the hybridization stringency and / or washing conditions, target sequences that will be 100% complementary to the probe (homologous probe) can be identified. Alternatively, stringency conditions can be adjusted to allow some mismatch in the sequences so that lower degrees of similarity are detected (heterologous probe). Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of Hybridization Principles and Nucleic Acid Probe Assay Strategy”, Elsevier: New York; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience: New York (1995), and also Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (5thEd. Cols Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0092] Specificity is typically a function of post-hybridization washes, with ionic strength and the temperature of the final wash solution being the critical factors. In general, high-stringency hybridization and washing conditions are selected because they are approximately 5°C below the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, under high-stringency conditions, a probe will hybridize to its target sequence but not to other sequences.

[0093] An example of high-stringency hybridization conditions for the hybridization of complementary nucleic acids having more than 100 complementary residues in a Southern Blot or Northern Blot filter is 50% formamide with 1 mg of Petition 870240068507, dated 12 / 08 / 2024, p. 41 / 96 26 / 58 heparin at 42°C, with hybridization performed overnight. An example of very high stringency washing conditions is 0.15M NaCl at 72°C for approximately 15 minutes. An example of high stringency washing conditions is a 0.2x SSC wash at 65°C for 15 minutes (see Sambrook, below, for a description of the SSC buffer).

[0094] A good example of hybridization conditions for the present invention includes hybridization in 7% SDS, 0.25 M NaPu4 pH 7.2 at 67°C overnight, followed by two washes in 5% SDS, 0.20 M NaPu4 pH 7.2 at 65°C for 30 minutes each wash, and two washes in 1% SDS, 0.20 M NaPu4 pH 7.2 at 65°C for 30 minutes each wash. A good example of a medium-stringence wash for a duplex of, for example, more than 100 nucleotides, is 1x SSC at 45°C for 15 minutes. A good example of a low-stringence wash for a duplex of, for example, more than 100 nucleotides, is 46x SSC at 40°C for 15 minutes.

[0095] For probes of approximately 10 to 50 nucleotides, high stringency conditions typically involve salt concentrations of less than approximately 1.0 M Na+ ions, typically concentrations of approximately 0.01 to 1.0 M Na+ ions (or other salts) at a pH of 7.0 to 8.3, and the temperature is typically at least approximately 30°C. High stringency conditions can also be achieved with the addition of destabilizing agents such as formamide. Generally, a signal-to-noise ratio of 2x (or greater) than that observed for an unrelated probe in the specific hybridization assay indicates the detection of a specific hybridization. Nucleic acids that do not hybridize with each other under high stringency conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created. Petition 870240068507, dated 12 / 08 / 2024, p. 42 / 96 27 / 58 using the maximum codon degeneracy allowed by the genetic code.

[0096] The following are some good examples of sets of hybridization / washing conditions that can be used to hybridize nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more preferably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4,1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. The sequences of the present invention can be detected using all the above conditions. For the purposes of defining the invention, high stringency conditions are used.

[0097] Throughout this document, words and expressions such as “promoter,” “promoter sequence,” and the like should be interpreted as a DNA sequence that, once operatively linked to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into RNA. A promoter is located 5' (or upstream) relative to the transcription start site of a nucleotide sequence of interest whose transcription into mRNA it controls and provides a site for the specific binding of RNA polymerase and other Petition 870240068507, dated 12 / 08 / 2024, p. 43 / 96 28 / 58 transcription factors for transcription initiation. May include other regulatory sequences known to a person skilled in the art. According to the present invention, the promoter may be heterologous or homologous to the respective cell or host. A nucleic acid sequence is “heterologous” to an organism or to a second nucleic acid sequence if it originates from a different species or, if from the same species, is modified from its original form.

[0098] As used in this document, the term unique for the ME240913 event means distinctive characteristics of the ME240913 event. Thus, nucleic acids unique to the ME240913 event are not found in other maize plants that are not ME240913.

[0099] As used in this patent application, the term “corn” refers to the species Zea mays and includes all varieties of plants that can be reproduced with corn, including wild species of corn.

[00100] “Detection Kit”, as used in this patent application, refers to a kit of parts useful in detecting the presence or absence of unique plant nucleic acids ME240913 in a sample, wherein the kit comprises nucleic acid probes and / or primers of the present invention, which form hybrids specifically under high stringency conditions with a target DNA sequence, and other materials necessary to enable nucleic acid amplification or hybridization methods.

[00101] Throughout this document, the term “transformation” and similar terms should be interpreted as a process for introducing heterologous DNA into a cell, plant tissue, or plant. It can occur under natural or artificial conditions, such as through the use of various well-known methods in the art, whether in a prokaryotic or eukaryotic host cell. The method is generally selected based on the cell. Petition 870240068507, dated 12 / 08 / 2024, p. 44 / 96 29 / 58 host that will be transformed and may include, but is not limited to, viral infection, electroporation, lipofection, particle bombardment (biolistics) and Agrobacterium-mediated.

[00102] Throughout this document, the term “transgene” should be interpreted as any nucleic acid sequence that is introduced into a cell through experimental manipulations, whether or not it is integrated into the genome. A transgene can be an “endogenous DNA sequence” or an “exogenous DNA sequence” (i.e., “heterologous”). The expression “endogenous DNA sequence” refers to a nucleotide sequence that is naturally found in the cell into which it is introduced. The expression “exogenous DNA sequence” refers to a nucleotide sequence that is not naturally found in the cell into which it is introduced. The term “transgenic,” when referring to a transformed organism, means an organism transformed with a recombinant DNA molecule that preferably comprises an operationally suitable promoter linked to a DNA sequence of interest.

[00103] Throughout this document, the term “vector” should be interpreted as a construct containing a DNA sequence that is operatively linked to one or more suitable control sequences capable of leading to the expression of said DNA sequence in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence for controlling such transcription, a coding sequence for the appropriate mRNA binding sites to the ribosome, and sequences that control the termination of transcription and translation, for example.

[00104] Several vectors may be suitable for carrying out the present invention. These vectors may be replicated autonomously in the host organism or replicated by the chromosome. The vector may also be a plasmid. According to the present Petition 870240068507, dated 12 / 08 / 2024, page 45 / 96 30 / 58 document, the terms “plasmid” and “vector” are sometimes used interchangeably. Preferably, the vector, according to the present invention, comprises the crylDa nucleic acid molecule comprising the nucleic acid sequence SEQ ID NO: 1, as defined in this document.

[00105] As used herein, the term transgenic event refers to a recombinant plant produced by the transformation and regeneration of a plant tissue or cell with heterologous DNA, for example, an expression cassette that includes a gene of interest. The term event refers to the original transformant and / or the offspring of the transformant that includes the heterologous DNA. The term event also refers to the offspring produced by a sexual cross between the transformant and another maize line. Furthermore, after repeated backcrossing with a parent, the introduced DNA and the flanking DNA of the transformed parents are present in the offspring of the cross at the same chromosomal location.The term event also refers to the original transformant DNA comprising the inserted DNA and the flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to offspring receiving the inserted DNA including the transgene of interest as a result of a sexual cross between a parent line that includes the inserted DNA (e.g., the original transformant and the offspring resulting from the cross) and a parent line that does not contain the inserted DNA. Typically, plant tissue transformation produces multiple events, each representing the insertion of a DNA construct within a different location in the genome of a plant cell. Based on transgene expression or other desirable characteristics, a particular event is selected. In this way, the terms event ME240913 and “event” can be used interchangeably. Petition 870240068507, dated 12 / 08 / 2024, page 46 / 96 31 / 58 interchangeable.

[00106] An insect-resistant ME240913 maize plant can be reproduced firstly by sexual crossing of a first parent maize plant consisting of a maize plant grown from a transgenic ME240913 maize plant, such as an ME240913 maize plant grown from seed deposited in the ATCC under accession number: PTA-126224, and the offspring thereof derived from transformation with the expression cassette of the means of implementation of the present invention that confer resistance to lepidopteran insect pests, with a second parent maize plant that may or may not exhibit resistance to lepidopteran insect pests, thus producing a plurality of first-generation plants; and then by selecting a first-generation plant that is resistant to lepidopteran insect pests; and self-pollination of the first-generation offspring plant, thus producing a plurality of second-generation offspring plants;and finally by selecting second-generation descendant plants resistant to lepidopteran insect pests. These steps may also include backcrossing the first-generation plant resistant to lepidopteran insect pests or the second-generation plant resistant to lepidopteran insect pests with the second parent maize plant or a third parent maize plant, thus producing a maize plant that is resistant to lepidopteran insect pests. Such methods can be used for the introgression of the ME240913 event into maize lines as well as for the pyramiding of the ME240913 event with other transgenic events.

[00107] Throughout this document, the terms “host cell”, “host organism” and similar terms should be interpreted as referring to the specific host organism or the Petition 870240068507, dated 12 / 08 / 2024, page 47 / 96 32 / 58 specific target cell, but also as being the descendants or potential descendants of these organisms or cells. Since, due to mutation or environmental effects, certain modifications may arise in successive generations, these descendants do not necessarily need to be identical to the parent cell. However, they are still included within the scope of protection of the present invention. According to the present invention, the host cells can be prokaryotic or eukaryotic. Preferably, the host cell according to the present invention is a plant host cell. Preferably, it comprises a nucleic acid sequence that is unique to the ME240913 event, which is selected from SEQ ID NO: 4, SEQ ID NO: 5 and complements thereof.

[00108] Throughout this document, words and expressions such as “transgenic plant cell”, “transgenic plant” and the like should be interpreted as cells or plants that possess and preferably express, through experimental manipulations, a transgene, as well as referring to the progeny of a transgenic plant and subsequent generations of plants, as above.

[00109] Throughout this document, the term "plant" and similar terms shall be interpreted as referring to the part or the whole of a plant organism. In this context, "part" means plant cells and tissues, organs and parts of plants in all their manifestations, such as seeds, leaves, anthers, fibers, ears, roots, root hairs, stems, embryos, calluses, cotyledons, petioles, collected material, plant tissue, reproductive tissue, and cell cultures. Transgenic plants, according to the present invention, can be generated and self-fertilized or crossed with other individuals in order to obtain additional transgenic plants. Transgenic plants can also be obtained by vegetative propagation of plant cells. Petition 870240068507, dated 12 / 08 / 2024, page 48 / 96 33 / 58 transgenic.

[00110] Throughout this document, words and expressions such as “pest”, “lepidopteran pest insects” and the like should be interpreted as insects of the order Lepidoptera, including, but not limited to, the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, Crambidae and Tineidae, more particularly noctuid Spodoptera sp., particularly S. frugiperda (Noctuidae) and cambid Diatraea sp., particularly D. saccharalis (Crambidae).

[00111] One embodiment of the present invention relates to a method for controlling lepidopteran insect pests of cultivated plants, including, but not limited to, caterpillars. Any method for controlling lepidopteran insect pests of cultivated plants is included within the scope of the present invention, and is not particularly relevant to obtaining the embodiments of the invention, provided that the cultivated plants, according to the present invention, comprise at least one nucleic acid sequence that is unique to event ME240913, which is selected from SEQ ID NO: 4, SEQ ID NO: 5, and complements thereof, wherein the method preferably comprises planting seeds obtained from a plant comprising at least one nucleic acid sequence that is unique to event ME240913, as defined herein, in a cultivated area of ​​plants susceptible to lepidopteran insect pests.

[00112] The “CrylDa” class of proteins also includes its homologs. “Homolog” means that the indicated protein or polypeptide maintains a defined relationship to other members of the Cry1Da class of proteins.

[00113] This invention relates to a genetically improved maize strain that produces a truncated Cry1Da protein, Petition 870240068507, dated 12 / 08 / 2024, page 49 / 96 34 / 58 modified for the control of lepidopteran insect pests. The invention is particularly designed for a transgenic maize event designated ME240913 comprising a novel genotype, as well as for compositions and methods for detecting nucleic acids unique to the ME240913 event in a biological sample. The invention is further designed for maize plants comprising the ME240913 genotype, for transgenic seeds of maize plants, and for methods for producing a maize plant comprising the ME240913 genotype by crossing a maize cross with itself comprising the ME240913 genotype or another maize line. Maize plants comprising the ME240913 genotype of the invention are useful in controlling lepidopteran insect pests including, but not limited to, the Noctuidae and / or Crambidae families, preferably S. frugiperda and D. saccharalis.Corn plants from event ME240913 show plant protection against susceptible lepidopteran pests, including, but not limited to, wild-type Cry1F and Cry1A resistant S. frugiperda.

[00114] In another embodiment, corn plants show high toxicity to susceptible lepidopteran pests, including but not limited to S. frugiperda.

[00115] In one embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence that is unique to the ME240913 event.

[00116] In another embodiment, the present invention relates to an isolated nucleic acid molecule that binds to a heterologous DNA molecule introduced into the genome of event ME240913 to the DNA genome in event ME240913 comprising at least 10 or more (for example 15, 20, 25, 30 or more) contiguous nucleotides of the heterologous DNA molecule and at least 10 or more (for example 15, 20, 25, 30 or more) contiguous nucleotides of the genome DNA. Petition 870240068507, dated 12 / 08 / 2024, p. 50 / 96 35 / 58 flanking the insertion site of the heterologous DNA molecule. Also included are nucleotide sequences comprising 10 or more nucleotides of the contiguous insertion sequence of the ME240913 event and at least one nucleotide of the flanking DNA of the ME240913 event adjacent to the insertion sequence. Such nucleotide sequences are unique and diagnose the ME240913 event. Hybridization or amplification of the nucleic acid of the genomic DNA of the ME240913 event produces an amplicon comprising such unique sequences that allows diagnosis of the ME240913 event. In one aspect of this embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 4, 5, and 8, and their complements.

[00117] In another embodiment, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence comprising at least one ME240913 event junction sequence, wherein a junction sequence transposes the junction between a heterologous expression cassette inserted within the maize genome and the maize genome DNA by pairing the insertion site which is unique to said ME240913 event and is diagnostic for the ME240913 event. In one aspect of this embodiment, the junction sequence is selected from the group consisting of SEQ ID NOs: 4 and 5, and their complements.

[00118] In another embodiment, the present invention relates to an isolated nucleic acid molecule that joins a heterologous DNA molecule to the maize plant genome at event ME240913, which comprises at least one sequence selected from the group consisting of SEQ ID NOS: 4, 5, and their complements.

[00119] In another embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence that is unique to the ME240913 event, wherein said nucleotide sequence encodes a protein comprising the Petition 870240068507, dated 12 / 08 / 2024, p. 51 / 96 36 / 58 amino acid sequence of SEQ ID NO: 3. In one aspect of this embodiment, the nucleotide sequence is SEQ ID NO: 8 and / or its complement.

[00120] In another embodiment, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of 4, 5 and 8, and the complements thereof. In one aspect of this embodiment, the isolated nucleic acid molecule is contained in a maize seed deposited in the “American Type Culture Collection” under accession number PTA-126224, or in plants grown from said seed.

[00121] In one embodiment of the present invention, an amplicon comprising a unique nucleotide sequence for the ME240913 event is provided. In one aspect of this embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 11 and 15, and their complements.

[00122] In another embodiment, the present invention encompasses flanking sequence primers for detecting the ME240913 event. Such flanking sequence primers comprise a nucleotide sequence of at least 10 contiguous nucleotides from the 5' or 3' flanking sequence. In one aspect of this embodiment, the contiguous nucleotides are selected from at least 10 contiguous nucleotides of SEQ ID NO: 6 (5' flanking sequence), or its complements. In another aspect of this embodiment, the 5' flanking sequence primer features the sequence of SEQ ID NO: 9 or its complement. In another aspect of this embodiment, the contiguous nucleotides are selected from at least 10 contiguous nucleotides of SEQ ID NO: 7 (the 3' flanking sequence), or its complements. Furthermore, in another aspect of this modality, the initiator of the flanking sequence 3' Petition 870240068507, dated 12 / 08 / 2024, p. 52 / 96 37 / 58 presents the SEQ ID NOs sequence: 12 or its complement.

[00123] In yet another embodiment, the present invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer that function together in the presence of a DNA template of the ME240913 event in a sample to produce a diagnostic amplicon for the ME240913 event. In one aspect of this embodiment, the first primer and / or the second primer is chosen from SEQ ID NO: 9, 10, or complements thereof. In another aspect of this embodiment, the first primer and / or the second primer is selected from the group consisting of SEQ ID NOs: 13, 14, and complements thereof. Still, in another aspect of this embodiment, the amplicon that is produced by the primer pair comprises SEQ ID NO: 11, 15, or complements thereof.

[00124] In another embodiment, the present invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer that function together in the presence of a DNA template of the ME240913 event in a sample to produce a diagnostic amplicon for the ME240913 event. Where the first primer is the same as or complementary to a maize plant genome sequence that pairs with the insertion point of a heterologous DNA sequence inserted into the genome of the ME240913 event, and the second polynucleotide primer sequence is the same as or complementary to the heterologous DNA sequence inserted into the genome of the ME240913 event.

[00125] In one aspect of this embodiment, the first polynucleotide primer comprises at least 10 contiguous nucleotides from positions 1-116 and 6307-6424 of SEQ ID NO: 8 and their complements. In another aspect of this embodiment, the first primer is Petition 870240068507, dated 12 / 08 / 2024, page 53 / 96 38 / 58 selected from the group consisting of SEQ ID NOs: 9, 13, and their complements. In another aspect of this embodiment, the second polynucleotide primer comprises at least 10 contiguous nucleotides from position 117-6306 of SEQ ID NO: 8, or its complements. Still, in another aspect of this embodiment, the second polynucleotide primer is selected from the group consisting of SEQ ID NOs: 10, 14, and their complements.

[00126] In another aspect of this embodiment, the first polynucleotide primer, which is presented in SEQ ID NO: 9, and the second polynucleotide primer, which is presented in SEQ ID NO: 10, work together in the presence of a DNA template of the ME240913 event in a sample to produce a diagnostic amplicon for the ME240913 event. In one embodiment of this aspect, the amplicon comprises the nucleotide sequence presented in SEQ ID NO: 11.

[00127] In yet another embodiment, the present invention relates to a method for detecting the presence of a nucleic acid molecule that is unique to the ME240913 event in a sample comprising corn nucleic acids, wherein the method comprises: (a) bringing the sample into contact with a pair of primers, (b) performing a nucleic acid amplification reaction so as to produce an amplicon, and (c) detecting the amplicon.

[00128] In another embodiment, the present invention relates to a method for detecting the presence of a nucleic acid molecule that is unique to the ME240913 event in a sample comprising corn nucleic acid, wherein the method comprises: (a) contacting the sample with a probe that hybridizes under high stringency conditions with genomic DNA of the ME240913 event and does not hybridize under high stringency conditions with DNA from a control corn plant, wherein the probe comprises at least Petition 870240068507, dated 12 / 08 / 2024, p. 54 / 96 39 / 58 minus 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and their complements; (b) subject the sample and probe to high-stringency hybridization conditions; and (c) detect hybridization of the probe to the nucleic acid molecule. Detection can be done by any means well known in the art including fluorescence, chemiluminescence, radiological, immunological and others. In the case where hybridization is used as a means for the amplification of a particular sequence to produce an amplicon that is diagnostic for the ME240913 event, the production and detection of the amplicon by any means well known in the art is indicative of hybridization with a target sequence where at least one probe or primer is used(a).

[00129] The term “biological sample” defines a sample derived from a maize plant that contains or is suspected of containing a nucleic acid comprising between five and ten nucleotides on either side of the point where one or the other of the two terminal points of the inserted heterologous DNA sequence is joined to the genomic DNA sequence within the chromosome into which the heterologous DNA sequence was inserted, herein also known as splicing sequences. Furthermore, the splicing sequence comprises as few as two nucleotides: those being the first nucleotide within the flanking or genomic DNA adjacent to the one covalently joined to the first nucleotide within the inserted heterologous DNA sequence. In one aspect of this embodiment, the probe comprises a nucleotide sequence comprising at least 10 contiguous nucleotides of SEQ ID NOS: 4, 5, and the complements thereof.

[00130] In yet another embodiment, the present invention relates to a kit for the detection of nucleic acids that are unique to the event. Petition 870240068507, dated 12 / 08 / 2024, p. 55 / 96 40 / 58 ME240913 in a biological sample. The kit comprises at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a method for nucleic acid detection. Amplification or hybridization with a target nucleic acid sequence in a sample followed by amplicon detection or hybridization with the target sequence diagnoses the presence of nucleic acid sequences unique to the ME240913 event in the sample. The kit further comprises other materials necessary to enable nucleic acid amplification or hybridization. In one aspect of this embodiment, a nucleic acid molecule contained in the kit comprises a nucleotide sequence selected from SEQ ID NO: 9, 10, 12, 13, 14, 16, and the complements thereof. In another aspect of this embodiment, the nucleic acid molecule is a primer selected from the group consisting of SEQ ID NOs: 9, 10, 13, 14, and their complements.Furthermore, in another aspect of this embodiment, the amplicon comprises SEQ ID NO: 11, 15, or complements thereof. A variety of detection methods can be used, including but not limited to TAQMAN, thermal amplification, ligase chain reaction, Southern blot, ELISA, and colorimetric and fluorescent detection methods. In particular, the present invention provides kits for detecting the presence of the target sequence, i.e., at least the SEQ ID NO: 4, 5 sequence, or a junction sequence in a sample containing genomic nucleic acid from ME240913. The kit comprises at least two polynucleotides capable of binding to the target site or substantially adjacent to the target site and at least one means for detecting the polynucleotide binding to the target site. The detection means may be by fluorescence, chemiluminescence, colorimetry, or isotopy and may be coupled with at least immunological methods to detect the binding. The kit may also... Petition 870240068507, dated 12 / 08 / 2024, p. 56 / 96 41 / 58 detect the presence of the target site in a sample, that is, at least the sequence SEQ ID NO: 4, 5, or a joining sequence of the ME240913 event, taking advantage of two or more polynucleotide sequences that together are capable of binding to adjacent nucleotide sequences or within approximately 100 base pairs of the target sequence and that can be extended between each other to form an amplicon that contains at least the target site.

[00131] In another embodiment, the present invention relates to a method for detecting the Cry1Da protein in a biological sample, the method comprising: (a) extracting protein from the tissue of the ME240913 event; (b) analyzing the extracted protein using an immunological method comprising antibodies specific for the Cry1Da protein produced by the ME240913 event; and (c) detecting the binding of said antibody to the Cry1Da protein.

[00132] In yet another embodiment, the present invention relates to a plant product derived from a corn plant of event ME240913, tissue, or seed, wherein the plant product comprises a nucleotide sequence that is equal to or complementary to the sequence that is unique to event ME240913, and wherein the sequence is detectable in the plant product using a nucleic acid amplification or hybridization method. In one aspect of this embodiment, the nucleotide sequence is equal to or complementary to at least one of SEQ ID NO: 4, 5 and complements thereof. In another aspect of this embodiment, the plant product is selected from the group consisting of corn flour, cornmeal, corn syrup, corn oil, cornstarch, and cereals manufactured wholly or in part that contain corn-based products.

[00133] In another embodiment, the present invention relates to an extract of a plant product derived from a corn plant ME240913, tissue or seed comprising a sequence of Petition 870240068507, dated 12 / 08 / 2024, p. 57 / 96 42 / 58 nucleotides that is equal to or complementary to a sequence that is unique to ME240913. In one aspect of this embodiment, the sequence is detectable in the extract using a nucleic acid amplification or hybridization method. In another aspect of this embodiment, the sequence is equal to or complementary to at least one of SEQ ID NO: 4 and 5. Still, in another aspect of this embodiment, the plant product is selected from the group consisting of corn flour, cornmeal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part that contain corn-based products.

[00134] Another embodiment of the present invention relates to a maize plant, or parts thereof, and seeds of a maize plant comprising the genotype of the transgenic event ME240913, wherein the genotype comprises at least one nucleotide sequence of SEQ ID NOS: 4, 5 or complements thereof. An example of a maize seed comprises the nucleic acid molecules of the invention that were deposited on 28 / 10 / 2019 and assigned the accession number PTA-126224. In one aspect of this embodiment, the maize plant is of the Hill maize lines. However, a person skilled in the art will recognize that the ME240913 genotype can be introduced into any plant variety that can be reproduced with maize, including wild maize species, and thus the list of reproduced lines of this embodiment should not be limited.

[00135] In another embodiment, the present invention relates to a maize plant comprising at least a first and a second DNA sequence linked to form a contiguous nucleotide sequence, wherein the first DNA sequence is within a joining sequence and comprises at least approximately 10 contiguous nucleotides selected from the group consisting of nucleotides 1-116 and 6307-6424 of SEQ ID NO: 8, and the Petition 870240068507, dated 12 / 08 / 2024, p. 58 / 96 43 / 58 complements thereof, wherein the second DNA sequence is within the inserted heterologous DNA sequence and comprises at least approximately 10 contiguous nucleotides selected from the group consisting of nucleotides 117-6306 of SEQ ID NO: 8, and complements thereof; and wherein the first and second DNA sequences are useful as probes or nucleotide primers to detect the presence of the ME240913 maize event nucleic acid sequences in a biological sample. In one aspect of this embodiment, the nucleotide primers are used in a DNA amplification method to amplify a target DNA sequence from standard DNA extracted from the maize plant, and the maize plant is identifiable among other maize plants by producing an amplicon corresponding to a DNA sequence comprising SEQ ID NO: 11, 15, and complements thereof.

[00136] In one embodiment, the present invention relates to a maize plant where the ME240913 genotype confers resistance to lepidopteran insect pests to the maize plant. In one aspect of this embodiment, the transgenic genotype conferring resistance to lepidopteran insect pests to the maize plant of the invention comprises a truncated or modified crylDa gene.

[00137] In another embodiment, the maize plant expresses adequate leaf concentrations of the truncated and modified cry1Da protein to confer high levels of leaf protection against damage by S. frugiperda. In another embodiment, the high level of leaf protection was found in several current S. frugiperda populations in Brazil, including populations known to have high frequencies of cry1F-resistant S. frugiperda. In another embodiment, insertion of the cry1Da gene from the ME20913 maize plant produces adequate expression of the truncated and modified Cry1Da protein in leaf tissue to produce high toxicity to Petition 870240068507, dated 12 / 08 / 2024, page 59 / 96 44 / 58 susceptible species of lepidopterans, including S. frugiperda. In yet another embodiment, the ME20913 maize plant is highly toxic to Cry1F-resistant S. frugiperda.

[00138] In yet another embodiment, the present invention provides a method for producing a maize plant resistant to lepidopteran insect pests comprising the steps of: sexually crossing a first parent maize plant with a second parent maize plant, wherein said first or second parent maize plant comprises the DNA event ME240913, so as to produce a plurality of first-generation offspring plants; selecting a first-generation offspring plant comprising the event ME240913. Preferably, the method further comprises self-pollinating the first-generation offspring plant so as to produce a plurality of second-generation offspring plants; and selecting from the second-generation offspring plants, a plant comprising the event ME240913.In one embodiment, the selection step can be based on evaluating resistance to lepidopteran insect pests, detecting the DNA of the ME240913 event according to the methods taught in the present invention, or herbicide treatment and selection of herbicide-resistant plants promoted by the PAT (bar) herbicide resistance gene contained in the ME240913 event. In a preferred embodiment, the method for producing a transgenic maize plant comprising the unique nucleic acids of the invention comprises the sexual crossing of a first parent maize plant containing the ME240913 event with a second non-transgenic parent maize plant to produce offspring plants, selection of a first-generation offspring plant that is resistant to lepidopteran insect pest infestation, repetition of the backcrossing cycle 4 times, and self-pollination of the parent plant containing the event. Petition 870240068507, dated 12 / 08 / 2024, pp. 60 / 96 45 / 58 ME240913 for obtaining homozygous plants.

[00139] In another embodiment, the present invention provides a method for producing hybrid maize seeds comprising the steps of: planting seeds of a first congenital maize line comprising the event ME240913 and seeds of a second congenital line having a different genotype; sexually crossing the two different congenital lines with each other; and harvesting the hybrid seed produced in this manner. In a preferred embodiment, the method comprises at least one of the steps of cultivating maize plants resulting from said plantings until flowering time and emasculating the flowers of the plants of one of the congenital maize lines. In one aspect of this embodiment, the first maize line reproduced provides the female offspring. In another aspect of this embodiment, the first maize line reproduced provides the male offspring.The present invention also relates to hybrid seeds produced by the incorporated method and to hybrid plants grown from seed.

[00140] The following examples are for the sole purpose of illustrating one or more preferred embodiments of the invention and are not to be construed as limiting the scope of the invention. EXAMPLES Example 1 - Gene construction

[00141] The gene construct containing the ubiquitin promoter, the nucleotide sequence of SEQ ID NO: 1 encoding the amino acid sequence of the Cry1Da insecticidal protein truncated from SEQ ID NO: 3, optimized for expression in maize, and the 3' region of the Agrobacterium nopalin synthase gene was synthesized at the DNA Cloning Service (http: / / www.dna-cloning.com / ) in the pUC vector flanked by the HindIII and EcoRI restriction sites. The construct was transferred from the pUC to the pTF101 vector (Paz et al., 2004) using restriction enzymes. Petition 870240068507, dated 12 / 08 / 2024, pp. 61 / 96 46 / 58 EcoRI and HindlII and T4 ligase, according to the manufacturer's instructions (LifeTech). The selection of the recombinant plasmid pTF101 UBI:: cry1Da::NOS was performed by transforming E. coli DH5a using spectinomycin, and cloning was confirmed by sequencing and cleavage with HindIII and BamHI enzymes. The commercial BigDye Terminator v3.1 kit (Applied Biosystems) was used for sequencing. Plasmid DNA from two bacterial colonies containing the gene construct was sequenced and compared with the sequence of interest, and they were found to be identical.

[00142] Once the cloning of the UBI::cry1Da::NOS gene into the pTF101 plasmid was confirmed, this gene construct was used to transform the Agrobacterium tumefaciens EHA101 strain using the electroporation methodology (BioRad / MicroPulser). The same transformation confirmation procedure described above was performed to verify the presence of the recombinant binary vector containing the crylDa gene in A. tumefaciens. Plasmid DNA was isolated from A. tumefaciens colonies and amplified with primers for detection of the bar gene.

[00143] Agrobacterium tumefaciens EHA 101 containing the gene constructs of interest (UBI::cry1Da::NOST and 35S::bar::35T) was used in the genetic transformation of maize. Example 2 - Genetic transformation of immature Hill maize embryos via Agrobacterium tumefaciens

[00144] The genotype used in this transformation protocol is maize HiII (Armstrong et al., 1991), according to the protocol by Frame et al. (2002), with minor modifications. Briefly, for the transformation of this genotype, immature embryos between 1.8 - 2.0 mm in length (10-12 days after pollination) were collected. Ears used for embryo collection were immersed in a 1:1 solution of commercial bleach (2.5% sodium hypochlorite) and distilled water with 1-2 drops of Tween 20 for 20 minutes. In Petition 870240068507, dated 12 / 08 / 2024, p. 62 / 96 47 / 58 then, they were rinsed with sterile distilled water for 5 minutes, twice.

[00145] Immature embryos were collected using a spatula from a superficial cut of the grains. For gene transfer to maize, Agrobacterium tumefaciens EHA101 was used. From a stock culture of A. tumefaciens containing the gene construct of interest, maintained in glycerol at -80 °C, a streak was made on YEP medium (5 g L-1 yeast extract; 10 g L-1 peptone; 5 g L-1 NaCl; 15 g L-1 bacto agar) containing the necessary antibiotics (spectinomycin 100 mg L-1 and 50 mg L-1 kanamycin) and the plate was incubated for 2 to 3 days at 28 °C (mother plate). For genetic transformation, a streak of Agrobacterium using a colony isolated from the mother plate was made on YEP medium containing the necessary antibiotics. The plate was incubated for 2 to 5 days at 19 °C. Then, Agrobacterium was resuspended in infection medium (4.0 gL-1 of N6 salts; 68.4 gL-1 of sucrose; 36.0 gL-1 of glucose; 0.7 gL-1 of proline; 1.5 mg.L-1 of 2,4-D; 1.0 mL.L-1N6 vitamins (1000X = 1.0 gL-1 of thiamine HCl; 0.5 gL-1 of pyridoxine HCl; 0.5 gL-1 of nicotinic acid); pH 5.2) supplemented with 100 μM of acetosyringone until an OD550 of 0.3-0.4 was achieved, and incubated on a shaker at ~150 rpm, 23 °C for 2 hours.

[00146] For infection of immature maize embryos, 50 to 100 embryos were collected in 1 mL of infection medium supplemented with acetosyringone. After collection, the embryos were rinsed twice, 1 mL of bacterial culture was added, and the suspension was incubated for five minutes at 23 °C. After infection, the embryos were transferred to the surface of co-culture medium (4.0 gL-1 of N6 salts; 1.5 mg.L-1 of 2,4-D; 30.0 gL-1 of sucrose; 0.7 gL-1 of proline; 1.0 mL.L-1 of N6 vitamins (1000X); 0.85 mg.L-1 of AgNOs; 100 μM of acetosyringone; 300 mg.L-1 of L-cysteine; 3.0 gL-1 of phytagel; pH 5.8). Petition 870240068507, dated 12 / 08 / 2024, pp. 63 / 96 48 / 58 with the scutellum facing upwards. The plates were incubated in the dark at 20 °C for 3 to 5 days. After co-culture, the embryos were transferred to resting medium (4.0 gL-1 of N6 salts; 1.5 mg.L-1 of 2,4-D; 30.0 gL-1 of sucrose; 0.5 gL-1 of MES; 0.7 gL-1 of proline; 1.0 mL.L-1 of N6 vitamins (1000X); 0.85 mg.L-1 of AgNOa; 100 mg.L-1 of Tioxin; 3.0 g.L-1 of phytagel; pH 5.8) at 28 °C (dark) for 7 to 15 days. Next, the embryos were transferred to the selection medium (4.0 gL-1 of N6 salts; 1.5 mg.L-1 of 2,4-D; 30.0 gL-1 of sucrose; 0.5 gL-1 of MES; 0.7 gL-1 of proline; 1.0 mL.L-1 of N6 vitamins (1000X); 0.85 mg.L-1 of AgNO3; 100 mg.L-1 of Tioxin; 1.5 and 3.0 mg / L of bialaphos; 3.0 gL-1 of phytagel; pH 5.8) (25 embryos / plate). Subcultures of these embryos in selective medium are performed every 15 days until vigorously growing calluses are selected.

[00147] Selected calluses were transferred to regeneration medium (4.62 gL-1 of MS salts; 60.0 gL-1 of sucrose; 100 mg.L-1 of myo-inositol; 1.0 mL.L-1 of MS vitamins (1000X); 1.5 mg / L of bialaphos; 4.0 gL-1 of phytagel; pH 5.8) and incubated at 26 ± 2 °C (dark) for 15 to 21 days. Calluses ready for germination, with a dry appearance and opaque white color, were transferred to germination medium (4.62 gL-1 of DM salts; 30.0 gL-1 of sucrose; 100 mg.L-1 of myo-inositol; 1.0 mL.L-1 of DM vitamins (1000X = 0.5 gL-1 of thiamine HCl; 0.5 gL-1 of pyridoxine HCl; 0.05 gL-1 of nicotinic acid); 3.0 gL-1 of phytagel; pH 5.8) (12 calluses per plate), 25 °C, 80-100 pE / m2 / sec light intensity, 16 hours photoperiod.

[00148] Seedlings with well-developed roots and leaf structures measuring approximately 5 cm in length (14 to 20 days) were transplanted into pots in a greenhouse containing a mixture of soil and organic matter (2 / 3 soil and 1 / 3 commercially produced organic matter (TDP 30 / 15)) undergoing an acclimatization phase. Petition 870240068507, dated 12 / 08 / 2024, pp. 64 / 96 49 / 58

[00149] After obtaining the Hill genotype with the ME240913 event, the event was introgressed from the HiII genotype into the tropical L3 lineage, using marker-assisted selection. Example 3 - Bioassays

[00150] In order to evaluate the susceptibility of transgenic maize expressing the truncated Cry1Da protein to S. frugiperda, bioassays were carried out in the laboratory.

[00151] Bioassays were conducted as follows: newly hatched caterpillars of the species S. frugiperda were used to infest leaves of cry1Da transgenic corn plants and the non-transgenic isoline (5 caterpillars per plant). The corn developmental stages used were V7 and V8, and the experiments were carried out in plastic containers and incubated in acclimatized growth chambers (28°C and 60% humidity, 12 hr light). Damage scores were assessed after 5 days. In each case, the experimental design consisted of: “experimental group” (transgenic corn event ME240913 containing the truncated cry1Da construct) and “control group” (non-transgenic corn).

[00152] The parameters evaluated were: injury score using the scale proposed by Carvalho, 1970 (0: plant with undamaged leaves; 1: plant with scraped leaves; 2: plant with perforated leaves; 3: plant with torn leaves; 4: plant with lesion in the whorl; and 5: plant with destroyed whorl); caterpillar survival (the number of surviving caterpillars in each pot was counted); and caterpillar biomass (using a precision balance with four decimal places). Example 4 - Bioassays for the control of S. frugiperda using the transgenic maize event ME240913

[00153] Trials with S. frugiperda: initially, the event ME240913 was tested for the control of this pest. Seeds from the event were Petition 870240068507, dated 12 / 08 / 2024, pp. 65 / 96 50 / 58 plants germinated in a greenhouse, and when the plants reached the V10-V12 leaf stage, the two newest leaves from each plant were used in bioassays with S. frugiperda. Three replicates were performed, with five caterpillars per replicate. Leaves from HiII and L3 corn were used as a negative control (caterpillars grow normally), and leaves from corn treated with Viptera® technology were used as a positive control (caterpillars cannot grow). In this first test, it was verified that the ME240913 event had a good capacity to control caterpillar development, reaching 100% mortality (Table 1). Table 1 - Evaluation of event ME240913 in relation to the control of S. frugiperda Event / Repetition Live caterpillars (After 05 days) Dead caterpillars Total weight of live caterpillars (mg) ME240913 (crylDa) / 1 0 5 0 ME240913 (crylDa) / 2 0 5 0 ME240913 (crylDa) / 3 0 5 0 Corn Viptera Control + / 1 0 5 0 Corn Viptera Control + / 2 0 5 0 Corn Viptera Control + / 3 0 5 0 Corn HiII Control - / 1 5 0 70.3 Corn HiII Control - / 2 4 1 77.3 Corn HiII Control - / 3 5 0 68.7 Corn L3 Control - / 1 4 1 39.5 Corn L3 Control - / 2 5 0 84.4 Corn L3 Control - / 3 5 0 61.6 Petition 870240068507, dated 12 / 08 / 2024, pp. 66 / 96 51 / 58

[00154] The bioassay with this event was repeated, using four replicates with 20 caterpillars per replicate, and the results confirmed that this event has the ability to control the development of S. frugiperda (Table 2). Figure 3 is representative of the feeding bioassays with S. frugiperda on non-transgenic and transgenic corn of the present invention. In this experiment, the treatment with the Viptera corn genotype was not used. Table 2 - Evaluation of transgenic maize events with respect to the control of S.[rugiperdaJb] assay 2) Event Live caterpillars Dead caterpillars Total weight of live caterpillars (mg) ME240913 (crylDa) / 1 0 20 0 ME240913 (crylDa) / 2 0 20 0 ME240913 (crylDa) / 3 0 20 0 ME240913 (crylDa) / 4 0 20 0 HiII / 1 18 02 176.7 HiII / 2 18 02 131.7 HiII / 3 15 05 137.0 HiII / 4 17 03 212.7 L3 / 1 19 01 131.9 L3 / 2 19 01 132.5 L3 / 3 19 01 133.3 L3 / 4 18 02 115.6 Example 5 - Exposure to fresh leaf tissue from two genetic backgrounds containing the ME240913 event.

[00155] In this experiment, hybrid 1 of the Helix lineage was used. Petition 870240068507, dated 12 / 08 / 2024, pp. 67 / 96 52 / 58 Hybrid 1 was HiII ME240913 X Embrapa L3 lineage (leaves V8 and V9). Hybrid 2 was HiII ME240913 X Embrapa L3 lineage (leaves V5 and V6), and the control was the hybrid of Helix L85 X Embrapa L3 lineages (leaves V8 and V9). Both hybrids were heterozygous for the ME240913 event.

[00156] Fresh leaf discs 1.8 cm in diameter were cut using a metal cutter and placed in 2.0% agar (1 mL / well) in 128-well plastic bioassay trays (BioBa-128, CD International, Pitman, NJ, USA).

[00157] For the tests, a susceptible laboratory population of fall armyworm, S. frugiperda, from Embrapa Maize and Sorghum was used as a standard, the same one used by Omoto et al 2016, as a susceptibility standard (Omoto, C., Bernardi, O., Salmeron, E., Sorgatto, RJ, Dourado, PM, Crivellari, A., Carvalho, RA, Willse, A., Martinelli, S., & Head, GP (2016). Field-evolved resistance to Cry1Ab maize by Spodoptera frugiperda in Brazil. Pest Management Science, 72(9), 1727-1736.). maintained on an artificial diet and without selective pressure from insecticides or Bt.

[00158] A newly hatched larva (0 to 24 hours) was placed in each well containing a leaf disc using a fine brush. The plates were sealed with Bio-CV-16 adhesives (CD International, Pitman, NJ, USA) and placed in a climate-controlled chamber (temperature 26 ± 1 °C; relative humidity of 60 ± 10%; photoperiod 14:10h light:dark). One hundred and twenty larvae were used for each treatment.

[00159] Mortality was assessed after 24 hours of exposure and subsequently daily until 100% mortality. Caterpillars that did not respond to brush touch were considered dead.

[00160] Fresh corn leaves expressing leaf tissue from event ME240913 produced mortality by S. frugiperda starting on day two after feeding and 100% mortality after three days. The same pattern of rapid and complete mortality was observed in both Petition 870240068507, dated 12 / 08 / 2024, pp. 68 / 96 53 / 58 hybrids were tested. The result is illustrated in Figure 4. Example 6 - Exposure to lyophilized leaf tissue diluted 1:25 of Event ME240913 in the artificial diet

[00161] This experiment evaluated the ME240913 event in the cross between the Helix L85 line and the Embrapa L3 line containing the ME240913 event (leaves collected at the V8 and V9 stages) and, as a control, the hybrid of the Helix L85 x Embrapa L3 lines (V8 and V9 leaves). Results were compared with those obtained using leaves from the control hybrid Helix L85 x Embrapa L3, also collected at the V8 and V9 stages.

[00162] Approximately seven plants of the two corn hybrids described above were harvested after 27 days of growth. Leaves between the V8 and V9 developmental stages were placed in plastic bags frozen with liquid nitrogen and transferred to an ultra-low temperature freezer at -80°C. The leaf tissues were freeze-dried using freeze-drying. After freeze-drying, the material was ground using a tissue grinder (IKA A11 Basic). Samples of the freeze-dried and ground leaves were stored in plastic cups with sealed lids at room temperature.

[00163] Freeze-dried transgenic corn leaves were prepared in a 1:25 ratio in the artificial diet of S. frugiperda containing 4% (w / w). The negative control contained 4% freeze-dried non-transgenic tissue. Approximately 1 L of the artificial diet of S. frugiperda was prepared using an adapted protocol containing only 56% of the total amount of agar compared to the regular protocol. The diet was cooled and maintained at 55 °C in a water bath as needed. For each treatment, 160 g of S. frugiperda diet were added to the plastic cup containing pre-weighed freeze-dried tissue. The leaf tissue was mixed with a spatula until visually uniform. The mixture was transferred to a thick plastic bag with a hole in Petition 870240068507, dated 12 / 08 / 2024, pp. 69 / 96 54 / 58 one of the ends and the diet was added to each of the wells in a bioassay tray (CD-International 128-well trays) by pressing the diet through the entire bag (like a piping bag). Approximately 0.8 ml of diet powder / leaf mixture was dispensed into each of the 128 individual wells for each transgenic and non-transgenic material (total of 256).

[00164] The standard susceptible S. frugiperda population described in the aforementioned assay was used.

[00165] A newly hatched larva (0 to 24 hours) was placed in each of the wells using a fine brush. The plates were sealed with Bio-CV-16 adhesives (CD International, Pitman, NJ, USA) and placed in a climate chamber (temperature 26 ± 1 °C; relative humidity 60 ± 10%; photoperiod 14:10 h light:dark).

[00166] Mortality was recorded on days 3, 6-10, and 13-14 after exposure. Visibly inactive larvae that did not move when touched with a fine brush were considered dead.

[00167] In this experiment, mortality of 67% was observed on day 7 and 97% on day 14 (125 / 128 larvae). The three remaining larvae showed significant growth inhibition compared to the larvae that fed on control leaf tissue (control).

[00168] Figure 5 illustrates the survival rate of newly hatched S. frugiperda caterpillars (%), evaluated up to 14 days after exposure to freeze-dried leaves in a 1:25 ratio with an artificial diet obtained from leaves of event ME240913 and the control. Example 7 - Protection against leaf damage in maize plants from Event ME240913 in fields infested by six different populations of S. frugiperda from different origins.

[00169] The control plants were obtained from the hybrid between the Helix L85 x Embrapa L3 lines. The plants containing the event were obtained from the hybrid between the Helix L85 x Embrapa L3 lines. Petition 870240068507, dated 12 / 08 / 2024, pp. 70 / 96 55 / 58 ME240913.

[00170] Naturally occurring populations of S. frugiperda were collected at the larval stage in six distinct maize-producing locations in Brazil, as described: two populations were collected in the state of Paraná (Palotina and Ivatuba), two in the state of Mato Grosso (Rondonópolis and Campo Verde), and two in the state of Minas Gerais (Paracatu and Sete Lagoas). The larvae were maintained under laboratory conditions with an artificial diet until the adult stage (cycle around ~30 days) without selective pressure from insecticides or Bt. The newly hatched larvae were then infested on plants at the V4 growth stage under field conditions.

[00171] The treatments consisted of a combination of two hybrids and six insect populations with three replicates. Plots of five rows, five meters long, were planted; infestation was carried out in the three central rows, which were used for evaluation. The two remaining rows were used as a buffer zone (border).

[00172] The assessment of damage caused by caterpillar feeding on corn plants was carried out according to Davis et al. 1992. In summary, a scale of 0 to 9 was used, where 0 represented plants with no damage and 9 represented plants with destroyed expanded leaves. Thus, an increase in the scale represented higher levels of leaf damage.

[00173] Field scores were recorded 7, 14, and 21 days after exposure.

[00174] The average visual damage to the leaves of the control treatment using the L85XL3 corn hybrid produced an infestation score for the six different populations of S. frugiperda ranging from 3.4 to 5.19 in the control treatment and close to zero in the hybrid expressing Cry1Da. That is, average scores of the plants evaluated in the hybrid Petition 870240068507, dated 12 / 08 / 2024, pp. 71 / 96 56 / 58 containing the event with CrylDa were considerably smaller (<0.1).

[00175] Figure 7 illustrates the results of damage scores (Davis et al. scale, 1992) from 0 to 9, caused by feeding on different populations of S. frugiperda (± Confidence Interval, at 5% probability), in conventional hybrid (conv) that does not express the ME240913 event and in corn hybrids (ME240913) in the field. Evaluated 14 days after infestation.

[00176] Figures 8A to 8F show the conventional control hybrid obtained from the cross between the Helix-L85 x Embrapa-L3 lines (left in the photos) and its respective isogenic version containing the event, Helix-L85 x Embrapa-L3 with event ME240913 (right in the photos). Each photo shows the conventional version and the version containing the ME240913 event, individually infested by each of six different populations of S. frugiperda collected in Brazil, in the following locations: population collected in Palotina-PR (8A), Rondonópolis-MT (8B), Rondonópolis+Campo Verde-MT (8C), Paracatu-MG (8D), Sete Lagoas-MG (8E) and Ivatuba-PR (8F). Leaf damage is visible on the controls on the left in each photo, while the hybrid containing the ME240913 event shows no damage (hybrid on the right in each photo). Example 8 - Bioassays using caterpillar populations resistant to transgenic corn containing the CrylF gene.

[00177] Tests were performed to verify the potential of the ME240913 event in controlling populations of S. frugiperda resistant to the Cry1F protein.

[00178] The experiment was conducted in a greenhouse, planting transgenic corn containing the ME240913 event and non-transgenic corn (negative control). Newly hatched caterpillars belonging to two distinct populations of S. frugiperda (one Petition 870240068507, dated 12 / 08 / 2024, pp. 72 / 96 Two populations (57 / 58 resistant to the CrylF gene and another population sensitive to the same gene) were inoculated into corn plants (15 caterpillars per plant) at stages V7 and V8. After infestation, the pots were isolated with voile cages, and injury scores were assessed after 7, 14, and 21 days. The experimental design consisted of 4 treatments, with 5 pots each, containing 2 to 3 corn plants per pot.

[00179] Treatment 1: Transgenic event ME240913 infested with the S. frugiperda population resistant to the Cry1 F protein according to Leite et al. 2016.

[00180] Treatment 2: Isogenic L3 non-transgenic strain infested with the Cry1F protein-resistant S. frugiperda population according to Leite et al 2016.

[00181] Treatment 3: Transgenic event ME240913 infested with susceptible caterpillar population originating from maintenance rearing at the entomology laboratory of Embrapa Maize and Sorghum.

[00182] Treatment 4: Isogenic non-transgenic line infested with susceptible caterpillar population originating from maintenance rearing at the entomology laboratory of Embrapa Maize and Sorghum.

[00183] The results indicated that the transgenic plant with the ME240913 event was able to control infestation with the Cry1 F protein-resistant S. frugiperda population as well as with the susceptible population, inhibiting its development (Figure 9) and protecting the plant from attack by this pest, as observed by the injury score (±CI, P=0.05). The survival percentage of S. frugiperda, evaluated 21 days after caterpillar release in different treatments, was 0% for treatments 1 and 3, and approximately 65% ​​and 35% for treatments 2 and 4, respectively. The biomass of S. frugiperda, evaluated 21 days after caterpillar release in different Petition 870240068507, dated 12 / 08 / 2024, pp. 73 / 96 58 / 58 treatments, was 0% for treatments 1 and 3, and approximately 260 mg and 300 mg for treatments 2 and 4, respectively. In both cases, the means are not overlapped by the CI, differing from each other (P=0.05).

[00184] This example 8 describes the use of an optimized Cry1Da codon sequence to produce maize plants expressing a truncated Cry1Da sequence with a high level of toxicity (100% mortality) for both wild populations and Cry1F-resistant S. frugiperda populations. The fact that 100% mortality was identified in Cry1F-resistant S. frugiperda populations when fresh leaves from the ME240913 event were used to feed these S. frugiperda populations confirms that the truncated protein and modified codon expressed from the Cry1Da gene used acts through a different mechanism than that existing in the commercial event containing the Cry1F gene. DEPOSIT

[00185] Corn seeds from the event ME240913 disclosed above were deposited on 10 / 28 / 2019 in accordance with the Budapest Treaty at the American Type Culture Collection (ATCC), 1801 University Boulevard, Manassas, VA 20110, under accession number PTA-126224.

Claims

1. Nucleic acid molecule, characterized in that it comprises a nucleotide sequence that is unique to event ME240913 of SEQ ID NO: 8, wherein maize seeds containing a nucleic acid molecule of event ME240913 were deposited in the American Type Culture Collection (ATCC) under accession number PTA126224.

2. Nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence encodes a truncated Cry1Da protein comprising the amino acid sequence of SEQ ID NO:

3.

3. Nucleic acid molecule according to claim 1 or 2, characterized in that the nucleic acid molecule is comprised in a maize seed deposited in the American Type Culture Collection (ATCC) under accession number PTA-126224.

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