Herbicide tolerance in a controlled environment assessment protocol

Abstract

The embodiments provide for methods for controlled environment screening of herbicides, including but not limited to, HPPD inhibiting herbicide tolerance in plants. Further embodiments provide a rating system for grading said tolerance. Yet other embodiments provide for controlled environment screening of pesticides, fungicides, insecticides, and / or nematicides. Further embodiments provide for methods for correlating the tolerance to said herbicides, to those results obtained in the field to screen and eliminate susceptible phenotypes to herbicides, in the controlled environment prior to future planting. Further embodiments provide for a recommended post-emergent treatment of herbicide based on the controlled environment screening results. Further embodiments provide for methods of using the plants and their progeny derived from the methods in a breeding program. Yet further embodiments provide for a novel marker associated with increased tolerance to said herbicides.

Description

231490W001TITLEHERBICIDE TOLERANCE IN A CONTROLLED ENVIRONMENT ASSESSMENT PROTOCOLCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 727,106 filed on December 2, 2024, the entire contents of which are hereby incorporated by reference.SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format and hereby incorporated by reference into the specification in its entirety. The name of the “xml” file containing the Sequence Listing is 231490WO01_SEQLISTING_St26.xml. The size of the xml file is 3 KB, and the xml file was created on November 26, 2025.BACKGROUND

[0003] Herbicide tolerance in plants and crops is very complex and can be affected by many interacting factors. Susceptible plants and crops to herbicides have a great impact on crop productivity.SUMMARY

[0004] Various embodiments provide new methods of correlating an assay for predicting herbicide tolerance of plants in a controlled environment, such as a greenhouse or a growth chamber, to herbicide tolerance of plants in a field.

[0005] One embodiment provides for a method for screening plants in a controlled environment to determine tolerance to an herbicide, wherein said method comprises: sowing one or more plant seeds in one or more containers filled with growth media; placing the one or more containers in a controlled environment under suitable growth conditions; applying an herbicide to the one or more containers; applying water to the one or more containers so that the one or more plant seeds germinate and grow into plants; rating the resulting one or more plants for herbicide tolerance after the one or more plants’ emergence; and predicting the future performance in the field of the one or more resulting plants or their progeny. The steps in the screening method are not limited to the order recited. For example, the HPPD inhibiting herbicide can be applied after germination.Another example is that the sowing of the more or more plant seeds can take place after placing the one or more containers in a controlled environment. Another embodiment provides for the screening method, wherein a plant part can be used instead of seed. Such plant parts can include, for example, vegetative cuttings, whether rooted or unrooted.231490W001

[0006] One embodiment provides for a method for screening non-transgenic plants in a controlled environment to determine tolerance to an herbicide, wherein said method comprises: sowing one or more non-transgenic plant seeds in one or more containers filled with growth media; placing the one or more containers in a controlled environment under suitable growth conditions; applying an herbicide to the one or more containers; applying water to the one or more containers so that the one or more plant seeds germinate and grow into plants; rating the resulting one or more plants for herbicide tolerance after the one or more plants’ emergence; and predicting the future performance in the field of the one or more resulting plants or their progeny. The steps in the screening method are not limited to the order recited. For example, the HPPD inhibiting herbicide can be applied after germination. Another example is that the sowing of the more or more plant seeds can take place after placing the one or more containers in a controlled environment. Another embodiment provides for the screening method, wherein a plant part can be used instead of seed. Such plant parts can include, for example, vegetative cuttings, whether rooted or unrooted.

[0007] Another embodiment includes a method for developing a plant, comprising applying plant breeding techniques to the resulting plants or progeny of the claimed screening method, or a plant part thereof, comprising crossing, recurrent selection, mutation breeding, wherein said mutation breeding selects for a mutation that is spontaneous or artificially induced, backcrossing, genomic selection, pedigree breeding, marker enhanced selection, haploid / double haploid production, or transformation, wherein application of said techniques results in development of a new plant. In another embodiment, the plant includes but is not limited to soybean, com, cotton, and canola.

[0008] Another embodiment provides for a method of producing a new plant, wherein said method comprises crossing the resultant plants or progeny thereof of the claimed method with a different plant containing a transgene for tolerance to an HPPD inhibiting herbicide, thereby producing a plant having enhanced tolerance to an HPPD inhibiting herbicide. A further embodiment includes that the plant produced by said crossing is a soybean, corn, cotton or canola plant.

[0009] Another embodiment provides for the method further comprising regulating the controlled environment temperature to approximately 20°C to 23 °C.

[0010] Another embodiment provides for the method further comprising adding a supplemental light source to promote plant growth.

[0011] Another embodiment provides for the method, wherein the growth media is comprised of any one individually or in combination of the following: soil, soilless mixes such as peat moss, coco coir, perlite, vermiculite, pine bark mixes (often with peat, sand, or perlite), sand, and231490W001 specialty mixes that incorporate ingredients like compost, composted yard waste, rice hulls, or specific soil pH needs or a synthetic cultivation medium.

[0012] In another embodiment, the herbicide is an HPPD (4-hydroxyphenyl-pyruvate dioxygenase) inhibiting herbicide.

[0013] In yet another embodiment, the HPPD inhibiting herbicide is a triketone. The triketone comprises mesotrione, sulcatrione, or tembotrione.

[0014] In yet another embodiment, the HPPD inhibiting herbicide is a pyrazole. The pyrazole includes but is not limited to topramezone.

[0015] In another embodiment, the HPPD inhibiting herbicide is an isoxazole. The isoxazole includes but is not limited to isoxaflutole.

[0016] Another embodiment provides for the method, wherein the rate of HPPD inhibiting herbicide application is at least two times greater than the recommended application rate. The herbicide can be applied pre or post emergence.

[0017] Another embodiment provides for the method, wherein the rate of HPPD inhibiting herbicide application is at least three times greater than the recommended application rate. The herbicide can be applied pre or post emergence.

[0018] Another embodiment provides for the method, wherein the rate of HPPD inhibiting herbicide application is at least four times greater than the recommended application rate. The herbicide can be applied pre or post emergence.

[0019] The method of claim 1, wherein the controlled environment screening for herbicide tolerance correlates with the herbicide tolerance in the field in a statistically significant way.

[0020] The method of claim 1, wherein the controlled environment screening to determine field tolerance to an herbicide correlates by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more with the herbicide tolerance in the field.

[0021] Another embodiment provides for the method, wherein the controlled environment screening for herbicide tolerance correlates by at least 50% with the herbicide tolerance in the field.

[0022] Another embodiment provides for the method, wherein said method provides for further post-application herbicide treatments.

[0023] Another embodiment provides for a method of producing a soybean plant having tolerance to an HPPD inhibiting herbicide as compared to a control plant, wherein the method comprises: isolating nucleic acids from one or more soybean plants; detecting in the nucleic acid, the presence of a genetic marker that is associated with tolerance to an HPPD inhibiting herbicide, wherein said genetic marker comprises SEQ ID NO: 1; selecting a first soybean plant based on the presence of231490W001 the marker associated with HPPD inhibiting herbicide tolerance; crossing a second soybean plant with said first soybean plant, wherein the second soybean plant does not comprise in its genome the marker associated with tolerance to an HPPD inhibiting herbicide; producing seed from said crossing; and selecting a soybean plant grown from said seed. The method further provides for crossing the progeny from said method with a transgenic soybean plant having tolerance to an HPPD inhibiting herbicide and to produce progeny with an enhanced tolerance to an HPPD inhibiting herbicide. A further embodiment provides for a soybean plant produced by the method having an increased tolerance to HPPD herbicide when compared to a soybean plant that has no tolerance to HPPD herbicide, wherein said increased tolerance is linked to a QTL comprising SEQ ID NO: 1.

[0024] Another embodiment provides for a method of determining the genotype of a soybean plant, wherein said method comprises obtaining a sample of nucleic acids from a soybean plant and detecting in the nucleic acids, a polymorphism, wherein said polymorphism is identified in SEQ ID NO:1.

[0025] Another embodiment provides for a marker for detecting tolerance to an HPPD inhibiting herbicide, wherein said marker comprises a single nucleotide polymorphism relative to a reference soybean genome for Glycine max at nucleotide position 17,065,990 on chromosome 14, wherein the reference genome is the Glycine max Williams82a2.75 reference genome.

[0026] Other aspects and embodiments will become apparent to those skilled in the art from a study of the following description and non-limiting examples.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0027] SEQ ID NO: 1 discloses the nucleotide sequence of marker Chrl4_17065990.BRIEF DESCRIPTION OF THE FIGURES

[0028] FIGs. la-le show the ranges of plant phenotypes when sprayed with an HPPD inhibiting herbicide. Ratings and phenotypes are collectively shown in Table 1.

[0029] FIG. la depicts a photograph of conventional soybean plants in a pot in the greenhouse that was sprayed with an HPPD inhibiting herbicide and having albino leaves, where the plant is dying and unlikely to recover.

[0030] FIG. lb depicts a photograph of soybean plants in a pot in the greenhouse that was sprayed with an HPPD inhibiting herbicide and having severe leaf symptoms, but the plant is more likely to survive.

[0031] FIG. 1c depicts a photograph of soybean plants in a pot in the greenhouse that was sprayed with an HPPD imhibiting herbicide and has moderate signs or symptoms of leaf damage.231490W001

[0032] FIG. Id depicts a photograph of soybean plants in a pot in the greenhouse that was sprayed with an HPPD inhibiting herbicide and has light leaf signs and symptoms of leaf damage.

[0033] FIG. le depicts shows a photograph of soybean plants in a pot in the greenhouse that was sprayed with an HPPD inhibiting herbicide and has no symptoms of leaf damage.

[0034] FIG. 2 is a graph showing the correlation of the assay among conventional soybean plants tested in the field (x axis) and the greenhouse (y axis). The field data is the mean from 2 years (2022 and 2023) and 2 locations with 1 rep per location. The GH data is from one experiment with 3 replications (2023) using the assay of the embodiments.

[0035] FIG. 3 is a graph showing the correlation of the assay among conventional soybean plants tested in the field (x axis) and the greenhouse (y axis). The field data is the mean of 3 locations with 2 replications per location (in 2024). The greenhouse data is from one expermient with 3 replications (2024) using the assay of the embodiments.

[0036] FIG. 4a depicts a Manhattan Plot generated using GWAS and shows a QTL for HPPD inhibiting tolerance on Chromosome 14 from the greenhouse data from FIG. 3.

[0037] FIG. 4b depicts the distributions (histogram) of HPPD injury ratings for lines in the greenouse assay from 2024 in FIG. 3 used in the GWAS.

[0038] FIG. 4c depicts a bar chart of the phenotypic distribution of the peak marker among the alternate alleles among the greenhouse assay results. The middle bar represents the lines that are heterozygous for the QTL region on Chromosome 14.DETAILED DESCRIPTION

[0039] Before explaining the various embodiments of the disclosure, it is to be understood that the embodiments are not limited in their application to the details of construction and the arrangement of the components set forth in the following description. Other embodiments can be practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0040] Throughout this disclosure, various publications, patents and published patent specifications are referenced. Where permissible, the disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in their entirety into the present disclosure to more fully describe the state of the art. Unless otherwise indicated, the disclosure encompasses conventional techniques of plant breeding, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols in Molecular Biology [(F. M. Ausubel, et al. eds., (1987)]; Plant Breeding: Principles and231490W001Prospects (Plant Breeding, Vol 1) M. D. Hayward, N, O. Bosemark, I. Romagosa; Chapman & Hall, (1993.); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) CURRENT Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Flames and G. R. Taylor eds. (1995)], Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture [R. I. Freshney, ed. (1987)]. Genome-wide association study (GWAS) has emerged in the past decade as a viable tool for identifying beneficial alleles from a genomic diversity panel (Priyanatha, C. et al., Genome-Wide Association Study of Soybean Germplasm Derived From Canadian x Chinese Crosses to Mine for Novel Alleles to Improve Seed Yield and Seed Quality Traits. Front. Plant Sci., (27 March 2022) Sec. Plant Breeding (13).

[0041] Unless otherwise noted, technical terms are used according to conventional usage in the art. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience, 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; Ausubel et al. (1987) Current Protocols in Molecular Biology, Green Publishing; Sambrook and Russell. (2001) Molecular Cloning: A Laboratory Manual 3rd. edition.

[0042] In order to facilitate understanding of the disclosure, the following definitions are provided:

[0043] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as lack of combinations when interpreted in the alternative (or).

[0044] “Applying” refers to the application of a substance or composition to a seed, plant, soil, or growth media. The method can include a step of contacting the seed, plant, soil, or growth media with the substance or composition to coat (completely or incompletely) the surface of the seed, plant, soil, or growth media or to adhere to the seed, plant, soil, or growth media. The substance or composition may be applied using methods including but not limited to mixing in a container, mechanical application, tumbling, spraying, misting, and immersion. Thus, the composition may be applied as a powder, a crystalline, a ready-to-use, a slurry, a mist, and / or a soak. For a general discussion of techniques used to apply fungicides to seeds, see “Seed Treatment,” 2d ed., (1986), edited by K. A Jeffs (chapter 9). The composition to be used as a seed treatment can comprise one or more of an herbicide, pesticide, a fungicide, an insecticide, a nematicide, an antimicrobial, an inoculant, a growth promoter, a polymer, a flow agent, a coating, or any combination thereof.231490W001

[0045] A “gene” is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

[0046] As used herein, the term “genotype” refers to the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable and / or detectable and / or manifested trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual’s genetic constitution at a single locus, at multiple loci, or more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome. Genotypes can be indirectly characterized, e.g., using markers and / or directly characterized by nucleic acid sequencing.

[0047] Nucleic acids are polynucleotides, long chainlike molecules composed of a series of nucleotides. An “isolated” nucleic acid molecule or an isolated protein or toxin is a nucleic acid molecule or protein or toxin that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or protein or toxin may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell or a transgenic plant.

[0048] As used herein, the terms “phenotype,” “phenotypic trait” or “trait” refer to one or more traits of an organism. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait.” In other cases, a phenotype is the result of several genes.

[0049] A “plant” is any plant at any stage of development, particularly a seed plant.

[0050] A “plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

[0051] “Plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0052] A “plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.231490W001

[0053] A “plant part” may be any part of a plant and include a plant cell, plant material, plant organ or plant tissue.

[0054] “Plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0055] “Quantitative Trait Loci (QTL)” as used herein refers to genetic loci that control to some degree, numerically representable traits that are usually continuously distributed.

[0056] “ Tolerance” is used to describe plants showing some symptoms to for example, a specified herbicide, fungicide, nematicide, biotic pest, pathogen, abiotic influence, or environmental condition, but are still able to produce marketable product with an acceptable yield.Some plants that are referred to as tolerant are only so in the sense that they may still produce a crop, even though the plants are symptomatic, and the yield may be reduced or minimized.

[0057] The materials and methods of the one or more embodiments are useful for determining susceptibility, and / or tolerance of plants, including non-transgenic plants, to HPPD inhibiting herbicides in a controlled environment as a predictor for field performance when treated with an HPPD inhibiting herbicides. Various embodiments of the method are useful, including a preemergence application of herbicide in a controlled environment to predict field performance with pre-emergence application in the field; a post-emergence application of herbicide in a controlled environment to predict field performance with pre-emergence application in the field; a preemergence application of herbicide in a controlled environment to predict field performance with post-emergence application in the field; and a post-emergence application of herbicide in a controlled environment to predict field performance with post-emergence application in the field.

[0058] One embodiment provides for a method for screening plants in a controlled environment to determine tolerance to an herbicide, wherein said method comprises: sowing one or more plant seeds in one or more containers filled with growth media; placing the one or more containers in a controlled environment under suitable growth conditions; applying an herbicide to the one or more containers; applying water to the one or more containers so that the one or more plant seeds germinate and grow into plants; rating the resulting one or more plants for herbicide tolerance after the one or more plants’ emergence; and predicting the future performance in the field of the one or more resulting plants or their progeny. The “controlled environment” is a created environment231490W001 where the parameters affecting the growth cycle of a plant can be regulated. Such parameters include but are not limited to, growth media, photoperiod, light intensity, watering, pests, temperature, etc., as for example, compared to plants growing in an open field. The controlled environment can be stationary or moveable. In other embodiments, the controlled environment is a growth chamber or a greenhouse.

[0059] A further embodiment provides for where the plant seeds are row crop seeds, including but not limited to, soybean, wheat, cotton, canola, com, sorghum or vegetable and fruit seeds including but not limited to, tomatoes, cucumbers, peppers, carrots, melons, watermelon, squash, lettuce and beet.

[0060] In certain embodiments, water is applied to the one or more containers in a controlled manner.

[0061] In certain embodiments, water is applied to the one or more containers daily.

[0062] In certain embodiments, the plants are rated within one week of the plants’ emergence from the soil. In certain embodiments, the plants are rated within two weeks of the plants’ emergence from the soil. In certain embodiments, the plants are rated within three weeks of the plants’ emergence from the soil. In certain embodiments, the plants are rated within four weeks of the plants’ emergence from the soil. In certain embodiments, the plants are rated within five or six weeks of the plants’ emergence from the soil. Thus, the method provides for rating the resulting plants and after germination. The plants can be rated at one week, two weeks, three weeks, four weeks, five weeks, and at least six weeks after sowing, whether with a pre or post germination herbicide treatment.

[0063] In other embodiments, the growth medium may be comprised in a container, such as a pot, which may be comprised of any type of plastic, paper, clay or natural material.

[0064] In another embodiment, the application of water can include water itself, or water and other additions, such as plant growth supplements (i.e., fertilizers).

[0065] Another embodiment provides for the method further comprising regulating the controlled environment temperature to approximately 20°C to 23°C (Celsius).

[0066] Another embodiment provides for the method further comprising adding a supplemental light source to the controlled environment to promote plant growth. The supplemental light source can consist of LED (light emitting diode) lights.

[0067] Another embodiment provides for the method, wherein the herbicide is an HPPD inhibiting herbicide. However, in addition to herbicide screening, another embodiment also provides for the controlled environment screening of pesticides, fungicides, insecticides, and / or nematicides.231490W001

[0068] Another embodiment provides for the method, wherein the rate of herbicide application is at least two times greater, three times greater, or four times greater than the recommended application rate. Another embodiment provides for wherein the herbicide is an HPPD inhibiting herbicide. The herbicide can be applied either pre or post germination.

[0069] Another embodiment provides for the method, wherein the rate of herbicide application is at least five, six, seven, eight, nine, or ten times greater than the recommended application rate. Another embodiment provides for wherein the herbicide is an HPPD inhibiting herbicide. The herbicide can be applied either pre or post germination.

[0070] Another embodiment provides for the method, wherein the controlled environment assay for herbicide tolerance correlates with the herbicide tolerance in the field in a statistically significant way.

[0071] Another embodiment provides for the method, wherein the controlled environment assay for herbicide tolerance correlates by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more with the herbicide tolerance in the field.

[0072] Another embodiment provides for the method, wherein the controlled environment assay for herbicide tolerance correlates by at least 50% with the herbicide tolerance in the field.

[0073] Another embodiment provides for the method, wherein said method provides for further post-application herbicide treatments. Such post-application treatment can be applied on the growth media or any part of the plant.

[0074] Another embodiment provides for a method for screening non-transgenic plants in a controlled environment to determine tolerance to an herbicide, wherein said method comprises: sowing one or more non-transgenic plant seeds in one or more containers filled with growth media; placing the one or more containers in a controlled environment under suitable growth conditions; applying an herbicide to the one or more containers; applying water to the one or more containers so that the one or more plant seeds germinate and grow into plants; rating the resulting one or more plants for herbicide tolerance after the one or more plants’ emergence; and predicting the future performance in the field of the one or more resulting plants or their progeny.

[0075] Transgenes and transformation methods facilitate engineering of the genome of plants to contain and express heterologous genetic elements, such as foreign genetic elements, or additional copies of endogenous elements, or modified versions of native or endogenous genetic elements in order to alter at least one trait of a plant in a specific manner. Any sequences, such as DNA, whether from a different species or from the same species, which have been stably inserted into a231490W001 genome using transformation are referred to herein collectively as “transgenes” and / or “transgenic events.”

[0076] Another embodiment provides for a method of improving plant yield by utilizing the method of the embodiments such that the most tolerant and resistant lines are chosen via the controlled environment screening for planting in the field. In some embodiments, the most tolerant and / or resistant lines are selected via the controlled environment screening method during the breeding process, thus increasing the efficiency of the selection of plants to move to the next stage of breeding. In some embodiments, the most tolerant and / or resistant lines are selected via the controlled environment screening method during the breeding process and then crossed with a plant containing a transgene for HPPD inhibiting herbicide tolerance to increase the overall HPPD inhibiting herbicide tolerance. In some embodiments, the transgene may be a 4-dihydroxyphenyl- pyruvate dioxygenase variant or a triketone deoxygenase, pyrazole or isoxaxole variant.

[0077] Another embodiment provides for the using a method of the embodiments to produce a new plant, wherein said method comprises crossing the resultant plants or progeny thereof of using a method of the embodiments with a different plant containing a transgene for tolerance to an HPPD inhibiting herbicide, thereby producing a plant having enhanced tolerance to an HPPD inhibiting herbicide.

[0078] Plants and their progeny selected for under the embodiments disclosed herein, which include row crop plants such as soybean, cotton, com, wheat and sorghum, can provide a source of breeding material that may be used to develop new plant varieties. Plant breeding techniques known in the art and used in a plant breeding programs include, but are not limited to, recurrent selection, mass selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, making double haploids, and transformation. Often combinations of these techniques are used. One embodiment is directed to methods for producing a plant by crossing a first parent plant with a second parent plant, wherein the first or second plant is the plant or progeny produced from the embodiments of the methods disclosed herein. Therefore, any breeding methods may include, selfing, backcrosses, hybrid breeding, and crosses to populations. These methods are well known in the art and some of the more commonly used breeding methods are described herein. Descriptions of breeding methods can be found in one of several reference books (e.g., Allard, Principles of Plant Breeding (1960); Simmonds, Principles of Crop Improvement (1979); Sneep, et al. (1979); Fehr, “Breeding Methods for Cultivar Development,” Chapter 7, Soybean Improvement, Production and Uses, 2. sup. nd ed., Wilcox editor (1987)).231490W001

[0079] Another embodiment comprises a method of determining the genotype of a soybean plant, wherein said method comprises obtaining a sample of nucleic acids from a soybean plant and detecting in the nucleic acids, a polymorphism or a SNP, wherein said polymorphism or SNP is identified in SEQ ID NO: 1.

[0080] The term “marker,” “genetic marker,” “molecular marker,” “marker nucleic acid,” and “marker locus” refer to a nucleotide sequence or encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A marker can be derived from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide, and can be represented by one or more particular variant sequences, or by a consensus sequence. In another sense, a marker is an isolated variant or consensus of such a sequence. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A “marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. A “marker locus” is a locus that can be used to track the presence of a second linked locus, e.g., a linked locus that encodes or contributes to expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a locus, such as a QTL, that are genetically or physically linked to the marker locus. Thus, a “marker allele,” alternatively an “allele of a marker locus” is one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population that is polymorphic for the marker locus. Other examples of such markers are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), microsatellite markers (e.g. SSRs), sequence-characterized amplified region (SCAR) markers, cleaved amplified polymorphic sequence (CAPS) markers, insertion / deletion (InDei) markers, or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. The term “single nucleotide polymorphism (SNP)” refers to a change in which a single base in the DNA differs from the usual base at that position. These single base changes are commonly abbreviated as “SNPs.”

[0081] The plants include, but are not limited to, any of the following and can also be treated with one or more chemical compositions, including one or more herbicides. Exemplary chemical231490W001 compositions that can be used with any crop but are often used as follows include but are not limited to:

[0082] Soybean Herbicides: Triketones such as Mesotrione, Topramezone, Sulcatrione, Tembotrione and Isoxaflutole, Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam- Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-) Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, and Glufosinate.

[0083] Fruits / Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam.

[0084] Cereals Herbicides: 2.4-D, Amidosulfuron, Bromoxynil, Carfentrazone-E, Chlorotoluron, Chlorsulfuron, Clodinafop-P, Clopyralid, Dicamba, Diclofop-M, Diflufenican, Fenoxaprop, Florasulam, Flucarb azone-N A, Flufenacet, Flupyrosulfuron-M, Fluroxypyr, Flurtamone, Glyphosate, lodosulfuron, Ioxynil, Isoproturon, MCPA, Mesosulfuron, Metsulfuron, Pendimethalin, Pinoxaden, Propoxycarbazone, Prosulfocarb, Pyroxsulam, Sulfosulfuron, Thifensulfuron, Tralkoxydim, Triasulfuron, Tribenuron, Trifluralin, Tritosulfuron.

[0085] Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S- )Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarb azone, Flufenacet, Pyroxasulfon.

[0086] Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan.

[0087] Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac- sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, FlonicamidFlubendiamide, Triflumuron,Rynaxypyr,Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram,231490W001 gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor.

[0088] Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiam ethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, B-Cyfluthrin, gamma / lambda Cyhalothrin, 4- [[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran.

[0089] Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Cy proconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flusilazole, Fluxapyroxad, Iprodione, Isopyrazam, Mepiquat-chloride, Metconazole, Metominostrobin, Paclobutrazole, Penthiopyrad., Picoxystrobin, Prochloraz, Prothioconazole, Pyraclostrobin, Tebuconazole, Thiophanate-methyl, Trifloxystrobin, Vinclozolin.

[0090] A further embodiment provides for a soybean plant having increased tolerance to HPPD herbicide when compared to a soybean plant that has no tolerance to HPPD herbicide, wherein said increased tolerance is linked to a QTL comprising SEQ ID NO: 1 on Chromosome 14 of Glycine max.

[0091] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1. Sowing soybean plants in a greenhouse

[0092] Soybean seeds were sown in 6-8-inch pots filled with either river sand or Pro-Mix® BX as growth media, and 3-5 plants were targeted for each. The temperature within the greenhouse was set between 20-23 °C, with light supplemented by SOLARSYSTEM® 550 grow lights (California Lightworks, Commercial Series). All soybean plants used were conventional (i.e., non-transgenic) lines that were HPPD inhibiting herbicide susceptible (controls), HPPD inhibiting herbicide tolerant soybean lines, or HPPD inhibiting herbicide sensitive soybean lines.Example 2, Spraying and watering of soybean plants in a greenhouse after sowing

[0093] Within 24 hours after planting, the pots were moved to an open outdoor space and sprayed with CALLISTO® at a calibrated rate of 4X (24 oz / acre product, or 0.76 lb ai / Acre for a 4X application rate - the recommended application rate is 6 oz / acre product, or about 0.19 lb ai / Acre), with a spray volume of ~15 gallons per acre, and then moved back to the greenhouse after drying.231490W001Plants were subsequently hand-watered daily with a controlled amount of water in order to avoid overwatering.Example 3, Rating scale the soybean plants in a greenhouse

[0094] Four weeks after sowing, the soybean plants were rated on a scale of 1-5, using the scale in Table 1.Table 1 : Rating scale of plants affected by HPPD inhibiting herbicide applicationRating Phenotype1 Plant shows no symptoms of leaf damage2 Plant shows light leaf signs and symptoms of leaf damage3 Plant shows moderate signs or symptoms of leaf damage4 Plant shows severe leaf symptoms, but the plant is more likely to survive5 Plant has albino leaves, is dying and unlikely to recoverExample 4, Results from greenhouse screening / assay

[0095] FIGs. la-le show photographs of the soybean plants from the greenhouse assay. FIG. la shows soybean plants having albino leaves, dying and unlikely to recover and having a rating score of 5. FIG. lb shows soybean plants having severe leaf symptoms, however, the plants are expected to recover and having a rating score of 4. FIG. 1c shows soybean plants having moderate signs or symptoms of leaf damage and having a rating score of 3. FIG. Id shows soybean plants with light leaf signs and symptoms of leaf damage and having a rating score of 2. FIG. le shows soybean plants with no symptoms of leaf damage and having a rating score of 1.

[0096] FIG. 2 shows a graph of the correlation of the greenhouse assay among soybean plants tested in the field (x axis) (mean of 2 locations across 2 years) and the greenhouse (y axis) and the greenhouse results are from one experiment in 2023 using the assay of the embodiments. As shown, the greenhouse assay for HPPD inhbiting herbicide tolerance where plants were observed post planting in the greenhouse after being sprayed with HPPD inhbiting herbicide and the field results of where soybean plants were sprayed with HPPD exhibited a 0.69 R2value, indicating a substantial correlation of greenhouse versus trial results for HPPD inhbiting herbicide.

[0097] FIG. 3 shows a graph of the correlation of the greenhouse assay among soybean plants tested in the field (x axis) and the greenhouse (y axis) across three locations: Champaign, Illinois, Lincoln, Nebraska and Nevada, Iowa. FIG. 2 shows the correlation between the greenhouse assay and field HPPD inhbiting herbicide tolerance ratings when using river sand as the growth media, while FIG. 3 shows the correlation between greenhouse assay and field HPPD inhbiting herbicide231490W001 tolerance ratings when using potting mix as a growth media. Both experiments show a positive trend with the correlation between field results and greenhouse results, however, when using the river sand, the correlation appears to be significantly higher.

[0098] As can be shown by the data in FIG. 3, there is approximately a 25% correlation in phenotypes between the greenhouse assay protocol and the field trials for tolerance to HPPD inhbiting herbicide treatment. The results suggest that the assay enables breeders to effectively eliminate 25% of the worst performing (i.e., most susceptible) soybean lines to HPPD inhbiting herbicide treatment, and allows the greenhouse to act as pre-screening tool for soybean lines prior to planting in the field. The greenhouse screening provides a further tool for the effective selection of future generations of soybean for increased tolerance to HPPD inhibiting herbicides and allows for the recommendation of secondary, post-treatment options based on the in initial greenhouse assay results.Table 2. Heritability of the Greenhouse HPPD inhbiting herbicide tolerance RatingsExperiment HeritabilityGH Champaign 0.65GH Lincoln 0.74GH Nevada 0.62Table 3. Correlation of greenhouse assay among 4 locations when compared to field trials of HPPD inhbiting herbicide sprayingCorrelation (R) GreenhouseFLD_Across 0.247FLD Champaign 0.197FLD Lincoln 0.189FLD_Nevada 0.264Example 5, HPPD inhbiting herbicide tolerance linked to Chromosome 14 and SEO ID NO: 1

[0099] FIGs. 4a-4c summarize results for a QTL for HPPD inhbiting herbicide tolerance linked to Chromosome 14. FIG. 4a depicts a Manhattan Plot generated using GWAS and shows a QTL for HPPD inhbiting herbicide tolerance on Chromosome 14 from the greenhouse data from FIG. 3. FIG. 4b depicts the distributions (histogram) of HPPD inhbiting herbicide injury ratings for lines in the greenouse assay from 2024 in FIG. 3 used in the GWAS, and FIG. 4c depicts a bar chart of the phenotypic distribution of the peak marker, SEQ ID NO: 1, among the alternate alleles among the231490W001 greenhouse assay results. After conducting GWAS using the Greenhouse assay HPPD inhbiting herbicide tolerance ratings, a significant QTL was seen for tolerance of HPPD inhbiting herbicide on Chromosome 14. Williams82a2.75 is the reference genome.

[0100] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the embodiments pertain. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0101] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

231490W001CLAIMSWhat is claimed is:Listing of Claims:

1. A method for screening plants in a controlled environment to determine field tolerance to at least one herbicide, wherein said method comprises: sowing one or more plant seeds in one or more containers filled with growth media; placing the one or more containers in a controlled environment under suitable growth conditions; applying an herbicide to the one or more containers; applying water to the one or more containers so that the one or more plant seeds germinate and grow into plants; rating the one or more resulting plants for herbicide tolerance after the plant’s emergence from the soil; and predicting the future performance of herbicide tolerance in the field of the one or more resulting plants or their progeny.

2. A method for screening non-transgenic plants in a controlled environment to determine field tolerance to at least one herbicide, wherein said method comprises: sowing one or more non-transgenic plant seeds in one or more containers filled with growth media; placing the one or more containers in a controlled environment under suitable growth conditions; applying an herbicide to the one or more containers; applying water to the one or more containers so that the one or more plant seeds germinate and grow into plants; rating the one or more resulting plants for herbicide tolerance after the plant’s emergence from the soil; and predicting the future performance of herbicide tolerance in the field of the one or more resulting plants or their progeny.

3. The method of claim 1, further comprising regulating the controlled environment temperature to approximately 20°C to 23 °C.

4. The method of claim 1, further comprising adding a supplemental light source to the controlled environment to promote plant growth.

5. The method of claim 1, wherein the growth media is comprised of sand.231490W0016. The method of claim 1, wherein the growth media is comprised of any one or more of soil, peat moss, coco coir, perlite, vermiculite, pine bark mixes, or a synthetic cultivation medium.

7. The method of claim 1, wherein the herbicide is an HPPD inhibiting herbicide.

8. The method of claim 7, wherein the herbicide is an HPPD inhibiting herbicide is a triketone, pyrazole or isoxazole.

9. The method of claim 7, wherein the rate of HPPD inhibiting herbicide application is at least two times greater than the recommended application rate.

10. The method of claim 7, wherein the rate of HPPD inhibiting herbicide application is at least three times greater than the recommended application rate.

11. The method of claim 7, wherein the rate of HPPD inhibiting herbicide application is at least four times greater than the recommended application rate.

12. The method of claim 1, wherein the controlled environment screening to determine field tolerance to an herbicide correlates with the herbicide tolerance in the field in a statistically significant way.

13. The method of claim 1, wherein the controlled environment assay for herbicide tolerance correlates by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more with the herbicide tolerance in the field.

14. The method of claim 1, wherein the controlled environment screening for herbicide tolerance correlates by at least 50% with the herbicide tolerance in the field.

15. The method of claim 1, wherein said method provides for further post-application herbicide treatments.

16. The method of claim 1, wherein plant yield is improved as a result of choosing the most tolerant plant lines for planting in the field.

17. The method of claim 1, wherein the breeding process of plants lines is improved as a result of the breeding process, choosing the most herbicide tolerant plant lines to move to the next stage of breeding.

18. A method of producing a soybean plant having tolerance to an HPPD inhibiting herbicide as compared to a control plant, wherein the method comprises: isolating nucleic acids from one or more soybean plants; detecting in the nucleic acids, the presence of a genetic marker that is associated with tolerance to an HPPD inhibiting herbicide, wherein said genetic marker comprises SEQ ID NO: 1;231490W001 selecting a first soybean plant based on the presence of the marker associated with HPPD inhibiting herbicide tolerance; crossing a second soybean plant with said first soybean plant, wherein the second soybean plant does not comprise in its genome the marker associated with tolerance to an HPPD inhibiting herbicide; producing seed from said crossing; and selecting a soybean plant grown from said seed.

19. A method of determining the genotype of a soybean plant, wherein said method comprises obtaining a sample of nucleic acids from a soybean plant and detecting in the nucleic acids, a polymorphism, wherein said polymorphism is identified in SEQ ID NO: 1.

20. A marker for detecting tolerance to an HPPD inhibiting herbicide, wherein said marker comprises a single nucleotide polymorphism relative to a reference soybean genome for Glycine max at nucleotide position 17,065,990 on chromosome 14, wherein the reference genome is the Glycine max Williams82a2.75 reference genome.

21. A method of producing a new plant, wherein said method comprises crossing the resultant plants or progeny thereof of claim 1 with a different plant containing a transgene for tolerance to an HPPD inhibiting herbicide, thereby producing a plant having enhanced tolerance to an HPPD inhibiting herbicide.

22. A plant produced by the method of claim 21, wherein said plant is a soybean, corn, cotton or canola plant.

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